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Cui H, Xin Y, Cao F, Gan Z, Tian Y, Liu W, Shi P. The correlation between CpG island methylation of hTERT promoter and human age prediction. Leg Med (Tokyo) 2023; 63:102270. [PMID: 37207612 DOI: 10.1016/j.legalmed.2023.102270] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Revised: 04/28/2023] [Accepted: 05/09/2023] [Indexed: 05/21/2023]
Abstract
DNA methylation is an epigenetic modification that occurs during the life cycle of individuals. Its degree is closely associated with the methylation status of CpG sites in its promoter region. Based on the previous screening that the hTERT methylation is both related to tumors and age, we suspected that the age inference based on hTERT methylation would be disturbed by the disease of the tested person. Herein, eight CpG sites in the hTERT promoter region were analyzed by real-time methylation-specific PCR, and we found that CpG2, CpG5, and CpG8 were closely related to the tumor (P < 0.05). The remaining five CpG sites had a large error in predicting age alone. Combining them to establish a model yielded better results, with an average age error of 4.35 years. This study provides a reliable and accurate detection method for the DNA methylation status of multiple CpG sites on the hTERT gene promoter, which can be used for the prediction of forensic age and assistant diagnosis of clinical diseases.
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Affiliation(s)
- Hanyue Cui
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
| | - Ye Xin
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
| | - Fangqi Cao
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Zhongshan North No 1 Road, Shanghai 200083, China
| | - Ziye Gan
- Ulink College of Shanghai, 559 Laiting South Road, Shanghai 201615, China
| | - Yuxiang Tian
- Department of Clinical Laboratory, Shanghai Xuhui District Dahua Hospital, Shanghai 200237, China
| | - Wenbin Liu
- Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Zhongshan North No 1 Road, Shanghai 200083, China.
| | - Ping Shi
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
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Rad FT, Gargari BN, Ghorbian S, Farsani ZS, Sharifi R. Inhibiting the growth of melanoma cells via hTERT gene editing using CRISPR-dCas9-dnmt3a system. Gene 2022; 828:146477. [PMID: 35398175 DOI: 10.1016/j.gene.2022.146477] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 03/21/2022] [Accepted: 04/01/2022] [Indexed: 12/24/2022]
Abstract
CRISPR-Cas9 gene-editing technology has pushed the boundaries of genetic modification. The principle of this method is based on the purposeful defense system of DNA degradation and will be one of the most powerful instruments for gene editing shortly. The purpose of this study was to evaluate the capability of this approach to manage melanoma cells. The present study used EF1a-hsaCas9-U6-gRNA as a hybrid vector of sgRNA and Cas9 for the transfection of A-375 melanoma cells. Transfection efficiency was enhanced by examining the two concentrations of 4 and 8 µg/mL of hexadimethrine bromide (trade name Polybrene). The existence of Cas9 in transfected cells was detected by flow cytometry. The expression level of the metabisulfite-modified hTERT gene was measured by real-time PCR technique. The presence of telomerase in cells was determined by flow cytometry and western blotting analysis. The hTERT gene promoter methylation was also evaluated by HRM assay. Finally, the induction of apoptosis in transfected A375 cells was assessed using flow cytometry. The results showed that the presence of gRNA significantly increased the transfection efficiency (up to about 7.75 times higher). The hTERT expression levels in A-375 cells were significantly decreased at different concentrations of Polybrene (in a dose-dependent manner) and various amounts of transfection (P < 0.05). The expression of hTERT in basal cells was not significantly different from the group transfected without gRNA (P˃0.05) but was significantly higher than the group transfected with gRNA (P < 0.05). The results of flow cytometry and western blotting analysis showed a decrease in hTERT level compared to cells transfected without gRNA as well as basal cells. The methylation of hTERT gene promoter in the cells transfected with gRNA at a concentration of 80 μg/mL in the presence of both 4 μg/mL and 8 μg/mL of Polybrene was significantly increased compared to those transfected without sRNA (P < 0.05). The flow cytometry results indicated no significant difference in the induction of apoptosis in the transfected cells compared to the basal cells (P < 0.05). Evidence suggests that the designed CRISPR/Cas9 system reduces the expression of the hTERT gene and telomerase presence, thereby inhibiting the growth of melanoma cells.
