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1
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Wang F, Zhou K, Yu Y, Peng L, Ye Y, Lin C, Xu C, Shen Z. RNAi-based transgenic maize to control double-spotted leaf beetle (Monolepta hieroglyphica). PEST MANAGEMENT SCIENCE 2025; 81:1412-1421. [PMID: 39552424 DOI: 10.1002/ps.8543] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/03/2024] [Revised: 10/21/2024] [Accepted: 11/04/2024] [Indexed: 11/19/2024]
Abstract
BACKGROUND The double-spotted leaf beetle (DLB), Monolepta hieroglyphica, is becoming a significant corn pest in China. It mainly attacks corn silk and developing kernels during the adult stage and is causing significant corn yield loss in north-eastern China. The damage caused by DLB is expected to worsen as pesticide usage is likely to decrease along with the upcoming commercial planting of transgenic lepidopteran-resistant maize in China. Therefore, it is highly desirable to develop transgenic corn for DLB resistance. RESULTS Three target genes, MhSsj1, MhSnf7 and MhSec23A were cloned from DLB by their sequence similarity to their corresponding homologous genes known for their effectiveness as RNA interference (RNAi) targets in western corn rootworm (WCR, Diabrotica virgifera virgifera). Injection of the double-stranded RNAs (dsRNAs) of MhSsj1, MhSnf7 and MhSec23A to DLB adults was highly effective to suppress the messenger RNAs (mRNAs) of these genes and resulted in high mortality. Furthermore, a synergistic effect was observed among the dsRNAs of these three target genes. Transgenic maize plants simultaneously transcribing dsRNAs of any two of the three target genes were found to be highly resistant to DLB adults, showcasing the potential of utilizing RNAi-based strategy for transgenic DLB control. CONCLUSION MhSsj1, MhSnf7 and MhSec23A are effective RNAi target genes and transgenic corn based on suppression of these genes by RNAi are effective for controlling adult DLB. © 2024 Society of Chemical Industry.
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Affiliation(s)
- Fahao Wang
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
| | - Kangdi Zhou
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
| | - Yifan Yu
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
| | - Luyao Peng
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
| | - Yuxuan Ye
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
- Zhejiang University Zhongyuan Institute, Zhengzhou, China
| | - Chaoyang Lin
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
| | - Chao Xu
- Hangzhou Ruifeng Biosciences Ltd, Hangzhou, China
| | - Zhicheng Shen
- State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
- Hangzhou Ruifeng Biosciences Ltd, Hangzhou, China
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2
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Zhang Y, Yang T, Yang Y, Xu D, Hu Y, Zhang S, Luo N, Ning L, Ren L. siRNAEfficacyDB: An experimentally supported small interfering RNA efficacy database. IET Syst Biol 2024; 18:199-207. [PMID: 39541343 DOI: 10.1049/syb2.12102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2024] [Revised: 09/26/2024] [Accepted: 10/19/2024] [Indexed: 11/16/2024] Open
Abstract
Small interfering RNA (siRNA) has revolutionised biomedical research and drug development through precise post-transcriptional gene silencing technology. Despite its immense potential, siRNA therapy still faces technical challenges, such as delivery efficiency, targeting specificity, and molecular stability. To address these challenges and facilitate siRNA drug development, we have developed siRNAEfficacyDB, a comprehensive database that integrates experimentally validated siRNA efficacy data. This database contains 3544 siRNA records, covering 42 target genes and 5 cell lines. It provides detailed information on siRNA sequences, target genes, inhibition efficiencies, experimental techniques, cell lines, siRNA concentrations, and incubation times. siRNAEfficacyDB offers a user-friendly web interface that makes it easy to query, browse and analyse data, enabling efficient access to siRNA-related information. In summary, siRNAEfficacyDB provides a useful data foundation for siRNA drug design and optimisation, serving as a valuable resource for advancing computer-aided siRNA design research and nucleic acid drug development. siRNAEfficacyDB is freely available at https://cellknowledge.com.cn/siRNAEfficacy for non-commercial use.
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Affiliation(s)
- Yang Zhang
- Innovative Institute of Chinese Medicine and Pharmacy, Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Ting Yang
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
| | - Yu Yang
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
| | - Dongsheng Xu
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
| | - Yucheng Hu
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
| | - Shuo Zhang
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
| | - Nanchao Luo
- School of Computer Science and Technology, Aba Teachers College, Aba, Sichuan, China
| | - Lin Ning
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
| | - Liping Ren
- School of Healthcare Technology, Chengdu Neusoft University, Chengdu, China
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3
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Zhao JH, Liu QY, Xie ZM, Guo HS. Exploring the challenges of RNAi-based strategies for crop protection. ADVANCED BIOTECHNOLOGY 2024; 2:23. [PMID: 39883232 PMCID: PMC11740845 DOI: 10.1007/s44307-024-00031-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Revised: 06/28/2024] [Accepted: 07/01/2024] [Indexed: 01/31/2025]
Abstract
RNA silencing (or RNA interference, RNAi) initiated by double-stranded RNAs is a conserved mechanism for regulating gene expression in eukaryotes. RNAi-based crop protection strategies, including host-induced gene silencing (HIGS), spray-induced gene silencing (SIGS) and microbe-induced gene silencing (MIGS), have been successfully used against various pests and pathogens. Here, we highlight the challenges surrounding dsRNA design, large-scale production of dsRNA and dsRNA delivery systems. Addressing these questions will accelerate the lab-to-field transition of RNAi-based strategies. Moreover, based on studies of exogenous dsRNA-induced RNAi inheritance in Caenorhabditis elegans, we speculate that RNAi-based strategies would confer longer-lasting protection for crops against pests or fungal pathogens.
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Affiliation(s)
- Jian-Hua Zhao
- State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
| | - Qing-Yan Liu
- State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
| | - Zong-Ming Xie
- Institute of Cotton Research, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi, China
| | - Hui-Shan Guo
- State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China.
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4
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Dalakouras A, Koidou V, Papadopoulou K. DsRNA-based pesticides: Considerations for efficiency and risk assessment. CHEMOSPHERE 2024; 352:141530. [PMID: 38401868 DOI: 10.1016/j.chemosphere.2024.141530] [Citation(s) in RCA: 12] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/15/2023] [Revised: 02/05/2024] [Accepted: 02/21/2024] [Indexed: 02/26/2024]
Abstract
In view of the ongoing climate change and the ever-growing world population, novel agricultural solutions are required to ensure sustainable food supply. Microbials, natural substances, semiochemicals and double stranded RNAs (dsRNAs) are all considered potential low risk pesticides. DsRNAs function at the molecular level, targeting specific regions of specific genes of specific organisms, provided that they share a minimal sequence complementarity of approximately 20 nucleotides. Thus, dsRNAs may offer a great alternative to conventional chemicals in environmentally friendly pest control strategies. Any low-risk pesticide needs to be efficient and exhibit low toxicological potential and low environmental persistence. Having said that, in the current review, the mode of dsRNA action is explored and the parameters that need to be taken into consideration for the development of efficient dsRNA-based pesticides are highlighted. Moreover, since dsRNAs mode of action differs from those of synthetic pesticides, custom-made risk assessment schemes may be required and thus, critical issues related to the risk assessment of dsRNA pesticides are discussed here.
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Affiliation(s)
| | - Venetia Koidou
- ELGO-DIMITRA, Institute of Industrial and Forage Crops, Larissa, Greece; University of Thessaly, Department of Biochemistry and Biotechnology, Larissa, Greece
| | - Kalliope Papadopoulou
- University of Thessaly, Department of Biochemistry and Biotechnology, Larissa, Greece
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5
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Li H, Mo P, Zhang J, Xie Z, Liu X, Chen H, Yang L, Liu M, Zhang H, Wang P, Zhang Z. Methionine biosynthesis enzyme MoMet2 is required for rice blast fungus pathogenicity by promoting virulence gene expression via reducing 5mC modification. PLoS Genet 2023; 19:e1010927. [PMID: 37733784 PMCID: PMC10547190 DOI: 10.1371/journal.pgen.1010927] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Revised: 10/03/2023] [Accepted: 08/20/2023] [Indexed: 09/23/2023] Open
Abstract
The emergence of fungicide resistance severely threatens crop production by limiting the availability and application of established fungicides. Therefore, it is urgent to identify new fungicidal targets for controlling plant diseases. Here, we characterized the function of a conserved homoserine O-acetyltransferase (HOA) from the rice blast fungus Magnaporthe oryzae that could serve as the candidate antifungal target. Deletion of the MoMET2 and MoCYS2 genes encoding HOAs perturbed the biosynthesis of methionine and S-adenyl methionine, a methyl group donor for epigenetic modifications, and severely attenuated the development and virulence of M. oryzae. The ∆Momet2 mutant is significantly increased in 5-methylcytosine (5mC) modification that represses the expression of genes required for pathogenicity, including MoGLIK and MoCDH-CYT. We further showed that host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 effectively controls rice blasts. Our studies revealed the importance of HOA in the development and virulence of M. oryzae, which suggests the potential feasibility of HOA as new targets for novel anti-rice blast measurements.
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Affiliation(s)
- Huimin Li
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
| | - Pengcheng Mo
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
| | - Jun Zhang
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
| | - Zhuoer Xie
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
| | - Xinyu Liu
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
| | - Han Chen
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
| | - Leiyun Yang
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
| | - Muxing Liu
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
| | - Haifeng Zhang
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
| | - Ping Wang
- Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America
| | - Zhengguang Zhang
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
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6
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Isenmann M, Stoddart MJ, Schmelzeisen R, Gross C, Della Bella E, Rothweiler RM. Basic Principles of RNA Interference: Nucleic Acid Types and In Vitro Intracellular Delivery Methods. MICROMACHINES 2023; 14:1321. [PMID: 37512632 PMCID: PMC10383872 DOI: 10.3390/mi14071321] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Revised: 06/23/2023] [Accepted: 06/26/2023] [Indexed: 07/30/2023]
Abstract
Since its discovery in 1989, RNA interference (RNAi) has become a widely used tool for the in vitro downregulation of specific gene expression in molecular biological research. This basically involves a complementary RNA that binds a target sequence to affect its transcription or translation process. Currently, various small RNAs, such as small interfering RNA (siRNA), micro RNA (miRNA), small hairpin RNA (shRNA), and PIWI interacting RNA (piRNA), are available for application on in vitro cell culture, to regulate the cells' gene expression by mimicking the endogenous RNAi-machinery. In addition, several biochemical, physical, and viral methods have been established to deliver these RNAs into the cell or nucleus. Since each RNA and each delivery method entail different off-target effects, limitations, and compatibilities, it is crucial to understand their basic mode of action. This review is intended to provide an overview of different nucleic acids and delivery methods for planning, interpreting, and troubleshooting of RNAi experiments.
