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Xu H, Xu D, Liu Y. Molecular Biology Applications of Psychrophilic Enzymes: Adaptations, Advantages, Expression, and Prospective. Appl Biochem Biotechnol 2024; 196:5765-5789. [PMID: 38183603 DOI: 10.1007/s12010-023-04810-5] [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] [Accepted: 12/09/2023] [Indexed: 01/08/2024]
Abstract
Psychrophilic enzymes are primarily produced by microorganisms from extremely low-temperature environments which are known as psychrophiles. Their high efficiency at low temperatures and easy heat inactivation property have attracted extensive attention from various food and industrial bioprocesses. However, the application of these enzymes in molecular biology is still limited. In a previous review, the applications of psychrophilic enzymes in industries such as the detergent additives, the food additives, the bioremediation, and the pharmaceutical medicine, and cosmetics have been discussed. In this review, we discuss the main cold adaptation characteristics of psychrophiles and psychrophilic enzymes, as well as the relevant information on different psychrophilic enzymes in molecular biology. We summarize the mining and screening methods of psychrophilic enzymes. We finally recap the expression of psychrophilic enzymes. We aim to provide a reference process for the exploration and expression of new generation of psychrophilic enzymes.
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Affiliation(s)
- Hu Xu
- Center for Pan-Third Pole Environment, Lanzhou University, Lanzhou, 730000, China
- CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing, 100190, China
| | - Dawei Xu
- CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing, 100190, China.
| | - Yongqin Liu
- Center for Pan-Third Pole Environment, Lanzhou University, Lanzhou, 730000, China.
- State Key Laboratory of Tibetan Plateau Earth System, Resources and Environment (TPESRE), Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100101, China.
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2
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Bertoli G, Fortunato F, Cava C, Manna I, Gallivanone F, Labate A, Panio A, Porro D, Gambardella A. Serum MicroRNAs as Predictors of Diagnosis and Drug-resistance in Temporal Lobe Epilepsy: A Preliminary Study. Curr Neuropharmacol 2024; 22:2422-2432. [PMID: 39403059 PMCID: PMC11451323 DOI: 10.2174/1570159x22666240516145823] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Revised: 07/18/2023] [Accepted: 07/21/2023] [Indexed: 10/19/2024] Open
Abstract
OBJECTIVE Temporal lobe epilepsy (TLE) is the most common form of refractory focal epilepsy, and the current clinical diagnosis is based on EEG, clinical neurological history and neuroimaging findings. METHODS So far, there are no blood-based molecular biomarkers of TLE to support clinical diagnosis, despite the pathogenic mechanisms underlying TLE involving defects in the regulation of gene expression. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression. RESULTS Recent studies show the feasibility of detecting miRNAs in body fluids; circulating miRNAs have emerged as potential clinical biomarkers in epilepsy, although the TLE miRNA profile needs to be addressed. Here, we analysed the diagnostic potential of 8 circulating miRNAs in sera of 52 TLE patients and 40 age- and sex-matched donor controls by RT-qPCR analyses. CONCLUSION We found that miR-34a-5p, -106b-5p, -130a-3p, -146a-5p, and -19a-3p are differently expressed in TLE compared to control subjects, suggesting a diagnostic role. Furthermore, we found that miR-34a-5p, -106b-5p, -146a-5p and miR-451a could become prognostic biomarkers, being differentially expressed between drug-resistant and drug-responsive TLE subjects. Therefore, serum miRNAs are diagnostic and drug-resistance predictive molecules of TLE.
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Affiliation(s)
- Gloria Bertoli
- Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, Segrate, Milan, Italy
- NBFC, National Biodiversity Future Center, Palermo 90133, Italy
| | - Francesco Fortunato
- Institute of Neurology, Department of Medical and Surgical Sciences, University “Magna Graecia”, Germaneto, Catanzaro, Italy
| | - Claudia Cava
- Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, Segrate, Milan, Italy
- NBFC, National Biodiversity Future Center, Palermo 90133, Italy
- IUSS, Scuola Universitaria Superiore Pavia, Pv, Italy
| | - Ida Manna
- IBFM-CNR, Section of Germaneto, Catanzaro, Italy
| | - Francesca Gallivanone
- Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, Segrate, Milan, Italy
- NBFC, National Biodiversity Future Center, Palermo 90133, Italy
| | - Angelo Labate
- Neurophysiopatology and Movement Disorders Clinic, University of Messina, Italy
| | - Antonella Panio
- Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, Segrate, Milan, Italy
| | - Danilo Porro
- Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, Segrate, Milan, Italy
- NBFC, National Biodiversity Future Center, Palermo 90133, Italy
- Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy
| | - Antonio Gambardella
- Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, Segrate, Milan, Italy
- Institute of Neurology, Department of Medical and Surgical Sciences, University “Magna Graecia”, Germaneto, Catanzaro, Italy
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3
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Silver Nanoparticles Modified by Carbosilane Dendrons and PEG as Delivery Vectors of Small Interfering RNA. Int J Mol Sci 2023; 24:ijms24010840. [PMID: 36614277 PMCID: PMC9820844 DOI: 10.3390/ijms24010840] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2022] [Revised: 12/26/2022] [Accepted: 12/28/2022] [Indexed: 01/06/2023] Open
Abstract
The fact that cancer is one of the leading causes of death requires researchers to create new systems of effective treatment for malignant tumors. One promising area is genetic therapy that uses small interfering RNA (siRNA). These molecules are capable of blocking mutant proteins in cells, but require specific systems that will deliver RNA to target cells and successfully release them into the cytoplasm. Dendronized and PEGylated silver nanoparticles as potential vectors for proapoptotic siRNA (siMCL-1) were used here. Using the methods of one-dimensional gel electrophoresis, the zeta potential, dynamic light scattering, and circular dichroism, stable siRNA and AgNP complexes were obtained. Data gathered using multicolor flow cytometry showed that AgNPs are able to deliver (up to 90%) siRNAs efficiently to some types of tumor cells, depending on the degree of PEGylation. Analysis of cell death showed that complexes of some AgNP variations with siMCL-1 lead to ~70% cell death in the populations that uptake these complexes due to apoptosis.
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4
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Geropoulos G, Psarras K, Papaioannou M, Giannis D, Meitanidou M, Kapriniotis K, Symeonidis N, Pavlidis ET, Pavlidis TE, Sapalidis K, Ahmed NM, Abdel-Aziz TE, Eddama MMR. Circulating microRNAs and Clinicopathological Findings of Papillary Thyroid Cancer: A Systematic Review. In Vivo 2022; 36:1551-1569. [PMID: 35738604 PMCID: PMC9301440 DOI: 10.21873/invivo.12866] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2022] [Revised: 04/24/2022] [Accepted: 04/26/2022] [Indexed: 02/08/2023]
Abstract
BACKGROUND/AIM Papillary thyroid cancer (PTC) is the most common endocrine malignancy with a rising incidence. There is a need for a non-invasive preoperative test to enable better patient counselling. The aim of this systematic review was to investigate the potential role of circulating microRNAs (miRNAs) in the diagnosis and prognosis of PTC. MATERIALS AND METHODS A systematic literature search was performed using MEDLINE, Cochrane, and Scopus databases (last search date was December 1, 2021). Studies investigating the expression of miRNAs in the serum or plasma of patients with PTC were deemed eligible for inclusion. RESULTS Among the 1,533 screened studies, 39 studies met the inclusion criteria. In total, 108 miRNAs candidates were identified in the serum, plasma, or exosomes of patients suffering from PTC. Furthermore, association of circulating miRNAs with thyroid cancer-specific clinicopathological features, such as tumor size (13 miRNAs), location (3 miRNAs), extrathyroidal extension (9 miRNAs), pre- vs. postoperative period (31 miRNAs), lymph node metastasis (17 miRNAs), TNM stage (9 miRNAs), BRAF V600E mutation (6 miRNAs), serum thyroglobulin levels (2 miRNAs), 131I avid metastases (13 miRNAs), and tumor recurrence (2 miRNAs) was also depicted in this study. CONCLUSION MiRNAs provide a potentially promising role in the diagnosis and prognosis of PTC. There is a correlation between miRNA expression profiles and specific clinicopathological features of PTC. However, to enable their use in clinical practice, further clinical studies are required to validate the predictive value and utility of miRNAs as biomarkers.