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Affiliation(s)
- Farbod Taghavi Rad
- Department of Molecular Genetics, Ahar Branch, Islamic Azad University, Ahar, Iran
| | - Bahar Naghavi Gargari
- Department of Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Basic Sciences, School of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
| | - Saied Ghorbian
- Department of Molecular Genetics, Ahar Branch, Islamic Azad University, Ahar, Iran.
| | - Zeinab Shirvani Farsani
- Department of Cell and Molecular Biology, Faculty of Life Science and Technology, Shahid Beheshti University, Tehran, Iran
| | - Rasoul Sharifi
- Department of Biology, Faculty of Basic Sciences, Ahar Branch, Islamic Azad University, Ahar, Iran
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Ricci C, Morandi L, Ambrosi F, Righi A, Gibertoni D, Maletta F, Agostinelli C, Corradini AG, Uccella S, Asioli S, Sessa F, La Rosa S, Papotti MG, Asioli S. Intron 4-5 hTERT DNA Hypermethylation in Merkel Cell Carcinoma: Frequency, Association with Other Clinico-pathological Features and Prognostic Relevance. Endocr Pathol 2021; 32:385-395. [PMID: 33909215 PMCID: PMC8370894 DOI: 10.1007/s12022-021-09669-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 02/15/2021] [Indexed: 12/23/2022]
Abstract
Merkel cell carcinoma (MCC) is an aggressive skin tumor with neuroendocrine differentiation, mainly affecting elderly population or immunocompromised individuals. As methylation of the human telomerase reverse transcriptase (mhTERT) has been shown to be a prognostic factor in different tumors, we investigated its role in MCC, in particular in intron 4-5 where rs10069690 has been mapped and recognized as a cancer susceptibility locus. DNA methylation analysis of hTERT gene was assessed retrospectively in a cohort of 69 MCC patients from the University of Bologna, University of Turin and University of Insubria. Overall mortality was evaluated with Kaplan-Meier curves and multivariable Royston-Parmar models. High levels of mhTERT (mhTERThigh) (HR = 2.500, p = 0.015) and p63 (HR = 2.659, p = 0.016) were the only two clinico-pathological features significantly associated with a higher overall mortality at the multivariate analysis. We did not find different levels of mhTERT between MCPyV (+) and (-) cases (21 vs 14, p = 0.554); furthermore, mhTERThigh was strongly associated with older age (80.5 vs 72 years, p = 0.026), no angioinvasion (40.7% vs 71.0%, p = 0.015), lower Ki67 (50 vs 70%, p = 0.005), and PD-L1 expressions in both tumor (0 vs 3%, p = 0.021) and immune cells (0 vs 10%, p = 0.002). mhTERT is a frequently involved epigenetic mechanism and a relevant prognostic factor in MCC. In addition, it belongs to the shared oncogenic pathways of MCC (MCPyV and UV-radiations) and it could be crucial, together with other epigenetic and genetic mechanisms as gene amplification, in determining the final levels of hTERT mRNA and telomerase activity in these patients.
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Affiliation(s)
- Costantino Ricci
- Pathology Unit, Maggiore Hospital, AUSL Bologna, Bologna, Italy
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy
| | - Luca Morandi
- Department of Biomedical and Neuromotor Sciences, Functional MR Unit, IRCCS Istituto delle Scienze Neurologiche, Bologna, Italy
| | | | - Alberto Righi
- Department of Pathology, IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy
| | - Dino Gibertoni
- Department of Biomedical and Neuromotor Sciences, Unit of Hygiene and Biostatistics, University of Bologna, Bologna, Italy
| | - Francesca Maletta
- Department of Oncology, University of Turin, Città Della Salute Hospital, Turin, Italy
| | - Claudio Agostinelli
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy
- Haematopathology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
| | - Angelo Gianluca Corradini
- Department of Biomedical and Neuromotor Sciences, Functional MR Unit, IRCCS Istituto delle Scienze Neurologiche, Bologna, Italy
| | - Silvia Uccella
- Unit of Pathology, Department of Medicine and Surgery, University of Insubria, Varese, Italy
| | - Silvia Asioli
- Unit of Pathology, Morgagni-Pierantoni Hospital, Forlì, 47121, Italy
| | - Fausto Sessa
- Unit of Pathology, Department of Medicine and Surgery, University of Insubria, Varese, Italy
| | - Stefano La Rosa
- Institute of Pathology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Mauro Giulio Papotti
- Department of Oncology, University of Turin, Città Della Salute Hospital, Turin, Italy
| | - Sofia Asioli
- Department of Biomedical and Neuromotor Sciences (DIBINEM) Surgical Pathology Section- Alma Mater Studiorum , University of Bologna , Bologna, Italy.