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Affiliation(s)
- Marie Isenmann
- Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany
- AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland
| | - Martin James Stoddart
- Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany
- AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland
| | - Rainer Schmelzeisen
- Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany
| | - Christian Gross
- Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany
| | - Elena Della Bella
- AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland
| | - René Marcel Rothweiler
- Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany
- AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland
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7
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Akbarimotlagh M, Azizi A, Shams-Bakhsh M, Jafari M, Ghasemzadeh A, Palukaitis P. Critical points for the design and application of RNA silencing constructs for plant virus resistance. Adv Virus Res 2023; 115:159-203. [PMID: 37173065 DOI: 10.1016/bs.aivir.2023.02.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/15/2023]
Abstract
Control of plant virus diseases is a big challenge in agriculture as is resistance in plant lines to infection by viruses. Recent progress using advanced technologies has provided fast and durable alternatives. One of the most promising techniques against plant viruses that is cost-effective and environmentally safe is RNA silencing or RNA interference (RNAi), a technology that could be used alone or along with other control methods. To achieve the goals of fast and durable resistance, the expressed and target RNAs have been examined in many studies, with regard to the variability in silencing efficiency, which is regulated by various factors such as target sequences, target accessibility, RNA secondary structures, sequence variation in matching positions, and other intrinsic characteristics of various small RNAs. Developing a comprehensive and applicable toolbox for the prediction and construction of RNAi helps researchers to achieve the acceptable performance level of silencing elements. Although the attainment of complete prediction of RNAi robustness is not possible, as it also depends on the cellular genetic background and the nature of the target sequences, some important critical points have been discerned. Thus, the efficiency and robustness of RNA silencing against viruses can be improved by considering the various parameters of the target sequence and the construct design. In this review, we provide a comprehensive treatise regarding past, present and future prospective developments toward designing and applying RNAi constructs for resistance to plant viruses.
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Affiliation(s)
- Masoud Akbarimotlagh
- Plant Pathology Department, Faculty of Agriculture, Tarbiat Modares University (TMU), Tehran, Iran
| | - Abdolbaset Azizi
- Department of Plant Protection, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran.
| | - Masoud Shams-Bakhsh
- Plant Pathology Department, Faculty of Agriculture, Tarbiat Modares University (TMU), Tehran, Iran
| | - Majid Jafari
- Department of Plant Protection, Higher Education Complex of Saravan, Saravan, Iran
| | - Aysan Ghasemzadeh
- Plant Pathology Department, Faculty of Agriculture, Tarbiat Modares University (TMU), Tehran, Iran
| | - Peter Palukaitis
- Department of Horticulture Sciences, Seoul Women's University, Seoul, Republic of Korea.
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Dong X, Zheng W. Cheminformatics Modeling of Gene Silencing for Both Natural and Chemically Modified siRNAs. Molecules 2022; 27:6412. [PMID: 36234948 PMCID: PMC9570765 DOI: 10.3390/molecules27196412] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 09/23/2022] [Accepted: 09/25/2022] [Indexed: 11/17/2022] Open
Abstract
In designing effective siRNAs for a specific mRNA target, it is critically important to have predictive models for the potency of siRNAs. None of the published methods characterized the chemical structures of individual nucleotides constituting a siRNA molecule; therefore, they cannot predict the potency of gene silencing by chemically modified siRNAs (cm-siRNA). We propose a new approach that can predict the potency of gene silencing by cm-siRNAs, which characterizes each nucleotide (NT) using 12 BCUT cheminformatics descriptors describing its charge distribution, hydrophobic and polar properties. Thus, a 21-NT siRNA molecule is described by 252 descriptors resulting from concatenating all the BCUT values of its composing nucleotides. Partial Least Square is employed to develop statistical models. The Huesken data (2431 natural siRNA molecules) were used to perform model building and evaluation for natural siRNAs. Our results were comparable with or superior to those from Huesken's algorithm. The Bramsen dataset (48 cm-siRNAs) was used to build and test the models for cm-siRNAs. The predictive r2 of the resulting models reached 0.65 (or Pearson r values of 0.82). Thus, this new method can be used to successfully model gene silencing potency by both natural and chemically modified siRNA molecules.
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Affiliation(s)
| | - Weifan Zheng
- BRITE Institute and Department of Pharmaceutical Sciences, College of Health and Sciences (CHAS), North Carolina Central University, Durham, NC 27707, USA
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Liu R, Zhang S, Wan R, Deng J, Fang W. Effect of Beclin-1 gene silencing on autophagy and apoptosis of the prostatic hyperplasia epithelial cells. Clinics (Sao Paulo) 2022; 77:100076. [PMID: 36088885 PMCID: PMC9468350 DOI: 10.1016/j.clinsp.2022.100076] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/26/2022] [Revised: 06/06/2022] [Accepted: 06/21/2022] [Indexed: 12/09/2022] Open
Abstract
OBJECTIVES This study aims to explore the effect of silencing Beclin-1 gene on autophagy and apoptosis of Benign Prostatic Hyperplasia (BPH) (BPH-1) cells under the condition of Androgen Deprivation (AD) and Autophagy Inhibition (AI). METHODS Control group (BPH-1 group), empty carrier group (sh-RNA-BPH-1 group) and Beclin-1 silenced group (sh-Beclin1-BPH-1 group) were set. The Beclin-1 gene silencing efficiency was detected by RT-PCR and Western blot. Autophagic flux was monitored by GFP-LC3 cleavage assay and cell apoptosis was analyzed by flow cytometry. The protein expression levels of LC3, Caspase-3, PARP-1, Bcl-2, and Bax were detected by Western blot. RESULTS The transfection of sh-Beclin-1 obviously down-regulated the expression of Beclin-1 at both mRNA and protein levels. Under the conditions of AD and AI, silencing of Beclin-1 restrained the autophagy of BPH-1 cells, as evidenced by a decreased number of autophagosomes and down-regulation of LC3-II protein (p < 0.001). The results of flow cytometry showed that the apoptotic rate of sh-Beclin-1 group was elevated significantly compared to the other two groups (p < 0.01). Western blot results showed that silencing of Beclin-1 promoted 89 kd fragmentation of PARP-1 (p < 0.001) and Caspase-3 activation (p < 0.01). Moreover, silencing of Beclin-1 resulted in declined Bcl-2 and augmented Bax protein expression in BPH-1 cells (p < 0.01), which ultimately led to a decreased Bcl-2/Bax ratio. CONCLUSIONS The results indicated that the silencing of Beclin-1 gene hampered autophagy while activating apoptosis in BPH-1 cells. Thus, Beclin-1 may participate in an antagonistic relationship between autophagy and apoptosis in BPH.
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Affiliation(s)
- Rongfu Liu
- Department of Urology, Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China; Department of Urology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China.
| | - Song Zhang
- Department of Urology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China
| | - Rui Wan
- Department of Urology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China
| | - Jiang Deng
- Department of Urology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China
| | - Wei Fang
- Department of Urology, Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China
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Chen MX, Zhu XD, Zhang H, Liu Z, Liu YN. SMRI: A New Method for siRNA Design for COVID-19 Therapy. JOURNAL OF COMPUTER SCIENCE AND TECHNOLOGY 2022; 37:991-1002. [PMID: 35992496 PMCID: PMC9374573 DOI: 10.1007/s11390-021-0826-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Accepted: 08/31/2021] [Indexed: 06/15/2023]
Abstract
UNLABELLED First discovered in Wuhan, China, SARS-CoV-2 is a highly pathogenic novel coronavirus, which rapidly spread globally and became a pandemic with no vaccine and limited distinctive clinical drugs available till March 13th, 2020. Ribonucleic Acid interference (RNAi) technology, a gene-silencing technology that targets mRNA, can cause damage to RNA viruses effectively. Here, we report a new efficient small interfering RNA (siRNA) design method named Simple Multiple Rules Intelligent Method (SMRI) to propose a new solution of the treatment of COVID-19. To be specific, this study proposes a new model named Base Preference and Thermodynamic Characteristic model (BPTC model) indicating the siRNA silencing efficiency and a new index named siRNA Extended Rules index (SER index) based on the BPTC model to screen high-efficiency siRNAs and filter out the siRNAs that are difficult to take effect or synthesize as a part of the SMRI method, which is more robust and efficient than the traditional statistical indicators under the same circumstances. Besides, to silence the spike protein of SARS-CoV-2 to invade cells, this study further puts forward the SMRI method to search candidate high-efficiency siRNAs on SARS-CoV-2's S gene. This study is one of the early studies applying RNAi therapy to the COVID-19 treatment. According to the analysis, the average value of predicted interference efficiency of the candidate siRNAs designed by the SMRI method is comparable to that of the mainstream siRNA design algorithms. Moreover, the SMRI method ensures that the designed siRNAs have more than three base mismatches with human genes, thus avoiding silencing normal human genes. This is not considered by other mainstream methods, thereby the five candidate high-efficiency siRNAs which are easy to take effect or synthesize and much safer for human body are obtained by our SMRI method, which provide a new safer, small dosage and long efficacy solution for the treatment of COVID-19. SUPPLEMENTARY INFORMATION The online version contains supplementary material available at 10.1007/s11390-021-0826-x.
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Affiliation(s)
- Meng-Xin Chen
- College of Software, Jilin University, Changchun, 130012 China
| | - Xiao-Dong Zhu
- Key Laboratory of Symbolic Computation and Knowledge Engineering of the Ministry of Education, Jilin University, Changchun, 130012 China
| | - Hao Zhang
- College of Computer Science and Technology, Jilin University, Changchun, 130012 China
| | - Zhen Liu
- College of Computer Science and Technology, Jilin University, Changchun, 130012 China
- Graduate School of Engineering, Nagasaki Institute of Applied Science, Nagasaki, 851-0193 Japan
| | - Yuan-Ning Liu
- College of Computer Science and Technology, Jilin University, Changchun, 130012 China
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11
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Xu H, Zhang H, Fan Y, Wang R, Cui R, Liu X, Chu S, Jiao Y, Zhang X, Zhang D. The purple acid phosphatase GmPAP17 predominantly enhances phosphorus use efficiency in soybean. PLANT SCIENCE : AN INTERNATIONAL JOURNAL OF EXPERIMENTAL PLANT BIOLOGY 2022; 320:111283. [PMID: 35643608 DOI: 10.1016/j.plantsci.2022.111283] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/18/2022] [Revised: 04/04/2022] [Accepted: 04/07/2022] [Indexed: 06/15/2023]
Abstract
Purple acid phosphatase (PAP) is an important plant acid phosphatase, which can secrete to the rhizosphere to decompose organophosphorus, promote phosphorus use efficiency, plant growth and development. However, little is known about the functions of intracellular PAP in plants, especially for soybean. Our previous study integrating QTL mapping and transcriptome analysis identified an promising low phosphorus (LP)-induced gene GmPAP17. Here, we determined that GmPAP17 was mainly expressed in roots and had a strong response to LP stress. Furthermore, and the relative expression in the root of LP tolerant genotypes NN94-156 was significantly greater than that of LP sensitive genotype Bogao after LP stress treatment. The overexpression of GmPAP17 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under LP stress condition, it was vice versa for RNAi interference of GmPAP17, indicating that GmPAP17 plays an important role in P use efficiency. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analysis showed that GmRAP2.2 was involved in the regulation network of GmPAP17. Taken together, our results suggest that GmPAP17 is a novel plant PAP that functions in the adaptation of soybean to LP stress, possibly through its involvement in P recycling in plants.