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Affiliation(s)
- Georgios Geropoulos
- Department of General and Endocrine Surgery, University College London Hospitals, London, U.K.;
- 2 Propedeutical Department of Surgery, Hippokration Hospital, School of Medicine,Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Kyriakos Psarras
- 2 Propedeutical Department of Surgery, Hippokration Hospital, School of Medicine,Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Maria Papaioannou
- Laboratory of Biological Chemistry, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, University Campus, Thessaloniki, Greece
| | - Dimitrios Giannis
- Department of Surgery, North Shore University Hospital, Manhasset, NY, U.S.A
| | - Maria Meitanidou
- 2 Propedeutical Department of Surgery, Hippokration Hospital, School of Medicine,Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | | | - Nikolaos Symeonidis
- 2 Propedeutical Department of Surgery, Hippokration Hospital, School of Medicine,Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Efstathios T Pavlidis
- 2 Propedeutical Department of Surgery, Hippokration Hospital, School of Medicine,Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Theodoros E Pavlidis
- 2 Propedeutical Department of Surgery, Hippokration Hospital, School of Medicine,Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Konstantinos Sapalidis
- 3 General Surgery Department, "AHEPA" University Hospital, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Nada Mabrouk Ahmed
- Department of General and Endocrine Surgery, University College London Hospitals, London, U.K
- Department of Pathology, University of Alexandria, Alexandria, Egypt
| | - Tarek Ezzat Abdel-Aziz
- Department of General and Endocrine Surgery, University College London Hospitals, London, U.K
| | - Mohammad M R Eddama
- Department of General and Endocrine Surgery, University College London Hospitals, London, U.K
- Research Department of Surgical Biotechnology, University College London, London, U.K
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Marin FR, Dávalos A, Kiltschewskij D, Crespo MC, Cairns M, Andrés-León E, Soler-Rivas C. RNA-Seq, Bioinformatic Identification of Potential MicroRNA-like Small RNAs in the Edible Mushroom Agaricus bisporus and Experimental Approach for Their Validation. Int J Mol Sci 2022; 23:4923. [PMID: 35563314 PMCID: PMC9100230 DOI: 10.3390/ijms23094923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Revised: 04/22/2022] [Accepted: 04/27/2022] [Indexed: 11/22/2022] Open
Abstract
Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases.
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Affiliation(s)
- Francisco R. Marin
- Department of Production and Characterization of Novel Foods, Institute of Food Science Research—CIAL (UAM + CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain;
| | - Alberto Dávalos
- Laboratory of Epigenetics of Lipid Metabolism, Madrid Institute for Advanced Studies (IMDEA)—Food, CEI UAM + CSIC, Pabellón Central del Antiguo Hospital de Cantoblanco, 28049 Madrid, Spain; (A.D.); (M.C.C.)
| | - Dylan Kiltschewskij
- School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW 2308, Australia; (D.K.); (M.C.)
| | - Maria C. Crespo
- Laboratory of Epigenetics of Lipid Metabolism, Madrid Institute for Advanced Studies (IMDEA)—Food, CEI UAM + CSIC, Pabellón Central del Antiguo Hospital de Cantoblanco, 28049 Madrid, Spain; (A.D.); (M.C.C.)
| | - Murray Cairns
- School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW 2308, Australia; (D.K.); (M.C.)
| | - Eduardo Andrés-León
- Bioinformatics Unit, Institute of Parasitology and Biomedicine “López Neyra”, Spanish National Research Council (CSIC), 18016 Granada, Spain;
| | - Cristina Soler-Rivas
- Department of Production and Characterization of Novel Foods, Institute of Food Science Research—CIAL (UAM + CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain;
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Yu X, Elfimova N, Müller M, Bachurski D, Koitzsch U, Drebber U, Mahabir E, Hansen HP, Friedman SL, Klein S, Dienes HP, Hösel M, Buettner R, Trebicka J, Kondylis V, Mannaerts I, Odenthal M. Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion. Cell Mol Gastroenterol Hepatol 2022; 13:1701-1716. [PMID: 35219894 PMCID: PMC9046234 DOI: 10.1016/j.jcmgh.2022.02.013] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 02/16/2022] [Accepted: 02/17/2022] [Indexed: 01/07/2023]
Abstract
BACKGROUND & AIMS Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis. METHODS Intracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model. RESULTS In our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets' expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease. CONCLUSIONS We provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.
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Affiliation(s)
- Xiaojie Yu
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Natalia Elfimova
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
| | - Marion Müller
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Daniel Bachurski
- Department I of Internal Medicine, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
| | - Ulrike Koitzsch
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Uta Drebber
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center of Integrative Oncology, University Clinic of Cologne and Bonn, Germany
| | - Esther Mahabir
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Hinrich P Hansen
- Department I of Internal Medicine, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center of Integrative Oncology, University Clinic of Cologne and Bonn, Germany
| | - Scott L Friedman
- Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Sabine Klein
- Department of Internal Medicine I, University Hospital Frankfurt, Frankfurt, Germany
| | - Hans Peter Dienes
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany
| | - Marianna Hösel
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Reinhard Buettner
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Center of Integrative Oncology, University Clinic of Cologne and Bonn, Germany
| | - Jonel Trebicka
- Department of Internal Medicine I, University Hospital Frankfurt, Frankfurt, Germany; European Foundation for the Study of Chronic Liver Failure - EF CLIF, Barcelona, Spain
| | - Vangelis Kondylis
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Inge Mannaerts
- Liver Cell Biology Research Group, Vrije Universiteit Brussel, Brussel, Belgium.
| | - Margarete Odenthal
- Institute for Pathology, Medical Faculty and University Hospital of Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Center of Integrative Oncology, University Clinic of Cologne and Bonn, Germany.
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Abstract
MicroRNAs (miRNAs) are considerably small yet highly important riboregulators involved in nearly all cellular processes. Due to their critical roles in posttranscriptional regulation of gene expression, they have the potential to be used as biomarkers in addition to their use as drug targets. Although computational approaches speed up the initial genomewide identification of putative miRNAs, experimental approaches are essential for further validation and functional analyses of differentially expressed miRNAs. Therefore, sensitive, specific, and cost-effective microRNA detection methods are imperative for both individual and multiplex analysis of miRNA expression in different tissues and during different developmental stages. There are a number of well-established miRNA detection methods that can be exploited depending on the comprehensiveness of the study (individual miRNA versus multiplex analysis), the availability of the sample and the location and intracellular concentration of miRNAs. This review aims to highlight not only traditional but also novel strategies that are widely used in experimental identification and quantification of microRNAs.
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8
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Afshari A, Yaghobi R, Rezaei G. Inter-regulatory role of microRNAs in interaction between viruses and stem cells. World J Stem Cells 2021; 13:985-1004. [PMID: 34567421 PMCID: PMC8422934 DOI: 10.4252/wjsc.v13.i8.985] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/11/2021] [Accepted: 07/13/2021] [Indexed: 02/06/2023] Open
Abstract
MicroRNAs (miRNAs) are well known for post-transcriptional regulatory ability over specific mRNA targets. miRNAs exhibit temporal or tissue-specific expression patterns and regulate the cell and tissue developmental pathways. They also have determinative roles in production and differentiation of multiple lineages of stem cells and might have therapeutic advantages. miRNAs are a part of some viruses' regulatory machinery, not a byproduct. The trace of miRNAs was detected in the genomes of viruses and regulation of cell reprograming and viral pathogenesis. Combination of inter-regulatory systems has been detected for miRNAs during viral infections in stem cells. Contraction between viruses and stem cells may be helpful in therapeutic tactics, pathogenesis, controlling viral infections and defining stem cell developmental strategies that is programmed by miRNAs as a tool. Therefore, in this review we intended to study the inter-regulatory role of miRNAs in the interaction between viruses and stem cells and tried to explain the advantages of miRNA regulatory potentials, which make a new landscape for future studies.
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Affiliation(s)
- Afsoon Afshari
- Shiraz Nephro-Urology Research Center, Shiraz University of Medical Sciences, Shiraz 7193711351, Iran
| | - Ramin Yaghobi
- Shiraz Transplant Research Center, Shiraz University of Medical Sciences, Shiraz 7193711351, Iran.
| | - Ghazal Rezaei
- Shiraz Transplant Research Center, Shiraz University of Medical Sciences, Shiraz 7193711351, Iran
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Pasquesi GIM, Perry BW, Vandewege MW, Ruggiero RP, Schield DR, Castoe TA. Vertebrate Lineages Exhibit Diverse Patterns of Transposable Element Regulation and Expression across Tissues. Genome Biol Evol 2021; 12:506-521. [PMID: 32271917 PMCID: PMC7211425 DOI: 10.1093/gbe/evaa068] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/02/2020] [Indexed: 12/11/2022] Open
Abstract
Transposable elements (TEs) comprise a major fraction of vertebrate genomes, yet little is known about their expression and regulation across tissues, and how this varies across major vertebrate lineages. We present the first comparative analysis integrating TE expression and TE regulatory pathway activity in somatic and gametic tissues for a diverse set of 12 vertebrates. We conduct simultaneous gene and TE expression analyses to characterize patterns of TE expression and TE regulation across vertebrates and examine relationships between these features. We find remarkable variation in the expression of genes involved in TE negative regulation across tissues and species, yet consistently high expression in germline tissues, particularly in testes. Most vertebrates show comparably high levels of TE regulatory pathway activity across gonadal tissues except for mammals, where reduced activity of TE regulatory pathways in ovarian tissues may be the result of lower relative germ cell densities. We also find that all vertebrate lineages examined exhibit remarkably high levels of TE-derived transcripts in somatic and gametic tissues, with recently active TE families showing higher expression in gametic tissues. Although most TE-derived transcripts originate from inactive ancient TE families (and are likely incapable of transposition), such high levels of TE-derived RNA in the cytoplasm may have secondary, unappreciated biological relevance.