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Romanowska K, Sobecka A, Rawłuszko-Wieczorek AA, Suchorska WM, Golusiński W. Head and Neck Squamous Cell Carcinoma: Epigenetic Landscape. Diagnostics (Basel) 2020; 11:diagnostics11010034. [PMID: 33375464 PMCID: PMC7823717 DOI: 10.3390/diagnostics11010034] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Revised: 12/21/2020] [Accepted: 12/24/2020] [Indexed: 02/06/2023] Open
Abstract
Head and neck squamous carcinoma (HNSCC) constitutes the sixth most prevalent cancer worldwide. The molecular pathogenesis of HNSCC includes disorders in cell cycle, intercellular signaling, proliferation, squamous cell differentiation and apoptosis. In addition to the genetic mutations, changes in HNSCC are also characterized by the accumulation of epigenetic alterations such as DNA methylation, histone modifications, non-coding RNA activity and RNA methylation. In fact, some of them may promote cancer formation and progression by controlling the gene expression machinery, hence, they could be used as biomarkers in the clinical surveillance of HNSCC or as targets for therapeutic strategies. In this review, we focus on the current knowledge regarding epigenetic modifications observed in HNSCC and its predictive value for cancer development.
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Affiliation(s)
- Kamila Romanowska
- Department of Head and Neck Surgery, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, 61-866 Poznan, Poland; (A.S.); (W.G.)
- Department of Medical Physics, Radiobiology Laboratory, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, 61-866 Poznan, Poland;
- Correspondence:
| | - Agnieszka Sobecka
- Department of Head and Neck Surgery, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, 61-866 Poznan, Poland; (A.S.); (W.G.)
- Department of Medical Physics, Radiobiology Laboratory, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, 61-866 Poznan, Poland;
| | | | - Wiktoria M. Suchorska
- Department of Medical Physics, Radiobiology Laboratory, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, 61-866 Poznan, Poland;
| | - Wojciech Golusiński
- Department of Head and Neck Surgery, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, 61-866 Poznan, Poland; (A.S.); (W.G.)
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Li S, Huang W, Li Y, Chen B, Li D. A Study of hTERT Promoter Methylation in Circulating Tumour DNAs of Patients with Ovarian Magnificent Tumour. Onco Targets Ther 2020; 13:12317-12323. [PMID: 33293825 PMCID: PMC7719343 DOI: 10.2147/ott.s274743] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2020] [Accepted: 10/24/2020] [Indexed: 02/01/2023] Open
Abstract
Objective Human telomerase reverse transcriptase (hTERT), a crucial enzyme for telomere maintenance, has been associated with the development of ovarian cancer (OC). The purpose of this study was to investigate the difference of methylation rates of hTERT promoter in tumour tissues and plasma samples of patients with ovarian magnificent tumour and those with ovarian benign tumour, as well as in plasma samples of healthy women. This study further aimed to establish a possible association between increased methylation rate of hTERT promoter and circulating tumour DNAs (ctDNA) amongst patients with ovarian magnificent tumour. Methods Tumour tissue samples and plasma samples were separately obtained from 17 patients with ovarian magnificent tumour (experiment group, group A) and from 15 patients with ovarian benign tumour (control group, group B). Another 15 plasma samples were acquired from healthy women (control group, group C). Promoter methylation was assessed by methylation-specific PCR (MSP). Statistical analysis was conducted using SPSS 22.0. Results Methylation of hTERT was observed in 76.5% of tumour tissue samples and in 70.6% of plasma samples from patients with ovarian magnificent tumour. It was also observed in 26.7% of tumour tissue samples and 20% of plasma samples from patients with ovarian benign tumour, and in 13.3% of plasma samples from healthy women. Comparing between plasmas and tissues, the respective rates of consistency, sensitivity and specificity were 70.59%, 76.9% and 50% in group A, and 80%, 50% and 90.9% in group B. Hence, the associations of hTERT methylation with ctDNAs (p=0.001) and tumour tissue samples (p=0.012) amongst patients with ovarian magnificent tumour were established. Conclusion An increased methylation of hTERT promoter is related to ctDNAs and tumour tissues of patients with ovarian magnificent tumour.