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Affiliation(s)
- Huanqing Xu
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
| | - Hengyou Zhang
- Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China
| | - Yukun Fan
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
| | - Ruiyang Wang
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
| | - Ruifan Cui
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
| | - Xiaoqian Liu
- Ministry of Agriculture and Rural Affairs Key Laboratory of Soybean Biology (Beijing), Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Shanshan Chu
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
| | - Yongqing Jiao
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
| | - Xingguo Zhang
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.
| | - Dan Zhang
- Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.
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12
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He W, Xu W, Fu K, Guo W, Kim DS, Zhang J. Positional effects of double-stranded RNAs targeting β-Actin gene affect RNA interference efficiency in Colorado potato beetle. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 2022; 184:105121. [PMID: 35715059 DOI: 10.1016/j.pestbp.2022.105121] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 05/06/2022] [Accepted: 05/11/2022] [Indexed: 06/15/2023]
Abstract
Pesticide resistance in pests drives the development of RNA interference (RNAi)-based technology as a novel approach for pest control. To investigate the effects of the positional dependency of double-stranded RNAs (dsRNAs), we newly designed four different 200 bp dsRNAs targeting Colorado potato beetle (CPB) β-Actin gene, termed as dsACT200-1 to dsACT200-4, to compare their insecticidal activity to CPB larvae together with our previously used 200 bp and 700 bp dsRNAs (dsACT200 and dsACT700), respectively (He et al., 2020a). Each of dsRNAs harbors different numbers of expected siRNAs predicted by sequence-based prediction platform, dsACT200 and dsACT200-2 have a relatively higher number of siRNA than other 200 bps dsRNAs. When CPB larvae were fed with in vitro synthesized dsRNA-painted potato leaves, all the tested dsRNAs showed significant effects to protect against CPB larvae. Combined with the survival rate of CPB larvae, β-Actin gene expression level and the surviving CPB larvae weight, various positional dsRNAs from the same allele showed different plant protection activity against CPB larvae and partially correlated with the predicted siRNA numbers and distribution on the target sequence. This study suggests the specific allelic locus for rational dsRNA design triggering RNAi efficiency for target gene silencing is an essential factor in enhancing the insecticidal activity.
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Affiliation(s)
- Wanwan He
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China; Wuhan Academy of Agricultural Sciences, Wuhan 430070, China
| | - Wenbo Xu
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China
| | - Kaiyun Fu
- Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi 830000, China
| | - Wenchao Guo
- Institute of Microbial Application, Xinjiang Academy of Agricultural Sciences, Urumqi 830000, China
| | - Dae Sung Kim
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China.
| | - Jiang Zhang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China.
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13
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Niu D, Hamby R, Sanchez JN, Cai Q, Yan Q, Jin H. RNAs - a new frontier in crop protection. Curr Opin Biotechnol 2021; 70:204-212. [PMID: 34217122 PMCID: PMC8957476 DOI: 10.1016/j.copbio.2021.06.005] [Citation(s) in RCA: 53] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Revised: 06/02/2021] [Accepted: 06/07/2021] [Indexed: 12/14/2022]
Abstract
Small RNA (sRNA)-mediated RNA interference (RNAi) is a regulatory mechanism conserved in almost all eukaryotes. sRNAs play a critical role in host pathogen interactions either endogenously or by traveling between the interacting organisms and inducing 'cross-Kingdom RNAi' in the counterparty. Cross-kingdom RNAi is the mechanistic basis of host-induced gene silencing (HIGS), which relies on genetically expressing pathogen-gene targeting RNAs in crops, and has been successfully utilized against both microbial pathogens and pests. HIGS is limited by the need to produce genetically engineered crops. Recent studies have demonstrated that double-stranded RNAs and sRNAs can be efficiently taken up by many fungal pathogens, and induce gene silencing in fungal cells. This mechanism, termed 'environmental RNAi', allows direct application of pathogen-gene targeting RNAs onto crops to silence fungal virulence-related genes for plant protection. In this review, we will focus on how we can leverage cross-kingdom RNAi and environmental RNAi for crop disease control.
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Affiliation(s)
- Dongdong Niu
- College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; Department of Microbiology & Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA
| | - Rachael Hamby
- Department of Microbiology & Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA
| | - Jonatan Nino Sanchez
- Department of Microbiology & Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA
| | - Qiang Cai
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Qin Yan
- College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
| | - Hailing Jin
- Department of Microbiology & Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA.
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14
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Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, Mohd-Azmi ML. Immediately early 2 (IE-2) and DNA polymerase SiRNA as virus-specific antiviral against novel transplacental cytomegalovirus strain ALL-03 in vitro. INFECTION GENETICS AND EVOLUTION 2021; 90:104783. [PMID: 33640483 DOI: 10.1016/j.meegid.2021.104783] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 02/06/2021] [Accepted: 02/23/2021] [Indexed: 10/22/2022]
Abstract
OBJECTIVE This study investigated the suitability of siRNA targeting specific genes that cause inhibition of virus replication in vitro especially for the virus that capable of crossing placenta and we employed a novel transplacental rat cytomegalovirus that mimics infection in human. METHODS Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells. FINDINGS Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300 pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P < 0.05). CONCLUSION In conclusion, this study clearly highlighted the feasibility of RNAi as an alternative antiviral therapy that could lead to controlling the CMV infection.
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Affiliation(s)
- Krishnan Nair Balakrishnan
- Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University Putra Malaysia, Selangor, Malaysia.
| | - Ashwaq Ahmed Abdullah
- Department of Microbiology, Faculty of Applied Science, Taiz University, Taiz, Yemen
| | - Jamilu Abubakar Bala
- Microbiology Unit, Department of Medical Laboratory Science, Faculty of Allied Health Sciences, Bayero University Kano, Nigeria
| | - Faez Firdaus Abdullah Jesse
- Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia
| | | | - Mustapha Mohamed Noordin
- Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University Putra Malaysia, Selangor, Malaysia
| | - Mohd Lila Mohd-Azmi
- Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University Putra Malaysia, Selangor, Malaysia.
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15
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Ali A, Swanepoel CM, Winger QA, Rozance PJ, Anthony RV. Chorionic somatomammotropin RNA interference alters fetal liver glucose utilization. J Endocrinol 2020; 247:251-262. [PMID: 33108344 PMCID: PMC7643541 DOI: 10.1530/joe-20-0375] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 09/22/2020] [Indexed: 12/20/2022]
Abstract
Chorionic somatomammotropin (CSH) is a placenta-specific hormone associated with fetal growth, and fetal and maternal metabolism in both humans and sheep. We hypothesized that CSH deficiency could impact sheep fetal liver glucose utilization. To generate CSH-deficient pregnancies, day 9 hatched blastocysts were infected with lentiviral particles expressing CSH-specific shRNA (RNAi) or scramble control shRNA (SC) and transferred to synchronized recipients. CSH RNAi generated two distinct phenotypes at 135 days of gestational age (dGA); pregnancies with IUGR (RNAi-IUGR) or with normal fetal weight (RNAi-NW). Fetal body, fetal liver and placental weights were reduced (P < 0.05) only in RNAi-IUGR pregnancies compared to SC. Umbilical artery plasma insulin and insulin-like growth factor 1 (IGF1) concentrations were decreased, whereas insulin receptor beta (INSR) concentration in fetal liver was increased (P < 0.05) in both RNAi phenotypes. The mRNA concentrations of IGF1, IGF2, IGF binding protein 2 (IGFBP2) and IGFBP3 were decreased (P < 0.05) in fetal livers from both RNAi phenotypes. Fetal liver glycogen concentration and glycogen synthase 1 (GYS1) concentration were increased (P < 0.05), whereas fetal liver phosphorylated-GYS (inactive GYS) concentration was reduced (P < 0.05) in both RNAi phenotypes. Lactate dehydrogenase B (LDHB) concentration was increased (P < 0.05) and IGF2 concentration was decreased (P < 0.05) in RNAi-IUGR fetal livers only. Our findings suggest that fetal liver glucose utilization is impacted by CSH RNAi, independent of IUGR, and is likely tied to enhanced fetal liver insulin sensitivity in both RNAi phenotypes. Determining the physiological ramifications of both phenotypes, may help to differentiate direct effect of CSH deficiency or its indirect effect through IUGR.
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Affiliation(s)
- Asghar Ali
- Colorado State University, Animal Reproduction and Biotechnology Lab, Fort Collins, CO, US
| | - Callie M. Swanepoel
- Colorado State University, Animal Reproduction and Biotechnology Lab, Fort Collins, CO, US
| | - Quinton A. Winger
- Colorado State University, Animal Reproduction and Biotechnology Lab, Fort Collins, CO, US
| | - Paul J. Rozance
- University of Colorado Anschutz Medical Campus, Perinatal Research Center, Aurora, CO, US
| | - Russell V. Anthony
- Colorado State University, Animal Reproduction and Biotechnology Lab, Fort Collins, CO, US
- Corresponding Author: 1683 Campus Delivery, 3107 Rampart Road, Colorado State University, Fort Collins, CO 80523-1683. Telephone: 970-491-2586; FAX: 970-491-3557;
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16
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Carli GJD, Rotela AT, Lubini G, Contiliani DF, Candia NB, Depintor TS, Abreu FCPD, Simões ZLP, Ríos DF, Pereira TC. SSD - a free software for designing multimeric mono-, bi- and trivalent shRNAs. Genet Mol Biol 2020; 43:e20190300. [PMID: 32141472 PMCID: PMC7197978 DOI: 10.1590/1678-4685-gmb-2019-0300] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2019] [Accepted: 01/13/2020] [Indexed: 02/06/2023] Open
Abstract
RNA interference (RNAi) is a powerful gene silencing technology, widely used in
analyses of reverse genetics, development of therapeutic strategies and
generation of biotechnological products. Here we present a free software tool
for the rational design of RNAi effectors, named siRNA and shRNA designer (SSD).