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Affiliation(s)
- Giulia I M Pasquesi
- Department of Biology, University of Texas at Arlington.,Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder
| | - Blair W Perry
- Department of Biology, University of Texas at Arlington
| | | | | | - Drew R Schield
- Department of Biology, University of Texas at Arlington.,Department of Ecology and Evolutionary Biology, University of Colorado, Boulder
| | - Todd A Castoe
- Department of Biology, University of Texas at Arlington
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10
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Cui X, Zhang S, Zhang Q, Guo X, Wu C, Yao M, Sun D. Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages. Front Genet 2020; 11:548268. [PMID: 33343617 PMCID: PMC7744623 DOI: 10.3389/fgene.2020.548268] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2020] [Accepted: 11/05/2020] [Indexed: 12/27/2022] Open
Abstract
A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3'UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.
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Affiliation(s)
- Xiaogang Cui
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, National Engineering Laboratory of Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China.,Key Lab of Medical Molecular Cell Biology of Shanxi Province, Institutes of Biomedical Sciences, Shanxi University, Taiyuan, China
| | - Shengli Zhang
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, National Engineering Laboratory of Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Qin Zhang
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, National Engineering Laboratory of Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Xiangyu Guo
- Center for Quantitative Genetics and Genomics, Aarhus University, Tjele, Denmark
| | - Changxin Wu
- Key Lab of Medical Molecular Cell Biology of Shanxi Province, Institutes of Biomedical Sciences, Shanxi University, Taiyuan, China
| | - Mingze Yao
- Key Lab of Medical Molecular Cell Biology of Shanxi Province, Institutes of Biomedical Sciences, Shanxi University, Taiyuan, China
| | - Dongxiao Sun
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, National Engineering Laboratory of Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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11
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Park HJ, Lee SS. Detection of miR‐155 Using Two Types of Electrochemical Approaches. B KOREAN CHEM SOC 2020. [DOI: 10.1002/bkcs.12128] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Affiliation(s)
- Hyeoun Ji Park
- Department of Pharmaceutical Engineering Soonchunhhyang University Chungnam 31538 South Korea
| | - Soo Suk Lee
- Department of Pharmaceutical Engineering Soonchunhhyang University Chungnam 31538 South Korea
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12
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Abstract
The complete genome sequence of the soft tick bunyavirus (STBV) was obtained using the Sanger sequencing technique. Comparison with other viral sequences revealed that STBV has unique sequences in the terminal regions that are highly conserved among the genus Orthonairovirus. The complete genome sequence of the soft tick bunyavirus (STBV) was obtained using the Sanger sequencing technique. Comparison with other viral sequences revealed that STBV has unique sequences in the terminal regions that are highly conserved among the genus Orthonairovirus.
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13
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Li L, Dai H, Nguyen AP, Gu W. A convenient strategy to clone small RNA and mRNA for high-throughput sequencing. RNA (NEW YORK, N.Y.) 2020; 26:218-227. [PMID: 31754076 PMCID: PMC6961543 DOI: 10.1261/rna.071605.119] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/23/2019] [Accepted: 10/24/2019] [Indexed: 06/10/2023]
Abstract
High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5' and 3' linkers. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 2'-O-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5' modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs ∼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.
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Affiliation(s)
- Lichao Li
- Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521, USA
| | - Hui Dai
- Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521, USA
| | - An-Phong Nguyen
- Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521, USA
| | - Weifeng Gu
- Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521, USA
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14
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Behrmann O, Hügle M, Bronsert P, Herde B, Heni J, Schramm M, Hufert FT, Urban GA, Dame G. A lab-on-a-chip for rapid miRNA extraction. PLoS One 2019; 14:e0226571. [PMID: 31856234 PMCID: PMC6922460 DOI: 10.1371/journal.pone.0226571] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Accepted: 11/28/2019] [Indexed: 12/16/2022] Open
Abstract
We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 μl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis.
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Affiliation(s)
- Ole Behrmann
- Department of Microbiology and Virology, Brandenburg Medical School Fontane, Neuruppin, Germany
- Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany
| | - Matthias Hügle
- Department of Microbiology and Virology, Brandenburg Medical School Fontane, Neuruppin, Germany
- Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany
| | - Peter Bronsert
- Institute for Surgical Pathology, Medical Center–University of Freiburg, Freiburg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
- German Cancer Consortium (DKTK), Partner Site Freiburg, Freiburg, Germany
| | - Bettina Herde
- Institute for Surgical Pathology, Medical Center–University of Freiburg, Freiburg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Julian Heni
- Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany
| | - Marina Schramm
- Department of Microbiology and Virology, Brandenburg Medical School Fontane, Neuruppin, Germany
| | - Frank T. Hufert
- Department of Microbiology and Virology, Brandenburg Medical School Fontane, Neuruppin, Germany
| | - Gerald A. Urban
- Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany
| | - Gregory Dame
- Department of Microbiology and Virology, Brandenburg Medical School Fontane, Neuruppin, Germany
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15
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Ochoa J, Valli A, Martín-Trillo M, Simón-Mateo C, García JA, Rodamilans B. Sterol isomerase HYDRA1 interacts with RNA silencing suppressor P1b and restricts potyviral infection. PLANT, CELL & ENVIRONMENT 2019; 42:3015-3026. [PMID: 31286514 DOI: 10.1111/pce.13610] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Accepted: 07/05/2019] [Indexed: 06/09/2023]
Abstract
Plants use RNA silencing as a strong defensive barrier against virus challenges, and viruses counteract this defence by using RNA silencing suppressors (RSSs). With the objective of identifying host factors helping either the plant or the virus in this interaction, we have performed a yeast two-hybrid screen using P1b, the RSS protein of the ipomovirus Cucumber vein yellowing virus (CVYV, family Potyviridae), as a bait. The C-8 sterol isomerase HYDRA1 (HYD1), an enzyme involved in isoprenoid biosynthesis and cell membrane biology, and required for RNA silencing, was isolated in this screen. The interaction between CVYV P1b and HYD1 was confirmed in planta by Bimolecular Fluorescence Complementation assays. We demonstrated that HYD1 negatively impacts the accumulation of CVYV P1b in an agroinfiltration assay. Moreover, expression of HYD1 inhibited the infection of the potyvirus Plum pox virus, especially when antiviral RNA silencing was boosted by high temperature or by coexpression of homologous sequences. Our results reinforce previous evidence highlighting the relevance of particular composition and structure of cellular membranes for RNA silencing and viral infection. We report a new interaction of an RSS protein from the Potyviridae family with a member of the isoprenoid biosynthetic pathway.
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Affiliation(s)
- Jon Ochoa
- Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain
| | - Adrián Valli
- Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain
| | - Mar Martín-Trillo
- Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain
| | - Carmen Simón-Mateo
- Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain
| | - Juan Antonio García
- Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain
| | - Bernardo Rodamilans
- Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain
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16
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Hong L, Liu R, Qiao X, Wang X, Wang S, Li J, Wu Z, Zhang H. Differential microRNA Expression in Porcine Endometrium Involved in Remodeling and Angiogenesis That Contributes to Embryonic Implantation. Front Genet 2019; 10:661. [PMID: 31402929 PMCID: PMC6677090 DOI: 10.3389/fgene.2019.00661] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2019] [Accepted: 06/24/2019] [Indexed: 11/13/2022] Open
Abstract
Background: In western swine breeds, up to 30% of embryonic losses occur during early pregnancy, and the majority of embryonic losses happens during implantation. In this period, maternal recognition of pregnancy begins to occur and blastocysts undergo dramatic morphologic changes. As with other species, changes in the uterine environment plays an important role in the process of embryo implantation in pigs. Erhualian (ER) pigs, one of the Chinese Taihu swine breeds, are known to have the highest litter size in the world. Experiments demonstrated that the greater embryonic survival on gestation day (GD) 12 in Chinese Taihu pigs is one important factor that contributes to enhanced litter size. This is largely controlled by maternal genes. In this study, endometrial samples were collected from pregnant Landrace×Large Yorkshire (LL) sows (parity 3) and ER sows (parity 3) on GD12 and the expression profiles of microRNAs (miRNAs) in the endometrium were compared between ER and LL using miRNA-seq technology. Results: A total of 288 miRNAs were identified in the pig endometrium, including 202 previously known and 86 novel miRNAs. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that highly abundant miRNAs might affect endometrial remodeling. Comparison between LL and ER sows revealed that 96 known miRNAs were differentially expressed between the two groups (including 78 up-regulated and 18 down-regulated miRNAs in ER compared to LL). Bioinformatics analysis showed that the target genes of some differentially expressed miRNAs were involved in pathways related to angiogenesis, proliferation, apoptosis, and tissue remodeling, which play critical roles in implantation by regulating endometrial structural changes and secretions of hormones, growth factors, and nutrients. Furthermore, the results demonstrated that insulin-like growth factor-1 protein expression was directly inhibited by miR-206. The lower expression of miR-206 in ER compared to LL might facilitate the angiogenesis of the endometrium during embryo implantation. Conclusions: The identified miRNAs that are differentially expressed in the endometrium of ER and LL pigs will contribute to the understanding of the role of miRNAs in embryonic implantation and the molecular mechanisms of the highest embryonic survival in Chinese ER pigs.