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Affiliation(s)
- Songyi Li
- Department of Gynecology, Hangzhou Women's Hospital, Hangzhou 310008, People's Republic of China
| | - Wei Huang
- Department of Gynecology, Hangzhou Women's Hospital, Hangzhou 310008, People's Republic of China
| | - Yinghua Li
- Department of Gynecology, Hangzhou Women's Hospital, Hangzhou 310008, People's Republic of China
| | - Beibei Chen
- Department of Gynecology, Hangzhou Women's Hospital, Hangzhou 310008, People's Republic of China
| | - Dingheng Li
- Department of Gynecology, Hangzhou Women's Hospital, Hangzhou 310008, People's Republic of China
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Zheng J, Mei Y, Zhai G, Zhao N, Jia D, Fan Y. Downregulation of RUNX3 has a poor prognosis and promotes tumor progress in kidney cancer. Urol Oncol 2020; 38:740.e11-740.e20. [PMID: 32600926 DOI: 10.1016/j.urolonc.2020.05.021] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2020] [Revised: 05/07/2020] [Accepted: 05/19/2020] [Indexed: 10/24/2022]
Abstract
BACKGROUND Kidney cancer usually shows no symptoms until the tumor is relatively large, and current drugs fail to stop the tumor recurrence. The transcriptional factor Runt-related transcription factor 3 (RUNX3) has been reported to function as a tumor suppressor in many types of cancers. METHODS Kidney cancer and adjacent normal tissues were collected from 12 patients to test the expression of RUNX3 by real-time quantitative PCR, immunoblotting, and immunohistochemistry. Promoter methylation status of RUNX3 was determined using methylation analysis from 103 patient samples. Kidney cancer cell lines and xenograft mouse model were used to investigate the promoter methylation and cancer progression through inhibitor treatment and loss/gain-of-function experiments. RESULTS RUNX3 was significantly downregulated in kidney cancer tissues and cells, which could be elevated by higher methylation status at its promoter region. RUNX3 promoter methylation was positively correlated with poor prognosis of kidney cancer. RUNX3 loss-of-function promoted the cell proliferation, migration, and invasion of kidney cancer cells, in contrast, RUNX3 overexpression inhibited the cancer cell progression. This study provides the first instance of the effect of RUNX3 expression and its promoter methylation status on kidney cancer. CONCLUSION Targeting RUNX3 pathway and its promoter methylation are potential therapeutic strategies to treat kidney cancer.
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Affiliation(s)
- Jianbo Zheng
- Department of Urology, Qilu Hospital of Shandong University, Jinan, Shandong, China; Department of Urology, Central Hospital of Zibo, Zibo, Shandong, China
| | - Yanhui Mei
- Department of Urology, Binzhou Medical University Hospital, Binzhou, Shandong, China
| | - Guangsheng Zhai
- Department of Radiotherapy, Central Hospital of Zibo, Zibo, Shandong, China
| | - Ning Zhao
- Department of Urology, Central Hospital of Zibo, Zibo, Shandong, China
| | - Dongsheng Jia
- Department of Urology, Central Hospital of Zibo, Zibo, Shandong, China
| | - Yidong Fan
- Department of Urology, Qilu Hospital of Shandong University, Jinan, Shandong, China.
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Xin Y, Dong K, Cao F, Tian Y, Sun J, Peng M, Liu W, Shi P. Studies of hTERT DNA methylation assays on the human age prediction. Int J Legal Med 2019; 133:1333-1339. [PMID: 31165262 DOI: 10.1007/s00414-019-02076-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2018] [Accepted: 05/17/2019] [Indexed: 11/25/2022]
Abstract
As an important aspect of epigenetics, DNA methylation has been proven to be suitable for forensic DNA analysis. By detecting changes in DNA methylation, it is desirable to construct a model of age patterns associated with it to infer the age of the individual. The hTERT gene methylation is closely related to tumors, but there are few reports on the relationship between hTERT gene promoter methylation and age. In this study, we utilized the methylation-specific polymerase chain reaction and real-time PCR (relative quantification and absolute quantification) approach to explore the connection between hTERT DNA methylation and age prediction. We fit three models for age prediction based on methylation assay for 90 blood samples from donors aged 1-79 years old. Among them, the model of absolute quantification of real-time enabled the age prediction with R2 = 0.9634. We verified the linear regression model with a validation set of 30 blood samples where prediction average error was 4.29 years. Generally, this reliable method improves the DNA methylation analysis of forensic samples.
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Affiliation(s)
- Ye Xin
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China
| | - Kaikai Dong
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China
| | - Fangqi Cao
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China
- Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Zhongshan North No 1 Road, Shanghai, 200083, China
| | - Yuxiang Tian
- Department of Clinical Laboratory, Shanghai Xuhui District Dahua Hospital, Shanghai, 200237, China
| | - Jing Sun
- Qinghai Provincial Key Laboratory of Qinghai-Tibet Plateau Biological Resources, Northwest Institute of Plateau Biology, The Chinese Academy of Sciences, Xiguan Avenue 59, Xining, 11 Qinghai Province, 810001, China
| | - Min Peng
- Qinghai Provincial Key Laboratory of Qinghai-Tibet Plateau Biological Resources, Northwest Institute of Plateau Biology, The Chinese Academy of Sciences, Xiguan Avenue 59, Xining, 11 Qinghai Province, 810001, China
| | - Wenbin Liu
- Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Zhongshan North No 1 Road, Shanghai, 200083, China.
| | - Ping Shi
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
- Qinghai Provincial Key Laboratory of Qinghai-Tibet Plateau Biological Resources, Northwest Institute of Plateau Biology, The Chinese Academy of Sciences, Xiguan Avenue 59, Xining, 11 Qinghai Province, 810001, China.
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