SSD incorporates our previously developed software Strand Analysis to construct
template DNAs amenable for the large scale production of mono-, bi- and
trivalent multimeric shRNAs, via in vitro rolling circle
transcription. We tested SSD by creating a trivalent multimeric shRNA against
the vitellogenin gene of Apis mellifera. RT-qPCR analysis
revealed that our molecule promoted a decrease in more than 50% of the target
mRNA, in a dose-dependent manner, when compared to the control group. Thus, SSD
software allows the easy design of multimeric shRNAs, for single or multiple
simultaneous knockdowns, which is especially interesting for studies involving
large amounts of double-stranded molecules.
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Affiliation(s)
- Gabriel José de Carli
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.,Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
| | - Abdon Troche Rotela
- Universidad Nacional de Asunción, Facultad de Ciencias Exactas y Naturales, San Lorenzo, Paraguay.,Universidad Nacional de Asunción, Facultad Politécnica, San Lorenzo, Paraguay
| | - Greice Lubini
- Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
| | - Danyel Fernandes Contiliani
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.,Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
| | - Nidia Benítez Candia
- Universidad Nacional de Asunción, Facultad de Ciencias Exactas y Naturales, San Lorenzo, Paraguay
| | - Thiago S Depintor
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil
| | - Fabiano Carlos Pinto de Abreu
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.,Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
| | - Zilá Luz Paulino Simões
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.,Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
| | - Danilo Fernández Ríos
- Universidad Nacional de Asunción, Facultad de Ciencias Exactas y Naturales, San Lorenzo, Paraguay
| | - Tiago Campos Pereira
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.,Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
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17
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Cytoplasmic expression of EGFR shRNA using a modified T7 autogene-based hybrid mRNA/DNA system induces long-term EGFR silencing and prolongs antitumor effects. Biochem Pharmacol 2020; 171:113735. [DOI: 10.1016/j.bcp.2019.113735] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Accepted: 11/26/2019] [Indexed: 12/18/2022]
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18
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Posiri P, Thongsuksangcharoen S, Chaysri N, Panyim S, Ongvarrasopone C. PmEEA1, the early endosomal protein is employed by YHV for successful infection in Penaeus monodon. FISH & SHELLFISH IMMUNOLOGY 2019; 95:449-455. [PMID: 31678535 DOI: 10.1016/j.fsi.2019.10.054] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Revised: 10/22/2019] [Accepted: 10/27/2019] [Indexed: 06/10/2023]
Abstract
Yellow head disease (YHD) is an infectious disease of Penaeus monodon which is caused by the yellow head virus (YHV). YHV infection invariably leads to 100% shrimp mortality within 3-5 days. Currently, an effective method to prevent or cure shrimp from YHV infection has not been elucidated. Therefore, the molecular mechanism underlying YHV infection should be examined. In this study, early endosome antigen 1 (EEA1) protein that was involved in the tethering step of the vesicle and early endosome fusion was investigated during YHV infection. The open reading frame of P. monodon EEA1 (PmEEA1) was cloned and sequenced (3000 bp). It encoded a putative protein of 999 amino acids and contained the zinc finger C2H2 domain signature at the N-terminus and the FYVE domain at the C-terminus. Suppression of PmEEA1 by specific dsRNA in shrimp showed inhibition of YHV replication after 48 h post YHV injection (hpi). On the other hand, shrimp received only NaCl without any dsRNA showed high YHV levels at approximately one hundred thousand times at 24 hpi and 48 hpi. Moreover, silencing of PmEEA1 by specific dsRNA followed by YHV challenge demonstrated a delay in shrimp mortality from 60 hpi to 168 hpi when compared to the control. These results indicated that YHV required PmEEA1 for trafficking within the infected cells, strongly suggesting that PmEEA1 may be a potential target to control and prevent YHV infection in P. monodon.
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Affiliation(s)
- Pratsaneeyaporn Posiri
- Institute of Molecular Biosciences, Mahidol University (Salaya Campus), Nakhon Pathom, 73170, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, 12120, Thailand
| | | | - Nattawadee Chaysri
- Institute of Molecular Biosciences, Mahidol University (Salaya Campus), Nakhon Pathom, 73170, Thailand
| | - Sakol Panyim
- Institute of Molecular Biosciences, Mahidol University (Salaya Campus), Nakhon Pathom, 73170, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand
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19
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Kwak SY, Lee S, Han HD, Chang S, Kim KP, Ahn HJ. PLGA Nanoparticles Codelivering siRNAs against Programmed Cell Death Protein-1 and Its Ligand Gene for Suppression of Colon Tumor Growth. Mol Pharm 2019; 16:4940-4953. [DOI: 10.1021/acs.molpharmaceut.9b00826] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Affiliation(s)
- Seo Young Kwak
- Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, South Korea
| | | | - Hee Dong Han
- Department of Immunology, School of Medicine, Konkuk University, Chungju 27478, South Korea
| | - Suhwan Chang
- Department of Biomedical Sciences, Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, South Korea
| | | | - Hyung Jun Ahn
- Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, South Korea
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20
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Chen Z, Krishnamachary B, Pachecho-Torres J, Penet MF, Bhujwalla ZM. Theranostic small interfering RNA nanoparticles in cancer precision nanomedicine. WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY 2019; 12:e1595. [PMID: 31642207 DOI: 10.1002/wnan.1595] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/27/2019] [Revised: 09/10/2019] [Accepted: 09/11/2019] [Indexed: 12/24/2022]
Abstract
Due to their ability to effectively downregulate the expression of target genes, small interfering RNA (siRNA) have emerged as promising candidates for precision medicine in cancer. Although some siRNA-based treatments have advanced to clinical trials, challenges such as poor stability during circulation, and less than optimal pharmacokinetics and biodistribution of siRNA in vivo present barriers to the systemic delivery of siRNA. In recent years, theranostic nanomedicine integrating siRNA delivery has attracted significant attention for precision medicine. Theranostic nanomedicine takes advantage of the high capacity of nanoplatforms to ferry cargo with imaging and therapeutic capabilities. These theranostic nanoplatforms have the potential to play a major role in gene specific treatments. Here we have reviewed recent advances in the use of theranostic nanoplatforms to deliver siRNA, and discussed the opportunities as well as challenges associated with this exciting technology. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Implantable Materials and Surgical Technologies > Nanomaterials and Implants.
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Affiliation(s)
- Zhihang Chen
- Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Balaji Krishnamachary
- Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Jesus Pachecho-Torres
- Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Marie-France Penet
- Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland.,Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Zaver M Bhujwalla
- Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland.,Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland.,Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland
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21
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Yang X, Wu X, Yang Y, Gu T, Hong L, Zheng E, Xu Z, Zeng F, Shi J, Zhou R, Cai G, Wu Z, Li Z. Improvement of developmental competence of cloned male pig embryos by short hairpin ribonucleic acid (shRNA) vector-based but not small interfering RNA (siRNA)-mediated RNA interference (RNAi) of Xist expression. J Reprod Dev 2019; 65:533-539. [PMID: 31631092 PMCID: PMC6923154 DOI: 10.1262/jrd.2019-070] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Xist is an X-linked ribonucleic acid (RNA) gene responsible for the cis induction of X chromosome inactivation (XCI). In cloned mammalian embryos, Xist is
ectopically activated at the morula to blastocyst stage on the X chromosome that is supposed to be active, thus resulting in abnormal XCI. Suppression of erroneous Xist
expression by injecting small interfering RNA (siRNA) remarkably increased the developmental efficiency of cloned male mouse embryos by approximately 10-fold. However, injection of
anti-Xist siRNA resulted in only a slight increase in the developmental ability of injected cloned male pig embryos because the blocking effect of the injected siRNA was
not maintained beyond the morula stage, which is 5 days post-activation. To develop a more effective approach for suppressing the ectopic expression of Xist in cloned pig
embryos, we compared the silencing effect of short hairpin RNA (shRNA) and siRNA on Xist expression and the effects of these two Xist knockdown methods on
the developmental competence of cloned male pig embryos. Results indicated that an shRNA-based RNA interference (RNAi) has a longer blocking effect on Xist expression than
an siRNA-mediated RNAi. Injection of anti-Xist shRNA plasmid into two-cell-stage cloned male pig embryos effectively suppressed Xist expression, rescued XCI
at the blastocyst stage, and improved the in vitro developmental ability of injected cloned embryos. These positive effects, however, were not observed in cloned male pig
embryos injected with anti-Xist siRNA. This study demonstrates that vector-based rather than siRNA-mediated RNAi of Xist expression can be employed to
improve pig cloning efficiency.
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Affiliation(s)
- Xuqiong Yang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Xiao Wu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Yang Yang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Ting Gu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Linjun Hong
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Enqin Zheng
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Zheng Xu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Fang Zeng
- College of Marine Sciences, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Junsong Shi
- Guangdong Wens Pig Breeding Technology Co., Ltd., Wens Foodstuff Group Co., Ltd., Yunfu 527400, Guangdong, China
| | - Rong Zhou
- Guangdong Wens Pig Breeding Technology Co., Ltd., Wens Foodstuff Group Co., Ltd., Yunfu 527400, Guangdong, China
| | - Gengyuan Cai
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Zhenfang Wu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
| | - Zicong Li
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, China
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22
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Koizumi M, Hirota Y, Nakayama M, Tamura M, Obuchi W, Kurimoto A, Tsuchida H, Maeda H. Design of 2'- O-methyl RNA and DNA double-stranded oligonucleotides: naturally-occurring nucleotide components with strong RNA interference gene expression inhibitory activity. NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 2019; 39:292-309. [PMID: 31509065 DOI: 10.1080/15257770.2019.1663384] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.