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Affiliation(s)
- Linjun Hong
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Ruize Liu
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, United States
| | - Xiwu Qiao
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Xingwang Wang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Shouqi Wang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Jiaqi Li
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Zhenfang Wu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Hao Zhang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
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17
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Kong Q, Quan X, Du J, Tai Y, Liu W, Zhang J, Zhang X, Mu Y, Liu Z. Endo-siRNAs regulate early embryonic development by inhibiting transcription of long terminal repeat sequence in pig†. Biol Reprod 2019; 100:1431-1439. [PMID: 30883641 DOI: 10.1093/biolre/ioz042] [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: 12/11/2018] [Revised: 02/21/2019] [Accepted: 03/16/2019] [Indexed: 11/12/2022] Open
Abstract
Activity of some endogenous retroviruses (ERVs) has been proven to be important for development of early mammalian embryo. However, abnormal activation of ERVs can also cause genetic diseases due to their ability to retrotranspose, so the regulatory mechanism to limit transcription of ERVs needs to be clarified. Endogenous small interfering RNA (endo-siRNA) has been reported to protect cells against transposable elements (TEs). Here, we determined the role of ERVs long terminal repeat sequences (LTRs) derived endo-siRNAs (LTR-siRNAs) on inhibition of the activity of ERVs during early embryonic development in pig. Seven most highly expressed LTR-siRNAs were identified in porcine zygote by high-throughput small RNA sequencing. We verified that the biogenesis of the LTR-siRNAs was DICER-dependent and they were generated from double-stranded RNA (dsRNA) formed by sense and antisense transcripts of LTRs. And, the expression of sense and antisense of LTRs might be due to the loss of DNA methylation at some LTR loci. Furthermore, we showed that the LTR-siRNAs could regulate early embryonic development by repression of LTRs expression at a post-transcriptional level. So, we propose here, during early embryonic development when epigenetic reprogramming occurs, the endo-siRNA pathway acts as a sophisticated balance of regulatory mechanism for ERV activity.
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Affiliation(s)
- Qingran Kong
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Xue Quan
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Jiawei Du
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Yurong Tai
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Wanxin Liu
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Jiaming Zhang
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Xiaolei Zhang
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Yanshuang Mu
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
| | - Zhonghua Liu
- Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Harbin, China
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18
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Moro LN, Amin G, Furmento V, Waisman A, Garate X, Neiman G, La Greca A, Santín Velazque NL, Luzzani C, Sevlever GE, Vichera G, Miriuka SG. MicroRNA characterization in equine induced pluripotent stem cells. PLoS One 2018; 13:e0207074. [PMID: 30507934 PMCID: PMC6277106 DOI: 10.1371/journal.pone.0207074] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Accepted: 10/24/2018] [Indexed: 12/31/2022] Open
Abstract
Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka‘s factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells.
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Affiliation(s)
| | | | | | - Ariel Waisman
- LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina
| | - Ximena Garate
- LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina
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19
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Ekmekci CG, Coskunpinar E, Avci H, Farooqi AA, Orhan KS, Akbas F. Integrative analysis of mRNA and microRNA expression profiles in laryngeal squamous cell carcinoma. J Cell Biochem 2018; 120:3415-3422. [PMID: 30362598 DOI: 10.1002/jcb.27612] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2018] [Accepted: 08/09/2018] [Indexed: 12/12/2022]
Abstract
Larynx cancer is a therapeutically challenging disease. Rapidly evolving experimentally validated data have significantly improved our understanding of the complex role of numerous RNA, DNA, and proteins that play a role in the development and progression of cancer. Based on the insights from approximately two decades of research, it seems clear that microRNAs (miRNAs) have revolutionized our concepts related to the main role of noncoding RNAs in different cancers' progression, development, and metastasis. Mechanistically, miRNAs have been reported to regulate different RNAs and finally protein-coding genes. The expression profiling of miRNAs and messenger RNA (mRNAs) was conducted for a deeper analysis of the miRNAs and mRNAs which play an essential role in larynx cancer. Downregulation or upregulation over twofolds in the miRNAs was considered to be significant, and that of sixfolds or below was considered to be significant for the mRNAs. In accordance with this approach, the expression levels of 43 miRNAs were increased in this study, whereas the expression levels of 129 were decreased. Accordingly, all the genomic expression studies provided evidence of upregulation of 97 genes, whereas 128 genes were found to be downregulated. Among these miRNAs, hsa-miR-20a-3p and hsa-miR-1972 were noted to be important in the etiology of larynx cancer.
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Affiliation(s)
| | - Ender Coskunpinar
- Department of Medical Biology, Faculty of Medicine, University of Health Sciences, Istanbul, Turkey
| | - Hakan Avci
- Department of Otolaryngology and Head and Neck Surgery, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey
| | - Ammad Ahmad Farooqi
- Laboratory for Translational Oncology and Personalized Medicine, Rashid Latif Medical College, Lahore, Pakistan
| | - Kadir Serkan Orhan
- Department of Otolaryngology and Head and Neck Surgery, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey
| | - Fahri Akbas
- Department of Medical Biology, Faculty of Medicine, Bezmialem Vakıf University, Istanbul, Turkey
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20
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Ahn JH, Kwak J, Lee JH, Lee SS. Efficient and accurate analysis of microRNA using a specific extension sequence. Mol Biol Rep 2018; 45:611-619. [PMID: 29846878 DOI: 10.1007/s11033-018-4200-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Accepted: 05/23/2018] [Indexed: 01/01/2023]
Abstract
We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay consists of poly(A) tailing and reverse transcription followed by real-time PCR. In the first step of this reaction, target miRNAs are poly(A) tailed by poly(A) polymerase followed by cDNA synthesis using poly(T) adaptors. In the second step, cDNA is hybridized to the 3'-end of a specific extension sequence that contains part of a miRNA sequence; this cDNA-specific extension sequence hybrid forms the novel PCR template. The PCR template is amplified in a SYBR Green-based quantitative real-time PCR with universal forward and reverse primers. The miR-106b in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative extension-based assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms.
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Affiliation(s)
- Jae Hyun Ahn
- HeimBiotek, Inc., A-201 Pangyo Silicon Park, Bundang-gu, Senognam-si, Kyeonggi-do, 13486, Republic of Korea
| | - Jiwon Kwak
- Department of Pharmaceutical Engineering, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan-si, Chungcheongnam-do, 31538, Republic of Korea
| | - Jae-Hoon Lee
- HeimBiotek, Inc., A-201 Pangyo Silicon Park, Bundang-gu, Senognam-si, Kyeonggi-do, 13486, Republic of Korea
| | - Soo Suk Lee
- Department of Pharmaceutical Engineering, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan-si, Chungcheongnam-do, 31538, Republic of Korea.
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21
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Identification of the miRNAome of early mesoderm progenitor cells and cardiomyocytes derived from human pluripotent stem cells. Sci Rep 2018; 8:8072. [PMID: 29795287 PMCID: PMC5966391 DOI: 10.1038/s41598-018-26156-3] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Accepted: 05/01/2018] [Indexed: 12/21/2022] Open
Abstract
MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.
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22
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Choi C, Yoon S, Moon H, Bae YU, Kim CB, Diskul-Na-Ayudthaya P, Ngu TV, Munir J, Han J, Park SB, Moon JS, Song S, Ryu S. mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets. RNA Biol 2018; 15:763-772. [PMID: 29638187 PMCID: PMC6152462 DOI: 10.1080/15476286.2018.1451723] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method “mirRICH.” The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.
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Affiliation(s)
- Cheolwon Choi
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Seulgi Yoon
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Hyesu Moon
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Yun-Ui Bae
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Chae-Bin Kim
- b Department of Life Science and Biotechnology , Soonchunhyang University , South Korea
| | | | - Trinh Van Ngu
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Javaria Munir
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - JaeWook Han
- c Department of Life Science , Korea University , South Korea
| | | | - Jong-Seok Moon
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Sujung Song
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
| | - Seongho Ryu
- a Soonchunhyang Institute of Med-bioscience (SIMS), Soonchunhyang University , South Korea
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23
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Zhang H, Liu J, Tai Y, Zhang X, Zhang J, Liu S, Lv J, Liu Z, Kong Q. Identification and characterization of L1-specific endo-siRNAs essential for early embryonic development in pig. Oncotarget 2017; 8:23167-23176. [PMID: 28423565 PMCID: PMC5410294 DOI: 10.18632/oncotarget.15517] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Accepted: 02/08/2017] [Indexed: 01/17/2023] Open
Abstract
Small noncoding RNAs (sncRNAs) play important roles in RNA interference (RNAi). In addition to microRNA (miRNA) and Piwi-interacting RNA (piRNA), one key member of sncRNAs group is endogenous small interfering RNA (endo-siRNA). Some studies do show the role of endo-siRNAs in Dicer and/or Ago mutants, however, the biological functions of specific endo-siRNAs remains mostly unanswered. In the study, we have performed a comparative analysis of endo-siRNAs present in porcine sperms, oocytes and zygotes, identified by deep sequencing and bioinformatics analysis. Further, we observe a large amount of endo-siRNAs specific binding on ORF2 and 3′ UTR of porcine L1 (L1-siRNAs). And, 9 L1-siRNAs generated from a dsRNA formed between L1 transcript and a newly identified an antisense noncoding RNA was characterized. We show the L1-siRNAs regulate early embryonic development by inhibiting the activity of L1 retrotransposition. This work can contribute to understanding the functional role of abundant endo-siRNAs in embryonic development.
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Affiliation(s)
- Heng Zhang
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Jilong Liu
- College of Veterinary Medicine, South China Agricultural University, Guangzhou Province 510642, China
| | - Yurong Tai
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Xiaolei Zhang
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Jiaming Zhang
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Shichao Liu
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Jiawei Lv
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Zhonghua Liu
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
| | - Qingran Kong
- Laboratory of Embryo Biotechnology, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
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Kim KJ, Kwak J, Lee JH, Lee SS. Real-time qRT-PCR assay for the detection of miRNAs using bi-directional extension sequences. Anal Biochem 2017; 536:32-35. [DOI: 10.1016/j.ab.2017.08.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2017] [Revised: 08/08/2017] [Accepted: 08/11/2017] [Indexed: 02/06/2023]
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25
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Ayati SH, Fazeli B, Momtazi-Borojeni AA, Cicero AFG, Pirro M, Sahebkar A. Regulatory effects of berberine on microRNome in Cancer and other conditions. Crit Rev Oncol Hematol 2017; 116:147-158. [PMID: 28693796 DOI: 10.1016/j.critrevonc.2017.05.008] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Revised: 04/13/2017] [Accepted: 05/22/2017] [Indexed: 12/19/2022] Open
Abstract
Berberine (BBR) is an isoquinoline alkaloid found in different plant families such as Berberidaceae, Ranunculaceae, and Papaveraceae. BBR is well-known for its anti-inflammatory, lipid-modifying, anticancer, anti-diabetic, antibacterial, antiparasitic and fungicide activities. Multiple pharmacological actions of BBR stem from different molecular targets of this phytochemical. MicroRNAs (miRs) are single-stranded, evolutionary conserved, small non-coding RNA molecules with a length of 19-23 nucleotides that are involved in RNA silencing and post-transcriptional regulation of gene expression through binding to the 3'-untranslated region (3'UTR) of target mRNA. MiRs emerged as important regulatory elements in almost all biological processes like cell proliferation, apoptosis, differentiation and organogenesis, and numerous human diseases such as cancer and diabetes. BBR was shown to regulate the expression of miRs in several diseases. Here, we reviewed the target miRs of BBR and the relevance of their modulation for the potential treatment of serious human diseases like multiple myeloma, hepatocellular carcinoma, colorectal cancer, gastric cancer, ovarian cancer and glioblastoma. The role of miR regulation in the putative anti-diabetic effects of BBR is discussed, as well.
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Affiliation(s)
- Seyed Hasan Ayati
- Immunology Research Center, Department of Immunology, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Badrieh Fazeli
- Department of Biology, Faculty of Science, Isfahan University, Isfahan, Iran
| | - Amir Abbas Momtazi-Borojeni
- Nanotechnology Research Center, Student Research Committee, Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
| | - Arrigo F G Cicero
- Department of Medical and Surgical Sciences, University of Bologna, Via Albertoni 15, Bologna, Italy
| | - Matteo Pirro
- Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Medicine, University of Perugia, Perugia, Italy
| | - Amirhossein Sahebkar
- Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran.
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Worku T, Rehman ZU, Talpur HS, Bhattarai D, Ullah F, Malobi N, Kebede T, Yang L. MicroRNAs: New Insight in Modulating Follicular Atresia: A Review. Int J Mol Sci 2017; 18:ijms18020333. [PMID: 28208755 PMCID: PMC5343868 DOI: 10.3390/ijms18020333] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Revised: 01/13/2017] [Accepted: 01/23/2017] [Indexed: 01/12/2023] Open
Abstract
Our understanding of the post-transcriptional mechanisms involved in follicular atresia is limited; however, an important development has been made in understanding the biological regulatory networks responsible for mediating follicular atresia. MicroRNAs have come to be seen as a key regulatory actor in determining cell fate in a wide range of tissues in normal and pathological processes. Profiling studies of miRNAs during follicular atresia and development have identified several putative miRNAs enriched in apoptosis signaling pathways. Subsequent in vitro and/or in vivo studies of granulosa cells have elucidated the functional role of some miRNAs along with their molecular pathways. In particular, the regulatory roles of some miRNAs have been consistently observed during studies of follicular cellular apoptosis. Continued work should gradually lead to better understanding of the role of miRNAs in this field. Ultimately, we expect this understanding will have substantial benefits for fertility management at both the in vivo or/and in vitro levels. The stable nature of miRNA holds remarkable promise in clinical use as a diagnostic tool and in reproductive medicine to solve the ever-increasing fertility problem. In this review, we summarize current knowledge of the involvement of miRNAs in follicular atresia, discuss the challenges for further work and pinpoint areas for future research.
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Affiliation(s)
- Tesfaye Worku
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
- School of Veterinary Medicine, Wollega University, P.O. Box 395, Nekemte, Ethiopia.
| | - Zia Ur Rehman
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
| | - Hira Sajjad Talpur
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
| | - Dinesh Bhattarai
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
| | - Farman Ullah
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
| | - Ngabu Malobi
- State Key Laboratory of Agricultural Microbiology, Education Ministry of China, College of Veterinary Medicine Huazhong Agricultural University, Wuhan 430070, China.
| | - Tesfaye Kebede
- Departments of Animal and Aquaculture Sciences, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, Norway.
| | - Liguo Yang
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
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Sermersheim MA, Park KH, Gumpper K, Adesanya TMA, Song K, Tan T, Ren X, Yang JM, Zhu H. MicroRNA regulation of autophagy in cardiovascular disease. Front Biosci (Landmark Ed) 2017; 22:48-65. [PMID: 27814601 DOI: 10.2741/4471] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Autophagy, a form of lysosomal degradation capable of eliminating dysfunctional proteins and organelles, is a cellular process associated with homeostasis. Autophagy functions in cell survival by breaking down proteins and organelles and recycling them to meet metabolic demands. However, aberrant up regulation of autophagy can function as an alternative to apoptosis. The duality of autophagy, and its regulation over cell survival/death, intimately links it with human disease. Non-coding RNAs regulate mRNA levels and elicit diverse effects on mammalian protein expression. The most studied non-coding RNAs to-date are microRNAs (miRNA). MicroRNAs function in post-transcriptional regulation, causing profound changes in protein levels, and affect many biological processes and diseases. The role and regulation of autophagy, whether it is beneficial or harmful, is a controversial topic in cardiovascular disease. A number of recent studies have identified miRNAs that target autophagy-related proteins and influence the development, progression, or treatment of cardiovascular disease. Understanding the mechanisms by which these miRNAs work can provide promising insight and potential progress towards the development of therapeutic treatments in cardiovascular disease.
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Affiliation(s)
- Matthew A Sermersheim
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA
| | - Ki Ho Park
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA
| | - Kristyn Gumpper
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA
| | - T M Ayodele Adesanya
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA
| | - Kuncheng Song
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA
| | - Tao Tan
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA
| | - Xingcong Ren
- Department of Pharmacology, The Penn State Hershey Cancer Institute, The Pennsylvania State University, College of Medicine and Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA
| | - Jin-Ming Yang
- Department of Pharmacology, The Penn State Hershey Cancer Institute, The Pennsylvania State University, College of Medicine and Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA
| | - Hua Zhu
- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA,
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28
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Surges R, Kretschmann A, Abnaof K, van Rikxoort M, Ridder K, Fröhlich H, Danis B, Kaminski RM, Foerch P, Elger CE, Weinsberg F, Pfeifer A. Changes in serum miRNAs following generalized convulsive seizures in human mesial temporal lobe epilepsy. Biochem Biophys Res Commun 2016; 481:13-18. [PMID: 27833019 DOI: 10.1016/j.bbrc.2016.11.029] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Accepted: 11/07/2016] [Indexed: 01/25/2023]
Abstract
MicroRNAs (miRNAs) are key regulators of gene expression and are involved in the pathomechanisms of epilepsy. MiRNAs may also serve as peripheral biomarkers of epilepsy. We investigated the miRNA profile in the blood serum of patients suffering from mesial temporal lobe epilepsy (mTLE) following a single focal seizure evolving to a bilateral convulsive seizure (BCS) during video-EEG monitoring. Data of 15 patients were included in the final analysis. MiRNA expression was determined using Real Time-PCR followed by thorough bioinformatical analysis of expression levels. We found that more than 200 miRNAs were differentially expressed in the serum of patients within 30 min after a single seizure. Validation of the 20 top miRNA candidates confirmed that 4 miRNAs (miR-143, miR-145, miR-532, miR-365a) were significantly deregulated. Interestingly, in a sub-group of patients with seizures occurring during sleep, we found 10 miRNAs to be deregulated up to 20-28 h after the seizure. In this group of patients, miR-663b was significantly deregulated. We conclude that single seizures are associated with detectable transient miRNA alterations in blood serum in the early postictal phase. The significant upregulation of miR-663b following BCS arising during sleep indicates potential suitability of this miRNA as a potential biomarker for seizure diagnostics.