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Affiliation(s)
- Makoto Koizumi
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Yasuhide Hirota
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Makiko Nakayama
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Masakazu Tamura
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Wataru Obuchi
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Akiko Kurimoto
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Hiroshi Tsuchida
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
| | - Hiroaki Maeda
- R&D and Biologics Divisions, Daiichi Sankyo Co. Ltd, Shinagawa, Tokyo, Japan
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23
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Suresh A, Reddy IJ, Mishra A, Mondal S. Suppression of COX-2 mRNA abundance in in vitro cultured goat (Capra hircus) endometrial cells by RNA interference and effect on PGF2-α and PGE2 concentrations. Anim Reprod Sci 2019; 209:106146. [PMID: 31514936 DOI: 10.1016/j.anireprosci.2019.106146] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 06/07/2019] [Accepted: 08/02/2019] [Indexed: 01/22/2023]
Abstract
Cyclooxygenase-2 (COX-2) has important functions in the synthesis and release of endometrial prostaglandin F2α (PGF2α). Excessive production of COX-2 leads to an increase in endometrial PGF2α synthesis and subsequently causes luteolysis and early embryonic mortality. The aim of this study was to investigate in goats the effects of COX-2 small interference RNA (siRNA) on COX-2 mRNA abundance and the secretion of PGF2α and PGE2 in goat endometrial cells. Endometrial cells isolated from goat uteri were cultured at 38.5 °C and 5% CO2. The cells were treated with different concentrations (0, 10, 25, 50, 100, 250, 500, 750 and 1000 nM per well) of three different COX-2 siRNAs at confluency for 24 h. At 24 h post culture, COX-2 mRNA abundance was quantified using qPCR and PGF2α and PGE2 concentrations were quantified in the culture medium. There was a lesser relative abundance of COX-2 mRNA in endometrial cells at 100 to 1000 nM siRNA. The greatest extent of abundance suppression, however, was observed with 1000 nM siRNA. Transfection of COX-2 siRNA (1000 nM) to endometrial cells suppressed the COX-2 mRNA abundance by 77%, 82%, and 84% with siRNA 1, 2, 3, respectively. Furthermore, with COX-2 siRNA transfected cells, there was a lesser (P < 0.05) PGF2α concentration than in cells not transfected, whereas PGE2 secretion was not affected. The results of the study provide evidence that COX-2 siRNA used in this study suppresses COX-2 mRNA abundance and PGF2α secretion but there was no association between PGE2 concentrations and COX-2 mRNA abundance in goat endometrial epithelial cells.
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Affiliation(s)
- Arul Suresh
- Animal Physiology Division, ICAR-National Dairy Research Institute, SRS, Bengaluru, Karnataka 560030, India
| | - I J Reddy
- Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, Karnataka 560030, India.
| | - Ashish Mishra
- Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, Karnataka 560030, India
| | - S Mondal
- Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, Karnataka 560030, India
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24
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Lee S, Ahn HJ. Anti-EpCAM-conjugated adeno-associated virus serotype 2 for systemic delivery of EGFR shRNA: Its retargeting and antitumor effects on OVCAR3 ovarian cancer in vivo. Acta Biomater 2019; 91:258-269. [PMID: 31026519 DOI: 10.1016/j.actbio.2019.04.044] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Revised: 04/01/2019] [Accepted: 04/22/2019] [Indexed: 12/16/2022]
Abstract
Adeno-associated virus (AAV) is a promising vector for systemic delivery of siRNA because of its long-term expression ability without immunogenicity and pathogenicity. However, its broad host tropism and lack of tissue specificity have limited clinical applications such as cancer therapy. Therefore, redirecting the natural tropism of AAV vectors to unique cell surface antigens is an important requirement for in vivo RNAi-based cancer therapy. To use the overexpression property of epithelial cell adhesion molecule (EpCAM) in specific cancer types, we herein created anti-EpCAM antibody-conjugated AAV serotype 2 (AAV2) vectors through a streptavidin-biotin bridge. Upon intravenous injection, anti-EpCAM-conjugated AAV2 vectors showed prominent tumor-specific accumulation in EpCAM-positive tumor-bearing mice without undesirable sequestration in liver. In addition, when loaded with transgenes to express shRNA against epidermal growth factor receptor (EGFR), systemically injected anti-EpCAM-conjugated AAV2/shEGFR vectors induced significant downregulation of EGFR expression in tumors and eventually suppressed tumor growth even at the long dosing interval of two weeks. This in vivo antitumor effect represents the increased infection efficacy of tropism-modified AAV2 vectors and prolonged expression of EGFR shRNA in tumor tissues. Thus, this study suggests the great potential of anti-EpCAM-conjugated AAV2/shEGFR vectors as RNAi-based cancer therapeutics. STATEMENT OF SIGNIFICANCE: Adeno-associated virus (AAV) is a promising vector for systemic delivery of siRNA, but its broad host tropism has limited clinical applications. By using the overexpression property of epithelial cell adhesion molecule (EpCAM) on tumors, we demonstrate that anti-EpCAM-conjugated AAV2 vectors through a streptavidin-biotin bridge are redirected to EpCAM-positive tumors in vivo. In addition, when loaded with transgenes to express shRNA against epidermal growth factor receptor (EGFR), systemically injected anti-EpCAM-conjugated AAV2/shEGFR vectors significantly downregulate EGFR expression in tumors, eventually suppressing tumor growth for long periods. We herein suggest the potential of anti-EpCAM-AAV2/shEGFR vectors as an antitumor agent. Furthermore, redirection of AAV2 infection through EpCAM would provide a powerful means for systemic delivery of short hairpin RNA to tumor sites.
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25
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Guo XY, Li Y, Fan J, Xiong H, Xu FX, Shi J, Shi Y, Zhao JQ, Wang YF, Cao XL, Wang WM. Host-Induced Gene Silencing of MoAP1 Confers Broad-Spectrum Resistance to Magnaporthe oryzae. FRONTIERS IN PLANT SCIENCE 2019; 10:433. [PMID: 31024598 PMCID: PMC6465682 DOI: 10.3389/fpls.2019.00433] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/20/2018] [Accepted: 03/21/2019] [Indexed: 05/21/2023]
Abstract
Rice blast caused by Magnaporthe oryzae (M. oryzae) is a major threat to global rice production. In recent years, small interference RNAs (siRNAs) and host-induced gene silencing (HIGS) has been shown to be new strategies for the development of transgenic plants to control fungal diseases and proved a useful tool to study gene function in pathogens. We here tested whether in vitro feeding artificial siRNAs (asiRNAs) could compromise M. oryzae virulence and in vivo HIGS technique could improve rice blast resistance. Our data revealed that silencing of M. oryzae MoAP1 by feeding asiRNAs targeting MoAP1 (i.e., asiR1245, asiR1362, and asiR1115) resulted in inhibited fungal growth, abnormal spores, and decreased pathogenicity. Among the asiRNAs, asiR1115 was the most inhibitory toward the rice blast fungus. Conversely, the asiRNAs targeting three other genes (i.e., MoSSADH, MoACT, and MoSOM1) had no effect on fungal growth. Transgenic rice plants expressing RNA hairpins targeting MoAP1 exhibited improved resistance to 11 tested M. oryzae strains. Confocal microscopy also revealed profoundly restricted appressoria and mycelia in rice blast-infected transgenic rice plants. Our results demonstrate that in vitro asiRNA and in vivo HIGS were useful protection approaches that may be valuable to enhance rice blast resistance.
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Affiliation(s)
- Xiao-Yi Guo
- Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sciences/Key Laboratory of Southwest Rice Biology and Genetic Breeding, Ministry of Agriculture, Deyang, China
| | - Yan Li
- Rice Research Institute, Sichuan Agricultural University, Chengdu, China
| | - Jing Fan
- Rice Research Institute, Sichuan Agricultural University, Chengdu, China
| | - Hong Xiong
- Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sciences/Key Laboratory of Southwest Rice Biology and Genetic Breeding, Ministry of Agriculture, Deyang, China
| | - Fu-Xian Xu
- Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sciences/Key Laboratory of Southwest Rice Biology and Genetic Breeding, Ministry of Agriculture, Deyang, China
| | - Jun Shi
- Mianyang Academy of Agricultural Sciences, Mianyang, China
| | - Yi Shi
- Rice Research Institute, Sichuan Agricultural University, Chengdu, China
| | - Ji-Qun Zhao
- Rice Research Institute, Sichuan Agricultural University, Chengdu, China
| | - Yi-Fu Wang
- Mianyang Academy of Agricultural Sciences, Mianyang, China
| | - Xiao-Long Cao
- Mianyang Academy of Agricultural Sciences, Mianyang, China
| | - Wen-Ming Wang
- Rice Research Institute, Sichuan Agricultural University, Chengdu, China
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26
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Guru Vishnu P, Bhattacharya TK, Bhushan B, Kumar P, Chatterjee RN, Paswan C, Dushyanth K, Divya D, Prasad AR. In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference. Mol Biol Rep 2019; 46:2947-2959. [PMID: 30879273 DOI: 10.1007/s11033-019-04756-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Accepted: 03/08/2019] [Indexed: 12/26/2022]
Abstract
Gene silencing by RNA interference is extensively used reverse genetic approach to analyse the implications of any gene in mammalian systems. The silencing of the Activin type IIB receptor belonging to transforming growth factor beta superfamily has demonstrated increase in muscle growth in many species. We designed five short hairpin RNA constructs targeting coding region of chicken ACTRIIB. All the shRNAs were transfected into chicken embryo fibroblast cells and evaluated their silencing efficiency by real time PCR and western blotting. Initially the computational analysis of target region and shRNA constructs was undertaken to predict sequence based features (secondary structures, GC% and H-b index) and thermodynamic features (ΔGoverall, ΔGduplex, ΔGbreak-target, ΔGintra-oligomer, ΔGinter-oligomer and ΔΔGends). We determined that all these predicted features were associated with shRNA efficacy. The invitro analysis of shRNA constructs exhibited significant (P < 0.05) reduction in the levels of ACTRIIB at mRNA and protein level. The knock down efficiency of shRNAs varied significantly (P < 0.001) from 83% (shRNA 1) to 43% (shRNA 5). All the shRNAs up regulated the myogenic pathway associated genes (MyoD and MyoG) significantly (P < 0.05). There was significant (P < 0.05) up-regulation of IFNA, IFNB and MHCII transcripts. The ACTRIIB expression was inversely associated with the expression of myogenic pathway and immune response genes. The anti ACTRIIB shRNA construct 1 and 3 exhibited maximum knock down efficiency with minimal interferon response, and can be used for generating ACTRIIB knockdown chicken with higher muscle mass.