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Affiliation(s)
- Rainer Surges
- Department of Epileptology, University Hospital of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany
| | - Anita Kretschmann
- Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany
| | - Khalid Abnaof
- Bonn-Aachen International Center for Information Technology (B-IT), Algorithmic Bioinformatics, University of Bonn, Dahlmannstr. 2, 53113, Bonn, Germany
| | - Marijke van Rikxoort
- Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany
| | - Kirsten Ridder
- Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany
| | - Holger Fröhlich
- Bonn-Aachen International Center for Information Technology (B-IT), Algorithmic Bioinformatics, University of Bonn, Dahlmannstr. 2, 53113, Bonn, Germany
| | - Bénédicte Danis
- UCB Pharma GmbH, Alfred-Nobel-Straße 10, 40789, Monheim, Germany
| | - Rafal M Kaminski
- UCB Pharma GmbH, Alfred-Nobel-Straße 10, 40789, Monheim, Germany
| | - Patrik Foerch
- UCB Pharma GmbH, Alfred-Nobel-Straße 10, 40789, Monheim, Germany
| | - Christian E Elger
- Department of Epileptology, University Hospital of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany
| | - Frank Weinsberg
- UCB Pharma GmbH, Alfred-Nobel-Straße 10, 40789, Monheim, Germany.
| | - Alexander Pfeifer
- Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany.
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Matoušek J, Kocábek T, Patzak J, Bříza J, Siglová K, Mishra AK, Duraisamy GS, Týcová A, Ono E, Krofta K. The "putative" role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.). PLANT MOLECULAR BIOLOGY 2016; 92:263-77. [PMID: 27392499 DOI: 10.1007/s11103-016-0510-7] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Accepted: 07/02/2016] [Indexed: 05/20/2023]
Abstract
Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.
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Affiliation(s)
- Jaroslav Matoušek
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic.
| | - Tomáš Kocábek
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic
| | - Josef Patzak
- Hop Research Institute, Co. Ltd., Kadaňská 2525, 438 46, Žatec, Czech Republic
| | - Jindřich Bříza
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic
| | - Kristýna Siglová
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic
- Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1760, 370 05, České Budějovice, Czech Republic
| | - Ajay Kumar Mishra
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic
| | - Ganesh Selvaraj Duraisamy
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic
| | - Anna Týcová
- Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05, České Budějovice, Czech Republic
- Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1760, 370 05, České Budějovice, Czech Republic
| | - Eiichiro Ono
- Research Institute, Suntory Global Innovation Center (SIC) Ltd., 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka, 618-8503, Japan
| | - Karel Krofta
- Hop Research Institute, Co. Ltd., Kadaňská 2525, 438 46, Žatec, Czech Republic
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Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes. Appl Environ Microbiol 2016; 82:2263-2269. [PMID: 26826223 DOI: 10.1128/aem.04044-15] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2015] [Accepted: 01/20/2016] [Indexed: 11/20/2022] Open
Abstract
Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faeciumNCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold;P= 0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold;P= 0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold;P= 0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold;P= 0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anti correlated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding of E. faeciumon the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action of E. faeciumon immune cell regulation in the small intestine.
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Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease. MOLECULAR THERAPY. NUCLEIC ACIDS 2016; 5:e297. [PMID: 27003755 PMCID: PMC5014463 DOI: 10.1038/mtna.2016.7] [Citation(s) in RCA: 96] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Accepted: 01/14/2016] [Indexed: 12/29/2022]
Abstract
Huntington's disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.
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Seyhan AA. microRNAs with different functions and roles in disease development and as potential biomarkers of diabetes: progress and challenges. MOLECULAR BIOSYSTEMS 2016; 11:1217-34. [PMID: 25765998 DOI: 10.1039/c5mb00064e] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Biomarkers provide information on early detection of diseases, in determining individuals at risk of developing complications or subtyping individuals for disease phenotypes. In addition, biomarkers may lead to better treatment strategies, personalized therapy, and improved outcome. A major gap in the field of biomarker development is that we have not identified appropriate (minimally invasive, life-style independent and informative) biomarkers for the underlying disease process(es) that can be measured in readily accessible samples (e.g. serum, plasma, blood, urine). miRNAs function as regulators in wide ranging cellular and physiological functions and also participate in many physiopathological processes and thus have been linked to many diseases including diabetes, metabolic and cardiovascular diseases, cancer, neurodegenerative diseases, and autoimmunity. Many miRNAs have been shown to have predictive value as potential biomarkers in a variety of diseases including diabetes, which are detectable in some instances many years before the manifestation of disease. Although some technical challenges still remain, due to their availability in the circulation, relative stability, and ease of detection; miRNAs have emerged as a promising new class of biomarkers to provide information on early detection of disease, monitoring disease progression, in determining individual's risk of developing complications or subtyping individuals for disease phenotypes, and to monitor response to therapeutic interventions. As a final note, most of the miRNAs reported in the literature have not yet been validated in sufficiently powered and longitudinal studies for specificity for that particular disease.
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Affiliation(s)
- Attila A Seyhan
- Translational Research Institute for Metabolism and Diabetes, Florida Hospital, Sanford Burnham Institute, 301 E. Princeton Street, Orlando, FL 32804, USA.
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P elements and the determinants of hybrid dysgenesis have different dynamics of propagation in Drosophila melanogaster populations. Genetica 2015; 143:751-9. [PMID: 26530414 DOI: 10.1007/s10709-015-9872-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2015] [Accepted: 10/25/2015] [Indexed: 01/14/2023]
Abstract
Intraspecific hybrid dysgenesis (HD) appears after some strains of D. melanogaster are crossed. The predominant idea is that the movement of transposable P elements causes HD. It is believed that P elements appeared in the D. melanogaster genome in the middle of the last century by horizontal transfer, simultaneously with the appearance of HD determinants. A subsequent simultaneous expansion of HD determinants and P elements occurred. We analyzed the current distribution of HD determinants in natural populations of D. melanogaster and found no evidence of their further spread. However, full-sized P elements were identified in the genomes of all analyzed natural D. melanogaster strains independent of their cytotypes. Thus, the expansion of P elements does not correlate with the expansion of HD determinants. We found that the ovaries of dysgenic females did not contain germ cells despite the equal number of primordial germ cells in early stages in dysgenic and non-dysgenic embryos. We propose that HD does not result from DNA damage caused by P element transposition, but it would be the disruption in the regulation of dysgenic ovarian formation that causes the dysgenic phenotypes.
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Vatandoost N, Amini M, Iraj B, Momenzadeh S, Salehi R. Dysregulated miR-103 and miR-143 expression in peripheral blood mononuclear cells from induced prediabetes and type 2 diabetes rats. Gene 2015; 572:95-100. [DOI: 10.1016/j.gene.2015.07.015] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2014] [Revised: 06/28/2015] [Accepted: 07/01/2015] [Indexed: 12/25/2022]
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35
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Kaushik A, Saraf S, Mukherjee SK, Gupta D. miRMOD: a tool for identification and analysis of 5' and 3' miRNA modifications in Next Generation Sequencing small RNA data. PeerJ 2015; 3:e1332. [PMID: 26623179 PMCID: PMC4662591 DOI: 10.7717/peerj.1332] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Accepted: 09/27/2015] [Indexed: 12/27/2022] Open
Abstract
In the past decade, the microRNAs (miRNAs) have emerged to be important regulators of gene expression across various species. Several studies have confirmed different types of post-transcriptional modifications at terminal ends of miRNAs. The reports indicate that miRNA modifications are conserved and functionally significant as it may affect miRNA stability and ability to bind mRNA targets, hence affecting target gene repression. Next Generation Sequencing (NGS) of the small RNA (sRNA) provides an efficient and reliable method to explore miRNA modifications. The need for dedicated software, especially for users with little knowledge of computers, to determine and analyze miRNA modifications in sRNA NGS data, motivated us to develop miRMOD. miRMOD is a user-friendly, Microsoft Windows and Graphical User Interface (GUI) based tool for identification and analysis of 5′ and 3′ miRNA modifications (non-templated nucleotide additions and trimming) in sRNA NGS data. In addition to identification of miRNA modifications, the tool also predicts and compares the targets of query and modified miRNAs. In order to compare binding affinities for the same target, miRMOD utilizes minimum free energies of the miRNA:target and modified-miRNA:target interactions. Comparisons of the binding energies may guide experimental exploration of miRNA post-transcriptional modifications. The tool is available as a stand-alone package to overcome large data transfer problems commonly faced in web-based high-throughput (HT) sequencing data analysis tools. miRMOD package is freely available at http://bioinfo.icgeb.res.in/miRMOD.
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Affiliation(s)
- Abhinav Kaushik
- Bioinformatics Laboratory, SCB Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, India
| | - Shradha Saraf
- Bioinformatics Laboratory, SCB Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, India
| | - Sunil K Mukherjee
- Department of Genetics, University of Delhi, South Campus, Delhi, India
| | - Dinesh Gupta
- Bioinformatics Laboratory, SCB Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, India
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Liu Q, Shin Y, Kee JS, Kim KW, Mohamed Rafei SR, Perera AP, Tu X, Lo GQ, Ricci E, Colombel M, Chiong E, Thiery JP, Park MK. Mach-Zehnder interferometer (MZI) point-of-care system for rapid multiplexed detection of microRNAs in human urine specimens. Biosens Bioelectron 2015; 71:365-372. [PMID: 25950930 DOI: 10.1016/j.bios.2015.04.052] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2015] [Revised: 04/13/2015] [Accepted: 04/17/2015] [Indexed: 12/19/2022]
Abstract
MicroRNAs have been identified as promising biomarkers for human diseases. The development of a point-of-care (POC) test for the disease-associated miRNAs would be especially beneficial, since miRNAs are unexpectedly well preserved in various human specimens, including urine. Here, we present the Mach-Zehnder interferometer-miRNA detection system capable of detecting multiple miRNAs in clinical urine samples rapidly and simultaneously in a label-free and real-time manner. Through measurement of the light phase change, the MZI sensor provides an optical platform for fast profiling of small molecules with improved accuracy. We demonstrate that this system could specifically detect target miRNAs (miR-21, and let-7a), and even identify the single nucleotide polymorphism of the let-7 family of miRNAs from synthetic and cell line samples. The clinical applicability of this system is confirmed by simultaneously detecting two types of miRNAs in urine samples of bladder cancer patients in a single reaction, with a detection time of 15 min. The POC system can be expanded to detect a number of miRNAs of different species and should be useful for a variety of clinical applications requiring at or near the site of patient care.
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Affiliation(s)
- Qing Liu
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Yong Shin
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Jack Sheng Kee
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Kyung Woo Kim
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Siti Rafeah Mohamed Rafei
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Agampodi Promoda Perera
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Xiaoguang Tu
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Guo-Qiang Lo
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore
| | - Estelle Ricci
- Service d'Urologie et Chirurgie de la Transplantation, Hôpital Edouard Herriot, 5 Place d'Arsonval, 69437 Lyon Cedex 03, France
| | - Marc Colombel
- Service d'Urologie et Chirurgie de la Transplantation, Hôpital Edouard Herriot, 5 Place d'Arsonval, 69437 Lyon Cedex 03, France
| | - Edmund Chiong
- Department of Urology, National University Health System, Singapore
| | - Jean Paul Thiery
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, 138673, Singapore
| | - Mi Kyoung Park
- Institute of Microelectronics, A⁎STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, 117685, Singapore.
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Bowater RP, Cobb AM, Pivonkova H, Havran L, Fojta M. Biophysical and electrochemical studies of protein–nucleic acid interactions. MONATSHEFTE FUR CHEMIE 2015. [DOI: 10.1007/s00706-014-1405-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Erickson JR, Echeverri K. In vivo modulation and quantification of microRNAs during axolotl tail regeneration. Methods Mol Biol 2015; 1290:159-67. [PMID: 25740485 PMCID: PMC5896293 DOI: 10.1007/978-1-4939-2495-0_13] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
The ability to regenerate diseased, injured, or missing complex tissue is widespread throughout lower vertebrates and invertebrates; however, our knowledge of the molecular mechanisms that regulate this amazing ability is still in its infancy. Many recent papers have shown important roles for microRNAs in regulating regeneration in a number of species. The ability to detect and quantify miRNA expression fluctuations at a single cell level in vivo in different cell types during processes like regeneration is very informative. In this chapter, we describe how to use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time following miRNA mimic injection as well as during regeneration. In this bicistronic vector, the mRFP allows for verification of miRNA expression, while the GFP functions as an internal control to normalize miRNA expression and thus obtain quantitative results. In addition, we demonstrate how this technique revealed dynamic miR-23a expression and function during tail regeneration.
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Affiliation(s)
- Jami R. Erickson
- Dept. of Genetics, Cell Biology and Development, Stem Cell Institute, University of Minnesota, Minneapolis, USA
| | - Karen Echeverri
- Dept. of Genetics, Cell Biology and Development, Stem Cell Institute, University of Minnesota, Minneapolis, USA
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Esguerra JLS, Mollet IG, Salunkhe VA, Wendt A, Eliasson L. Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs. Genes (Basel) 2014; 5:1018-31. [PMID: 25383562 PMCID: PMC4276924 DOI: 10.3390/genes5041018] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2014] [Revised: 10/01/2014] [Accepted: 10/21/2014] [Indexed: 12/31/2022] Open
Abstract
Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs) is termed "stimulus-secretion coupling." Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D). The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs) are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.
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Affiliation(s)
- Jonathan L S Esguerra
- Unit of Islet Cell Exocytosis, Department of Clinical Sciences in Malmö, Lund University Diabetes Centre, Lund University, CRC 91-11, Jan Waldenströms gata 35, 205 02 Malmö, Sweden.
| | - Inês G Mollet
- Unit of Islet Cell Exocytosis, Department of Clinical Sciences in Malmö, Lund University Diabetes Centre, Lund University, CRC 91-11, Jan Waldenströms gata 35, 205 02 Malmö, Sweden.
| | - Vishal A Salunkhe
- Unit of Islet Cell Exocytosis, Department of Clinical Sciences in Malmö, Lund University Diabetes Centre, Lund University, CRC 91-11, Jan Waldenströms gata 35, 205 02 Malmö, Sweden.
| | - Anna Wendt
- Unit of Islet Cell Exocytosis, Department of Clinical Sciences in Malmö, Lund University Diabetes Centre, Lund University, CRC 91-11, Jan Waldenströms gata 35, 205 02 Malmö, Sweden.
| | - Lena Eliasson
- Unit of Islet Cell Exocytosis, Department of Clinical Sciences in Malmö, Lund University Diabetes Centre, Lund University, CRC 91-11, Jan Waldenströms gata 35, 205 02 Malmö, Sweden.
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Kretschmann A, Danis B, Andonovic L, Abnaof K, van Rikxoort M, Siegel F, Mazzuferi M, Godard P, Hanon E, Fröhlich H, Kaminski RM, Foerch P, Pfeifer A. Different microRNA profiles in chronic epilepsy versus acute seizure mouse models. J Mol Neurosci 2014; 55:466-79. [PMID: 25078263 PMCID: PMC4303710 DOI: 10.1007/s12031-014-0368-6] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2014] [Accepted: 06/26/2014] [Indexed: 11/29/2022]
Abstract
Epilepsy affects around 50 million people worldwide, and in about 65 % of patients, the etiology of disease is unknown. MicroRNAs are small non-coding RNAs that have been suggested to play a role in the pathophysiology of epilepsy. Here, we compared microRNA expression patterns in the hippocampus using two chronic models of epilepsy characterised by recurrent spontaneous seizures (pilocarpine and self-sustained status epilepticus (SSSE)) and an acute 6-Hz seizure model. The vast majority of microRNAs deregulated in the acute model exhibited increased expression with 146 microRNAs up-regulated within 6 h after a single seizure. In contrast, in the chronic models, the number of up-regulated microRNAs was similar to the number of down-regulated microRNAs. Three microRNAs—miR-142-5p, miR-331-3p and miR-30a-5p—were commonly deregulated in all three models. However, there is a clear overlap of differentially expressed microRNAs within the chronic models with 36 and 15 microRNAs co-regulated at 24 h and at 28 days following status epilepticus, respectively. Pathway analysis revealed that the altered microRNAs are associated with inflammation, innate immunity and cell cycle regulation. Taken together, the identified microRNAs and the pathways they modulate might represent candidates for novel molecular approaches for the treatment of patients with epilepsy.
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Affiliation(s)
- Anita Kretschmann
- Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany
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Eliasson L, Esguerra JLS. Role of non-coding RNAs in pancreatic beta-cell development and physiology. Acta Physiol (Oxf) 2014; 211:273-84. [PMID: 24666639 DOI: 10.1111/apha.12285] [Citation(s) in RCA: 64] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Revised: 02/25/2014] [Accepted: 03/17/2014] [Indexed: 12/15/2022]
Abstract
The progression of diabetes is accompanied by increasing demand to the beta-cells to produce and secrete more insulin, requiring complex beta-cell adaptations. Functionally active and ubiquitous non-coding RNAs (ncRNAs) have the capacity to take part in such adaptations as they have been shown to be key regulatory molecules in various biological processes. In the pancreatic islets, the function of ncRNAs and their contribution to disease development is beginning to be understood. Here, we review the different classes of ncRNAs, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), and their potential contribution to insulin secretion. A special focus will be on miRNAs and their regulatory function in beta-cell physiology and insulin exocytosis. As important players in gene regulation, ncRNAs have huge potential in opening innovative therapeutic avenues against diabetes and associated complications.