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Affiliation(s)
- P Guru Vishnu
- Sri Venkateswara Veterinary University, Tirupathi, A.P., India.
| | | | - Bharat Bhushan
- Division of Animal Genetics & Breeding, Indian Veterinary Research Institute, Izatnagar, U.P., India
| | - Pushpendra Kumar
- Division of Animal Genetics & Breeding, Indian Veterinary Research Institute, Izatnagar, U.P., India
| | | | | | - K Dushyanth
- ICAR-Directorate of Poultry Research, Hyderabad, India
| | - D Divya
- ICAR-Directorate of Poultry Research, Hyderabad, India
| | - A Rajendra Prasad
- Division of Animal Genetics & Breeding, Indian Veterinary Research Institute, Izatnagar, U.P., India
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27
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Hu X, Ke G, Jang SRJ. Modeling Pancreatic Cancer Dynamics with Immunotherapy. Bull Math Biol 2019; 81:1885-1915. [PMID: 30843136 DOI: 10.1007/s11538-019-00591-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2018] [Accepted: 02/22/2019] [Indexed: 12/11/2022]
Abstract
We develop a mathematical model of pancreatic cancer that includes pancreatic cancer cells, pancreatic stellate cells, effector cells and tumor-promoting and tumor-suppressing cytokines to investigate the effects of immunotherapies on patient survival. The model is first validated using the survival data of two clinical trials. Local sensitivity analysis of the parameters indicates there exists a critical activation rate of pro-tumor cytokines beyond which the cancer can be eradicated if four adoptive transfers of immune cells are applied. Optimal control theory is explored as a potential tool for searching the best adoptive cellular immunotherapies. Combined immunotherapies between adoptive ex vivo expanded immune cells and TGF-[Formula: see text] inhibition by siRNA treatments are investigated. This study concludes that mono-immunotherapy is unlikely to control the pancreatic cancer and combined immunotherapies between anti-TGF-[Formula: see text] and adoptive transfers of immune cells can prolong patient survival. We show through numerical explorations that how these two types of immunotherapies are scheduled is important to survival. Applying TGF-[Formula: see text] inhibition first followed by adoptive immune cell transfers can yield better survival outcomes.
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Affiliation(s)
- Xiaochuan Hu
- Department of Mathematics and Statistics, Texas Tech University, Lubbock, TX, 79409-1042, USA.,Department of Mathematics and Statistics, University of the Incarnate Word, San Antonio, TX, 78209, USA
| | - Guoyi Ke
- Department of Mathematics and Statistics, Texas Tech University, Lubbock, TX, 79409-1042, USA.,Department of Mathematics and Physical Sciences, Louisiana State University of Alexandria, Alexandria, LA, 71302, USA
| | - Sophia R-J Jang
- Department of Mathematics and Statistics, Texas Tech University, Lubbock, TX, 79409-1042, USA.
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28
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Kwak SY, Han HD, Ahn HJ. A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry. Sci Rep 2019; 9:2993. [PMID: 30816180 PMCID: PMC6395690 DOI: 10.1038/s41598-019-39407-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2018] [Accepted: 01/24/2019] [Indexed: 12/30/2022] Open
Abstract
The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.
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Affiliation(s)
- Seo Young Kwak
- Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, South Korea
| | - Hee Dong Han
- Department of Immunology, School of Medicine, Konkuk University, Chungju, South Korea
| | - Hyung Jun Ahn
- Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, South Korea.
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29
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Madureira TV, Pinheiro I, Malhão F, Castro LFC, Rocha E, Urbatzka R. Silencing of PPARαBb mRNA in brown trout primary hepatocytes: effects on molecular and morphological targets under the influence of an estrogen and a PPARα agonist. Comp Biochem Physiol B Biochem Mol Biol 2018; 229:1-9. [PMID: 30528668 DOI: 10.1016/j.cbpb.2018.12.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Accepted: 12/01/2018] [Indexed: 12/23/2022]
Abstract
The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol - EE2) and a PPARα agonist (Wy-14,643 - Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.
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Affiliation(s)
- Tânia Vieira Madureira
- Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U.Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal; Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U.Porto), Laboratory of Histology and Embryology, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal
| | - Ivone Pinheiro
- Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U.Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal; Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U.Porto), Laboratory of Histology and Embryology, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal
| | - Fernanda Malhão
- Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U.Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal; Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U.Porto), Laboratory of Histology and Embryology, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal
| | - L Filipe C Castro
- Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U.Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal; Department of Biology, University of Porto (U.Porto), Rua do Campo Alegre, P 4169-007 Porto, Portugal
| | - Eduardo Rocha
- Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U.Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal; Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U.Porto), Laboratory of Histology and Embryology, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal
| | - Ralph Urbatzka
- Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U.Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal.
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30
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In-vitro inhibition of spring viremia of carp virus replication by RNA interference targeting the RNA-dependent RNA polymerase gene. J Virol Methods 2018; 263:14-19. [PMID: 30336160 DOI: 10.1016/j.jviromet.2018.10.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2018] [Revised: 09/20/2018] [Accepted: 10/12/2018] [Indexed: 11/23/2022]
Abstract
Spring viremia of carp, a fatal viral disease, is caused by the spring viremia of carp virus (SVCV) and can result in up to 70% mortalities in common carps and significant economic losses in several other cyprinid aquaculture. The present study aimed to investigate the possible control of SVCV replication in Epithelioma papulosum cyprini (EPC) cells using the RNA interference technology targeting the RNA-dependent RNA polymerase (L) gene of the SVCV that is essential for its replication. Three stealth small interfering RNA (siRNA) sequences were designed to target three different regions on the SVCV-L gene. The specific siRNAs designed were investigated individually or in combinations to inhibit the SVCV-L gene expression and the virus replication. Results showed that the most effective siRNA sequence was the siRNA-602 that specifically reduced the SVCV replication by two logs as indicated by the virus titration and quantitative real-time PCR. Results, also, showed that the minimum effective concentration of siRNA-602 was 20 nM when used to transfect the EPC cells before the virus inoculation. Results of this study clearly indicate that targeting the SVCV-L gene by RNAi can reduce the SVCV replication in vitro, that may lead to the control of SVCV in fish.
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31
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Yan Y, Acevedo M, Mignacca L, Desjardins P, Scott N, Imane R, Quenneville J, Robitaille J, Feghaly A, Gagnon E, Ferbeyre G, Major F. The sequence features that define efficient and specific hAGO2-dependent miRNA silencing guides. Nucleic Acids Res 2018; 46:8181-8196. [PMID: 30239883 PMCID: PMC6144789 DOI: 10.1093/nar/gky546] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2018] [Revised: 05/10/2018] [Accepted: 06/05/2018] [Indexed: 01/18/2023] Open
Abstract
MicroRNAs (miRNAs) are ribonucleic acids (RNAs) of ∼21 nucleotides that interfere with the translation of messenger RNAs (mRNAs) and play significant roles in development and diseases. In bilaterian animals, the specificity of miRNA targeting is determined by sequence complementarity involving the seed. However, the role of the remaining nucleotides (non-seed) is only vaguely defined, impacting negatively on our ability to efficiently use miRNAs exogenously to control gene expression. Here, using reporter assays, we deciphered the role of the base pairs formed between the non-seed region and target mRNA. We used molecular modeling to reveal that this mechanism corresponds to the formation of base pairs mediated by ordered motions of the miRNA-induced silencing complex. Subsequently, we developed an algorithm based on this distinctive recognition to predict from sequence the levels of mRNA downregulation with high accuracy (r2 > 0.5, P-value < 10-12). Overall, our discovery improves the design of miRNA-guide sequences used to simultaneously downregulate the expression of multiple predetermined target genes.
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Affiliation(s)
- Yifei Yan
- Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Mariana Acevedo
- Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Lian Mignacca
- Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Philippe Desjardins
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Nicolas Scott
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
- Département d’informatique et de recherche opérationnelle, Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Roqaya Imane
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Jordan Quenneville
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Julie Robitaille
- Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Albert Feghaly
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Etienne Gagnon
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
- Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - Gerardo Ferbeyre
- Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
| | - François Major
- Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada
- Département d’informatique et de recherche opérationnelle, Université de Montréal, Montréal, Québec H3C 3J7, Canada
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Chen X, Zhu Z, Yu F, Huang J, Jia R, Pan J. Effect of shRNA-mediated Xist knockdown on the quality of porcine parthenogenetic embryos. Dev Dyn 2018; 248:140-148. [PMID: 30055068 DOI: 10.1002/dvdy.24660] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2018] [Revised: 06/27/2018] [Accepted: 07/13/2018] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Parthenogenetically activated oocytes exhibit poor embryo development and lower total numbers of cells per blastocyst accompanied by abnormally increased expression of Xist, a long noncoding RNA that plays an important role in triggering X chromosome inactivation during embryogenesis. RESULTS To investigate whether knockdown of Xist influences parthenogenetic development in pigs. We developed an anti-Xist short hairpin RNA (shRNA) vector, which can significantly inhibit Xist expression for at least seven days when injected at 12-13 hr after parthenogenetic activation. Embryonic cleavage, blastocyst formation, and total blastocyst cell numbers were compared during the blastocyst stage, as well as the expression of an X-linked gene and three pluripotent transcription factors. Knockdown of Xist significantly increases the total blastocyst cell number, but does not influence the rate of embryo cleavage and blastocyst formation. The expressions of Sox2, Nanog, and Oct4 were also significantly improved in the injected embryos compared with the control at the blastocyst stage, but the Foxp3 expression level was not increased significantly. CONCLUSIONS The present study provides valuable information for understanding the role of Xist in parthenogenesis and presents a new approach for improving the quality of porcine parthenogenetic embryos. Developmental Dynamics 248:140-148, 2019. © 2018 Wiley Periodicals, Inc.
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Affiliation(s)
- Xiaoyu Chen
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, Zhejiang, P.R. China
| | - Zhiwei Zhu
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, Zhejiang, P.R. China
| | - Fuxian Yu
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, Zhejiang, P.R. China
| | - Jing Huang
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, Zhejiang, P.R. China
| | - Ruoxin Jia
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, Zhejiang, P.R. China
| | - Jianzhi Pan
- Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agriculture Science, Hangzhou, Zhejiang, P.R. China
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Thümecke S, Schröder R. UTR-specific knockdown of Distal-less and Sp8 leads to new phenotypic variants in the flour beetle Tribolium. Dev Genes Evol 2018; 228:163-170. [PMID: 29855703 DOI: 10.1007/s00427-018-0614-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2018] [Accepted: 05/08/2018] [Indexed: 10/14/2022]
Abstract
RNA interference (RNAi)-mediated knockdown serves as an effective technique for the functional analysis of developmental genes that is well established in many organisms. In the beetle Tribolium castaneum, double-stranded RNA is applied by simple injection and distributes systemically within the tissue. Thus, systematic testing for RNAi specificity and efficiency is easily possible in this organism. Generally, the use of non-overlapping dsRNA fragments yielding qualitatively identical phenotypes is the method of choice to verify target-specific knockdown effects. Here, we show that UTR-specific RNAi results in different effects regarding quality, severity and penetrance when compared to RNAi fragments directed at the coding region. Furthermore, when using 3'UTR-specific dsRNA, we first describe the Distal-lessRNAi antenna-to-leg transformation phenotype in the Tribolium larva, which has only been observed in the adult beetle and Drosophila so far. In addition, we unexpectedly observed sterility effects caused by 3'UTR-specific knockdown of the Tribolium-Sp8 orthologue that is not seen when dsRNA targeted a sequence within the coding-region or the 5'UTR that itself led to early embryonic lethality. We conclude that targeting UTR sequences by region-specific RNAi can reveal unexpected new aspects of gene function applicable in basic research and crop protection.
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Affiliation(s)
- Susanne Thümecke
- Institut für Biowissenschaften, Universität Rostock, Albert-Einsteinstr. 3, 18059, Rostock, Germany
| | - Reinhard Schröder
- Institut für Biowissenschaften, Universität Rostock, Albert-Einsteinstr. 3, 18059, Rostock, Germany.
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34
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RNAi-mediated SLC7A11 knockdown inhibits melanogenesis-related genes expression in rabbit skin fibroblasts. J Genet 2018. [DOI: 10.1007/s12041-018-0945-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
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Yang N, Mu L, Zhao B, Wang M, Hu S, Zhao B, Chen Y, Wu X. RNAi-mediated SLC7A11 knockdown inhibits melanogenesis-related genes expression in rabbit skin fibroblasts. J Genet 2018; 97:463-468. [PMID: 29932066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Solute carrier family 7 member 11 (SLC7A11) is a cystine/glutamate exchanger, also known as xCT, has been found to play an important role in pheomelanin synthesis. Adjusting the cystine content of cells to influence pheomelanin synthesis affects the proportion of total melanin, changing mammalian coat colour. In our previous study, we used RNA-seq to show that SLC7A11 was involved in coat colour regulation in Rex rabbits. However, the precise role of SLC7A11 in rabbit coat colour formation has not been investigated. To better understand the functions of SLC7A11 in rabbits, we used RNA interference (RNAi) to explore the effects of small interfering RNA (siRNA)-mediated downregulation of SLC7A11 gene expression on the expression of melanogenesis-related genes in rabbit skin fibroblasts (RAB-9). The effects of siRNA treatment were measured by quantitative real-time polymerase chain reaction and the efficiency of RNAi was calculated. The expression levels of melanogenesis-related genes, including MITF, MC1R, Agouti, CREB1 and SLC24A5 were detected at 24 h after RNAi transfection. The results showed that MITF, MC1R, SLC24A5, Agouti and CREB1 expression was significantly downregulated after SLC7A11 inhibition. This suggested that changes in SLC7A11 gene expression could directly affect the transcription of genes related to melanin production. This provides a scientific basis for further study of the role of SLC7A11 in the formation of coat colour.
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Affiliation(s)
- Naisu Yang
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu, People's Republic of China.
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Xie N, Liao HW, Ou WS, Zhou X, Hu Y, Fu N, Yang XF, Liao DF. Construction of COX-2 short hairpin RNA expression vector and its inhibitory effect on hepatic fibrosis. BIOTECHNOL BIOTEC EQ 2018. [DOI: 10.1080/13102818.2018.1431569] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Affiliation(s)
- Ni Xie
- Department of Gastroenterology, Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan, PR China
| | - Hong Wu Liao
- Department of Gastroenterology, Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan, PR China
| | - Wen Sheng Ou
- Department of Gastroenterology, The First People's Hospital of Chenzhou, Chenzhou, Hunan, PR China
| | - Xu Zhou
- Department of Gastroenterology, Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan, PR China
| | - Yang Hu
- Department of Gastroenterology, Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan, PR China
| | - Nian Fu
- Department of Gastroenterology, Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan, PR China
| | - Xue Feng Yang
- Department of Gastroenterology, Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan, PR China
| | - Duan-Fang Liao
- Key Laboratory for Quality Evaluation of Bulk Herbs of Hunan Province, School of pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan, China
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Herrera-Carrillo E, Liu YP, Berkhout B. Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors. Hum Gene Ther Methods 2018; 28:177-190. [PMID: 28712309 DOI: 10.1089/hgtb.2017.036] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of novel RNA therapeutics. Transient induction of RNAi via transfection of synthetic small interfering RNAs can trigger the selective knockdown of a target mRNA. For durable silencing of gene expression, either artificial short hairpin RNA or microRNA encoding transgene constructs were developed. These miRNAs are based on the molecules that induce the natural RNAi pathway in mammals and humans: the endogenously expressed miRNAs. Significant efforts focused on the construction and delivery of miRNA cassettes in order to solve basic biology questions or to design new therapy strategies. Several viral vectors have been developed, which are particularly useful for the delivery of miRNA expression cassettes to specific target cells. Each vector system has its own unique set of distinct properties. Thus, depending on the specific application, a particular vector may be most suitable. This field was previously reviewed for different viral vector systems, and now the recent progress in the field of miRNA-based gene-silencing approaches using lentiviral vectors is reported. The focus is on the unique properties and respective limitations of the available vector systems for miRNA delivery.
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Affiliation(s)
- Elena Herrera-Carrillo
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam , Amsterdam, The Netherlands
| | - Ying Poi Liu
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam , Amsterdam, The Netherlands
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam , Amsterdam, The Netherlands
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Ruan D, Peng J, Wang X, Ouyang Z, Zou Q, Yang Y, Chen F, Ge W, Wu H, Liu Z, Zhao Y, Zhao B, Zhang Q, Lai C, Fan N, Zhou Z, Liu Q, Li N, Jin Q, Shi H, Xie J, Song H, Yang X, Chen J, Wang K, Li X, Lai L. XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer. Stem Cell Reports 2018; 10:494-508. [PMID: 29337117 PMCID: PMC5830944 DOI: 10.1016/j.stemcr.2017.12.015] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Revised: 12/16/2017] [Accepted: 12/18/2017] [Indexed: 01/28/2023] Open
Abstract
Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.
Normal and abnormal pig SCNT embryos featured different transcriptome patterns Disrupting XIST gene in donor cells could enhance pig NT embryo development capacity Kdm4A injection was able to improve development of pig pre-implantation stage embryos Kdm4A addition caused XIST derepression and failed to improve pig cloning efficiency
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Affiliation(s)
- Degong Ruan
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Jiangyun Peng
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Xiaoshan Wang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Zhen Ouyang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Qingjian Zou
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yi Yang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Fangbing Chen
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Weikai Ge
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Han Wu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Zhaoming Liu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yu Zhao
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Bentian Zhao
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Quanjun Zhang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Chengdan Lai
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Nana Fan
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Zhiwei Zhou
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Qishuai Liu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Nan Li
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Qin Jin
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Hui Shi
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Jingke Xie
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Hong Song
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Xiaoyu Yang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, China
| | - Jiekai Chen
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Kepin Wang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
| | - Xiaoping Li
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
| | - Liangxue Lai
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China.
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Herrera-Carrillo E, Berkhout B. Dicer-independent processing of small RNA duplexes: mechanistic insights and applications. Nucleic Acids Res 2017; 45:10369-10379. [PMID: 28977573 PMCID: PMC5737282 DOI: 10.1093/nar/gkx779] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Accepted: 08/24/2017] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs (miRNAs) play a pivotal role in the regulation of cellular gene expression via the conserved RNA interference (RNAi) mechanism. Biogenesis of the unusual miR-451 does not require Dicer. This molecule is instead processed by the Argonaute 2 (Ago2) enzyme. Similarly, unconventional short hairpin RNA (shRNA) molecules have been designed as miR-451 mimics that rely exclusively on Ago2 for maturation. We will review recent progress made in the understanding of this alternative processing route. Next, we describe different Dicer-independent shRNA designs that have been developed and discuss their therapeutic advantages and disadvantages. As an example, we will present the route towards development of a durable gene therapy against HIV-1.
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Affiliation(s)
- Elena Herrera-Carrillo
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, the Netherlands
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, the Netherlands
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40
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Wang X, Tao C, Yuan C, Ren J, Yang M, Ying H. AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma. Mol Med Rep 2017; 16:1964-1972. [PMID: 28656282 PMCID: PMC5561784 DOI: 10.3892/mmr.2017.6847] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Accepted: 04/19/2017] [Indexed: 01/15/2023] Open
Abstract
Aquaporin 3 (AQP3) and phospholipase D2 (PLD2) are abnormally expressed and/or localized in squamous cell carcinoma (SCC). AQP3 transports glycerol to PLD2 for the synthesis of lipid second messenger, which can mediate the effect of the AQP3/PLD2 signaling module in the regulation of keratinocyte proliferation and differentiation. However, the role of the AQP3/PLD2 signaling module in the pathogenesis of SCC remains to be fully elucidated. In the present study, the expression levels of AQP3 and PLD2 in tissue samples were examined using immunohistochemistry, it was found that the expression levels of AQP3 and PLD2 in tissue samples of actinic keratosis (AK), Bowen's disease (BD) and SCC were significantly increased. AQP3 small interfering RNA (siRNA) and PLD2 siRNA were constructed and used for transfection into the human A431 SCC cell line, and their anticancer effect on SCC was examined. The mRNA expression and protein expression levels of AQP3 and PLD2 were significantly downregulated following siRNA transfection. AQP3 siRNA and PLD2 siRNA inhibited the proliferation and promoted the apoptosis of A431 cells. Taken together, the findings of the present study suggested that increased levels of AQP3 and PLD2 were correlated with tumor progression and development in SCC. AQP3 siRNA and PLD2 siRNA significantly downregulated the mRNA and protein levels of AQP3 and PLD2 in the A431 cells; inhibiting proliferation and promoting apoptosis in vitro. The concomitant effects of AQP3/PLD2 signaling by inhibiting the expression of siRNA may be important for the treatment of SCC in the future.
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Affiliation(s)
- Xiaoyong Wang
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, P.R. China
| | - Chengjun Tao
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, P.R. China
| | - Chengda Yuan
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, P.R. China
| | - Jinping Ren
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, P.R. China
| | - Ming Yang
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, P.R. China
| | - Hangyu Ying
- Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, P.R. China
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Cambon K, Zimmer V, Martineau S, Gaillard MC, Jarrige M, Bugi A, Miniarikova J, Rey M, Hassig R, Dufour N, Auregan G, Hantraye P, Perrier AL, Déglon N. Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2017; 5:259-276. [PMID: 28603746 PMCID: PMC5453866 DOI: 10.1016/j.omtm.2017.05.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Accepted: 05/07/2017] [Indexed: 01/12/2023]
Abstract
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from a polyglutamine expansion in the huntingtin (HTT) protein. There is currently no cure for this disease, but recent studies suggest that RNAi to downregulate the expression of both normal and mutant HTT is a promising therapeutic approach. We previously developed a small hairpin RNA (shRNA), vectorized in an HIV-1-derived lentiviral vector (LV), that reduced pathology in an HD rodent model. Here, we modified this vector for preclinical development by using a tat-independent third-generation LV (pCCL) backbone and removing the original reporter genes. We demonstrate that this novel vector efficiently downregulated HTT expression in vitro in striatal neurons derived from induced pluripotent stem cells (iPSCs) of HD patients. It reduced two major pathological HD hallmarks while triggering a minimal inflammatory response, up to 6 weeks after injection, when administered by stereotaxic surgery in the striatum of an in vivo rodent HD model. Further assessment of this shRNA vector in vitro showed proper processing by the endogenous silencing machinery, and we analyzed gene expression changes to identify potential off-targets. These preclinical data suggest that this new shRNA vector fulfills primary biosafety and efficiency requirements for further development in the clinic as a cure for HD.