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Affiliation(s)
- L. Eliasson
- Department of Clinical Sciences-Malmö; Islet Cell Exocytosis; Lund University Diabetes Centre; Lund University; Malmö Sweden
| | - J. L. S. Esguerra
- Department of Clinical Sciences-Malmö; Islet Cell Exocytosis; Lund University Diabetes Centre; Lund University; Malmö Sweden
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Luo Z, Azencott R, Zhao Y. Modeling miRNA-mRNA interactions: fitting chemical kinetics equations to microarray data. BMC SYSTEMS BIOLOGY 2014; 8:19. [PMID: 24548346 PMCID: PMC3937077 DOI: 10.1186/1752-0509-8-19] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/10/2013] [Accepted: 02/12/2014] [Indexed: 01/22/2023]
Abstract
BACKGROUND The miRNAs are small non-coding RNAs of roughly 22 nucleotides in length, which can bind with and inhibit protein coding mRNAs through complementary base pairing. By degrading mRNAs and repressing proteins, miRNAs regulate the cell signaling and cell functions. This paper focuses on innovative mathematical techniques to model gene interactions by algorithmic analysis of microarray data. Our goal was to elucidate which mRNAs were actually degraded or had their translation inhibited by miRNAs belonging to a very large pool of potential miRNAs. RESULTS We proposed two chemical kinetics equations (CKEs) to model the interactions between miRNAs, mRNAs and the associated proteins. In order to reduce computational cost, we used a non linear profile clustering method named minimal net clustering and efficiently condensed the large set of expression profiles observed in our microarray data sets. We determined unknown parameters of the CKE models by minimizing the discrepancy between model prediction and data, using our own fast non linear optimization algorithm. We then retained only the CKE models for which the optimized fit to microarray data is of high quality and validated multiple miRNA-mRNA pairs. CONCLUSION The implementation of CKE modeling and minimal net clustering reduces drastically the potential set of miRNA-mRNA pairs, with a high gain for further experimental validations. The minimal net clustering also provides good miRNA candidates that have similar regulatory roles.
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Affiliation(s)
- Zijun Luo
- Department of Mathematics, University of Houston, 4800 Calhoun, Houston, TX, USA.
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Johnson BN, Mutharasan R. Biosensor-based microRNA detection: techniques, design, performance, and challenges. Analyst 2014; 139:1576-88. [DOI: 10.1039/c3an01677c] [Citation(s) in RCA: 113] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
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Abstract
MicroRNAs (miRNAs) have attracted ever-increasing interest in recent years. Since experimental approaches for determining miRNAs are nontrivial in their application, computational methods for the prediction of miRNAs have gained popularity. Such methods can be grouped into two broad categories (1) performing ab initio predictions of miRNAs from primary sequence alone and (2) additionally employing phylogenetic conservation. Most methods acknowledge the importance of hairpin or stem-loop structures and employ various methods for the prediction of RNA secondary structure. Machine learning has been employed in both categories with classification being the predominant method. In most cases, positive and negative examples are necessary for performing classification. Since it is currently elusive to experimentally determine all possible miRNAs for an organism, true negative examples are hard to come by, and therefore the accuracy assessment of algorithms is hampered. In this chapter, first RNA secondary structure prediction is introduced since it provides a basis for miRNA prediction. This is followed by an assessment of homology and then ab initio miRNA prediction methods.
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Affiliation(s)
- Jens Allmer
- Molecular Biology and Genetics, Izmir Institute of Technology, Izmir, Turkey
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Molecular markers for prostate cancer in formalin-fixed paraffin-embedded tissues. BIOMED RESEARCH INTERNATIONAL 2013; 2013:283635. [PMID: 24371818 PMCID: PMC3859157 DOI: 10.1155/2013/283635] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 08/29/2013] [Accepted: 10/10/2013] [Indexed: 12/14/2022]
Abstract
Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.
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Raabe CA, Tang TH, Brosius J, Rozhdestvensky TS. Biases in small RNA deep sequencing data. Nucleic Acids Res 2013; 42:1414-26. [PMID: 24198247 PMCID: PMC3919602 DOI: 10.1093/nar/gkt1021] [Citation(s) in RCA: 162] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias.
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Affiliation(s)
- Carsten A Raabe
- Institute of Experimental Pathology (ZMBE), University of Muenster, Von-Esmarch-Strasse 56, 48149 Muenster, Germany and Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, 13200 Penang, Malaysia
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Giles CB, Girija-Devi R, Dozmorov MG, Wren JD. mirCoX: a database of miRNA-mRNA expression correlations derived from RNA-seq meta-analysis. BMC Bioinformatics 2013; 14 Suppl 14:S17. [PMID: 24267917 PMCID: PMC3850996 DOI: 10.1186/1471-2105-14-s14-s17] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Background Experimentally validated co-expression correlations between miRNAs and genes are a valuable resource to corroborate observations about miRNA/mRNA changes after experimental perturbations, as well as compare miRNA target predictions with empirical observations. For example, when a given miRNA is transcribed, true targets of that miRNA should tend to have lower expression levels relative to when the miRNA is not expressed. Methods We processed publicly available human RNA-seq experiments obtained from NCBI's Sequence Read Archive (SRA) to identify miRNA-mRNA co-expression trends and summarized them in terms of their Pearson's Correlation Coefficient (PCC) and significance. Results We found that sequence-derived parameters from TargetScan and miRanda were predictive of co-expression, and that TargetScan- and miRanda-derived gene-miRNA pairs tend to have anti-correlated expression patterns in RNA-seq data compared to controls. We provide this data for download and as a web application available at http://wrenlab.org/mirCoX/. Conclusion This database of empirically established miRNA-mRNA transcriptional correlations will help to corroborate experimental observations and could be used to help refine and validate miRNA target predictions.
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Dong H, Lei J, Ding L, Wen Y, Ju H, Zhang X. MicroRNA: Function, Detection, and Bioanalysis. Chem Rev 2013; 113:6207-33. [PMID: 23697835 DOI: 10.1021/cr300362f] [Citation(s) in RCA: 883] [Impact Index Per Article: 73.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Haifeng Dong
- Research Center for Bioengineering and Sensing Technology, University of Science & Technology Beijing, Beijing 100083, P. R. China
| | - Jianping Lei
- State Key Laboratory of Analytical
Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China
| | - Lin Ding
- State Key Laboratory of Analytical
Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China
| | - Yongqiang Wen
- Research Center for Bioengineering and Sensing Technology, University of Science & Technology Beijing, Beijing 100083, P. R. China
| | - Huangxian Ju
- State Key Laboratory of Analytical
Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China
| | - Xueji Zhang
- Research Center for Bioengineering and Sensing Technology, University of Science & Technology Beijing, Beijing 100083, P. R. China
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Song A, Ye J, Zhang K, Sun L, Zhao Y, Yu H. Lentiviral vector-mediated siRNA knockdown of c-MYC: cell growth inhibition and cell cycle arrest at G2/M phase in Jijoye cells. Biochem Genet 2013; 51:603-17. [PMID: 23657834 DOI: 10.1007/s10528-013-9590-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2012] [Accepted: 10/16/2012] [Indexed: 12/25/2022]
Abstract
Inhibition of c-MYC has been considered as a potential therapy for lymphoma treatment. We explored a lentiviral vector-mediated small interfering RNA (siRNA) expression vector to stably reduce c-MYC expression in B cell line Jijoye cells and investigated the effects of c-MYC downregulation on cell growth, cell cycle, and apoptosis in vitro. The expression of c-MYC mRNA and protein levels were inhibited significantly by c-MYC siRNA. The c-MYC downregulation resulted in the inhibition of cell proliferation and cell cycle arrest at G2/M phase, which was associated with decreased expression of cyclin B and cyclin-dependent kinase 1 (CDK1) and increased expression of CDK inhibitor p21 proteins. In addition, downregulation of c-MYC induced cell apoptosis characterized by DNA fragmentation and caspase-3 activation. Taken together, these results suggest that lentiviral vector-mediated siRNA for c-MYC may be a promising approach for targeting c-MYC in the treatment of Burkitt lymphoma.
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Affiliation(s)
- Aiqin Song
- Department of Pediatric Hematology, Affiliated Hospital of Qingdao University Medical College, 16 Jiangsu Road, Qingdao, 266001 Shandong, China.
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Gao L, Ren W, Chang S, Guo B, Huang S, Li M, Guo Y, Li Z, Song T, Zhi K, Huang C. Downregulation of miR-145 expression in oral squamous cell carcinomas and its clinical significance. ACTA ACUST UNITED AC 2013; 36:194-9. [PMID: 23548968 DOI: 10.1159/000349956] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
BACKGROUND MicroRNAs have been reported to play roles as oncogenes or tumor suppressor genes in human cancers. However, the expression levels of miR-145 in oral squamous cell carcinoma (OSCC) are unclear. The purpose of this study was to investigate the status of miR-145 expression in OSCC and determine its clinical significance. PATIENTS AND METHODS We examined miR-145 levels in 62 OSCC tissue samples and cell lines by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The relationship between miR-145 expression and clinicopathologic factors of OSCC patients was analyzed. RESULTS The proportion of miR-145 low expression was 82.26% (51/62) among the 62 OSCC patients, and expression levels of miR-145 in OSCC tissue samples and cell lines were significantly lower than in non-tumor controls. miR-145 expression levels were not significantly associated with age (p = 0.607), sex (p = 0.213), location (p = 0.952), histology (p = 0.603), pT stage (p = 0.305), pTNM stage (p = 0.471), and lymphatic metastasis (p = 1.000). CONCLUSION miR-145 may be involved in the early tumorigenesis of oral squamous cells, and might be a potential biomarker in the early diagnosis of OSCC.
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Affiliation(s)
- Ling Gao
- Department of Oral Maxillofacial Surgery, Stomatological Hospital, College of Medicine, Xi'an, Shaanxi, P.R. China
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