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Affiliation(s)
- Karine Cambon
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Virginie Zimmer
- Department of Clinical Neurosciences, Laboratory of Cellular and Molecular Neurotherapies, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
- Neuroscience Research Center, Laboratory of Cellular and Molecular Neurotherapies, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
| | - Sylvain Martineau
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Marie-Claude Gaillard
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Margot Jarrige
- Institut National de la Santé et de la Recherche Médicale UMR861, I-Stem, AFM, 91100 Corbeil-Essonnes, France
- UEVE UMR861, I-STEM, AFM, 91100 Corbeil-Essonnes, France
- CECS, I-STEM, AFM, 91100 Corbeil-Essonnes, France
| | - Aurore Bugi
- CECS, I-STEM, AFM, 91100 Corbeil-Essonnes, France
| | - Jana Miniarikova
- Department of Research & Development, uniQure, 1105 Amsterdam, the Netherlands
| | - Maria Rey
- Department of Clinical Neurosciences, Laboratory of Cellular and Molecular Neurotherapies, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
- Neuroscience Research Center, Laboratory of Cellular and Molecular Neurotherapies, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
| | - Raymonde Hassig
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Noelle Dufour
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Gwenaelle Auregan
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Philippe Hantraye
- CEA, DRF, Institute of Biology Francois Jacob, Molecular Imaging Research Center, F-92265 Fontenay-aux-Roses, France
- CNRS, CEA, Paris-Sud University, University Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France
| | - Anselme L. Perrier
- Institut National de la Santé et de la Recherche Médicale UMR861, I-Stem, AFM, 91100 Corbeil-Essonnes, France
- UEVE UMR861, I-STEM, AFM, 91100 Corbeil-Essonnes, France
| | - Nicole Déglon
- Department of Clinical Neurosciences, Laboratory of Cellular and Molecular Neurotherapies, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
- Neuroscience Research Center, Laboratory of Cellular and Molecular Neurotherapies, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
- Corresponding author: Nicole Déglon, Lausanne University Hospital (CHUV), Laboratory of Cellular and Molecular Neurotherapies (LNCM), Pavillon 3, Avenue de Beaumont, 1011 Lausanne, Switzerland.
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Down-Regulation of MRP1 Expression in C6/VP16 Cells by Chitosan-MRP1-siRNA Nanoparticles. Cell Biochem Biophys 2016; 72:227-33. [PMID: 25543328 DOI: 10.1007/s12013-014-0442-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
High expression of multidrug resistance-associated protein 1 (MRP1) in tumor cells reduces effectiveness of chemotherapy drugs. In this study, we screened MRP1 interfering RNA (MRP1-siRNA) molecules that are able to reverse etoposide (VP16) resistance in multidrug resistance rat glioma cell line C6/VP16, and identified one siRNA molecule that is able to effectively deplete the expression of MRP1 gene and reverse tumor cells resistance to etoposide. Since siRNA instability limits its application in treatment of diseases, we next tested silencing effect of chitosan-MRP1-siRNA nanoparticles and found that the nanoparticles with N:P ratio 175 are able to effectively inhibit MRP1 mRNA and protein expression. Our data demonstrate that chitosan can be used as siRNA carrier for high efficient gene silencing in tumor cells.
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Munkácsy G, Sztupinszki Z, Herman P, Bán B, Pénzváltó Z, Szarvas N, Győrffy B. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments. MOLECULAR THERAPY. NUCLEIC ACIDS 2016; 5:e366. [PMID: 27673562 PMCID: PMC5056990 DOI: 10.1038/mtna.2016.66] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Accepted: 07/05/2016] [Indexed: 01/31/2023]
Abstract
No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.
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Affiliation(s)
- Gyöngyi Munkácsy
- MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary.,MTA-SE Pediatrics and Nephrology Research Group, Budapest, Hungary
| | - Zsófia Sztupinszki
- Second Department of Pediatrics, Semmelweis University, Budapest, Hungary
| | - Péter Herman
- MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary
| | - Bence Bán
- Second Department of Pediatrics, Semmelweis University, Budapest, Hungary
| | - Zsófia Pénzváltó
- MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary
| | - Nóra Szarvas
- MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary
| | - Balázs Győrffy
- MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary.,Second Department of Pediatrics, Semmelweis University, Budapest, Hungary
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Zeng F, Huang Z, Yuan Y, Shi J, Cai G, Liu D, Wu Z, Li Z. Effects of RNAi-mediated knockdown of Xist on the developmental efficiency of cloned male porcine embryos. J Reprod Dev 2016; 62:591-597. [PMID: 27569767 PMCID: PMC5177977 DOI: 10.1262/jrd.2016-095] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.
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Affiliation(s)
- Fang Zeng
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangdong 510642, P. R. China
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Rehman L, Su X, Guo H, Qi X, Cheng H. Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae. BMC Biotechnol 2016; 16:57. [PMID: 27455996 PMCID: PMC4960691 DOI: 10.1186/s12896-016-0287-4] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Accepted: 07/15/2016] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. RESULTS High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs-19-nt duplex with 2-nt 3' overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)-into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. CONCLUSION Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.
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Affiliation(s)
- Latifur Rehman
- Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081 China
| | - Xiaofeng Su
- Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081 China
| | - Huiming Guo
- Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081 China
| | - Xiliang Qi
- Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081 China
| | - Hongmei Cheng
- Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081 China
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Singhania R, Pavey S, Payne E, Gu W, Clancy J, Jubair L, Preiss T, Saunders N, McMillan NAJ. Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2016; 1859:1034-42. [PMID: 27321990 DOI: 10.1016/j.bbagrm.2016.06.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Revised: 06/14/2016] [Accepted: 06/15/2016] [Indexed: 11/30/2022]
Abstract
Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing.
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Affiliation(s)
- Richa Singhania
- The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia
| | - Sandra Pavey
- The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia
| | - Elizabeth Payne
- The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia
| | - Wenyi Gu
- The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia
| | - Jennifer Clancy
- The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Luqman Jubair
- Menzies Health Institute Queensland and School of Medical Science, Griffith University, Southport, Australia
| | - Thomas Preiss
- The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Nicholas Saunders
- The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia
| | - Nigel A J McMillan
- The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia; Menzies Health Institute Queensland and School of Medical Science, Griffith University, Southport, Australia.
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Mansoori B, Mohammadi A, Shir Jang S, Baradaran B. Mechanisms of immune system activation in mammalians by small interfering RNA (siRNA). ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2015; 44:1589-96. [DOI: 10.3109/21691401.2015.1102738] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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He LJ, Xie D, Hu PJ, Liao YJ, Deng HX, Kung HF, Zhu SL. Macrophage migration inhibitory factor as a potential prognostic factor in gastric cancer. World J Gastroenterol 2015; 21:9916-9926. [PMID: 26379396 PMCID: PMC4566384 DOI: 10.3748/wjg.v21.i34.9916] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Revised: 04/13/2015] [Accepted: 07/15/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate macrophage migration inhibitory factor (MIF) expression and its clinical relevance in gastric cancer, and effects of MIF knockdown on proliferation of gastric cancer cells.
METHODS: Tissue microarray containing 117 samples of gastric cancer and adjacent non-cancer normal tissues was studied for MIF expression by immunohistochemistry (IHC) semiquantitatively, and the association of MIF expression with clinical parameters was analyzed. MIF expression in gastric cancer cell lines was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Two pairs of siRNA targeting the MIF gene (MIF si-1 and MIF si-2) and one pair of scrambled siRNA as a negative control (NC) were designed and chemically synthesized. All siRNAs were transiently transfected in AGS cells with OligofectamineTM to knock down the MIF expression, with the NC group and mock group (OligofectamineTM alone) as controls. At 24, 48, and 72 h after transfection, MIF mRNA was analyzed by RT-PCR, and MIF and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot. The proliferative rate of AGS cells was assessed by methylthiazolyl tetrazolium (MTT) assay and colony forming assay.
RESULTS: The tissue microarray was informative for IHC staining, in which the MIF expression in gastric cancer tissues was higher than that in adjacent non-cancer normal tissues (P < 0.001), and high level of MIF was related to poor tumor differentiation, advanced T stage, advanced tumor stage, lymph node metastasis, and poor patient survival (P < 0.05 for all). After siRNA transfection, MIF mRNA was measured by real-time PCR, and MIF protein and PCNA were assessed by Western blot analysis. We found that compared to the NC group and mock group, MIF expression was knocked down successfully in gastric cancer cells, and PCNA expression was downregulated with MIF knockdown as well. The cell counts and the doubling times were assayed by MTT 4 d after transfection, and colonies formed were assayed by colony forming assay 10 d after transfection; all these showed significant changes in gastric cancer cells transfected with specific siRNA compared with the control siRNA and mock groups (P < 0.001 for all).
CONCLUSION: MIF could be of prognostic value in gastric cancer and might be a potential target for small-molecule therapy.
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Méndez C, Ahlenstiel CL, Kelleher AD. Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus. World J Virol 2015; 4:219-244. [PMID: 26279984 PMCID: PMC4534814 DOI: 10.5501/wjv.v4.i3.219] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2014] [Revised: 01/24/2015] [Accepted: 04/29/2015] [Indexed: 02/05/2023] Open
Abstract
While human immunodeficiency virus 1 (HIV-1) infection is controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference (RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by non-coding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing (TGS), as well as fine-tuning their expression through post-transcriptional gene silencing (PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.
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Ponthan F, Yusoff NM, Soria NM, Heidenreich O, Coffey K. Gene Silencing by RNAi in Mammalian Cells. ACTA ACUST UNITED AC 2015; 111:26.2.1-26.2.17. [DOI: 10.1002/0471142727.mb2602s111] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- Frida Ponthan
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne United Kingdom
| | - Narazah Mohd Yusoff
- Advanced Medical & Dental Institute, Universiti Sains Malaysia Pulau Pinang Malaysia
| | - Natalia Martinez Soria
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne United Kingdom
| | - Olaf Heidenreich
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne United Kingdom
| | - Kelly Coffey
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne United Kingdom
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