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Najafipour, R., Momeni, A., Yousefipour, F., Mousavi, S., & Moghbelinejad, S. (2021). Underexpression of hsa-miR-449 family and their promoter hypermethylation in infertile men: A case-control study. International Journal of Reproductive BioMedicine (IJRM), 19(1), 23–34. https://doi.org/10.18502/ijrm.v19i1.8177
https://doi.org/10.18502/ijrm.v19i1.8177
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Reza Najafipour
Reza Najafipour
Research Institute for Prevention of Non-Communicable Diseases, Cellular and Molecular Research Centre, Qazvin University of Medical Sciences, Qazvin, Iran
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Abdolmabood Momeni
Abdolmabood Momeni
Biology Department, School of Basic Science, Arak University, Arak, Iran
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Farideh Yousefipour
Farideh Yousefipour
National Institute of Engineering and Biotechnology, Tehran, Iran
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Shaghayegh Mousavi
Shaghayegh Mousavi
Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
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Sahar Moghbelinejad
Sahar Moghbelinejad
Research Institute for Prevention of Non-Communicable Diseases, Cellular and Molecular Research Centre, Qazvin University of Medical Sciences, Qazvin, Iran
Published Date
- Jan 25, 2021
Underexpression of hsa-miR-449 family and their promoter hypermethylation in infertile men: A case-control study
Abstract
Background: Post-transcriptional microRNAs (miRNAs) have an important pattern in the spermatogenesis process.
Objective: Study of the expression and methylation of hsa-miR-449 family in sperm samples of infertile men.
Materials and Methods: In this case-control study, we recruited 74 infertile men (with asthenozoospermia, teratozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia) and 30 control samples. Methylation-specific PCR (MSP) method was used for methylation evaluation of hsa-miR-449 a, b, c promoter. By Real time PCR (qRT-PCR) method,we showed downregulation of hsa-miR-449 a, b, c in the sperm samples of infertile men and compared it to their fertile counterparts.
Results: There was significant underexperssion, in hsa-miR-449-b in oligoasthenoteratospermic samples (p = 0.0001, F = 2.9). About the methylation pattern, infertile men showed high frequency of methylation in the promoter of hsa-miR-449 a, b, c in comparison to controls (60.8% vs 23.3%), the highest amount of methylation was observed in oligoasthenoteratospermia samples (81.2%).
Conclusion: In this study, low expression and high methylation of hsa-miR-449-b were observed in infertile men in compared to control samples, which can be one of the causes of defective spermatogenesis.
Key words: Spermatogenesis, miR-449, Expression, Epigenetic.
References
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[15] Pomper N, Liu Y, Hoye ML, Dougherty JD. CNS MicroRNA profiles: a database for cell type enriched microRNA expression across the mouse central nervous system. Sci Rep 2020; 20: 4914–4921.
[16] Zhang Zh, Zhuang L, Lin ChH. Roles of microRNAs in establishing and modulating stem cell potential. Int J Mol Sci 2019; 20: 3643–3673.
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[18] Yang X, Feng M, Jiang X, Wu Zh, Li Zh, Aau M, et al. miR- 449a and miR-449b are direct transcriptional targets of E2F1 and negatively regulate pRb-E2F1 activity through a feedback loop by targeting CDK6 and CDC25A. Genes Dev 2009; 23: 2388–2393.
[19] Czech B, Hannon GJ. Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 2011; 12: 19–31.
[20] Boeva V. Analysis of genomic sequence motifs for deciphering transcription factor binding and transcriptional regulation in eukaryotic cells. Front Genet 2016; 7: 1–16.
[21] Bao J, Li D, Wang L, Wu J, Hu Y, Wang Zh, et al. MicroRNA- 449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway. J Biol Chem 2012; 287: 21686–21698.
[22] Wu Q, Song R, Ortogero N, Zheng H, Evanoff R, Small CL, et al. The RNase III enzyme DROSHA is essential for MicroRNA production and spermatogenesis. J Biol Chem 2012; 287: 25173–25190.
[23] Li Q, LI H, Zhao X, Wang B, Zhang L, Zhang C, et al. DNA methylation mediated downregulation of miR-449c controls osteosarcoma cell cycle progression by directly targeting oncogene c-Myc. Int J Biol Sci 2017; 13: 1038–1050.
[24] Zhang Q, Yang Zh, Shan J, Liu L, Liu Ch, Shen J, et al. MicroRNA-449a maintains self-renewal in liver cancer stem-like cells by targeting Tcf3. Oncotarget 2017; 8: 110187–110200.
[25] Wu J, Bao J, Kim M, Yuan Sh, Tang Ch, Zheng H, et al. Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis. Proc Natl Acad Sci USA 2014; 111: E2851–E2857.
[26] World Health Organization Do RHaR. WHO Laboratory Manual for the Examination and Processing of Human Semen. Geneva: WHO Press; 2013.
[27] Momeni AM, Najafiour R, Hamta A, Jahani S, Moghbelinejad S. Experssion and methylation pattern of has_miR_34 family in sperm samples of infertile men. Reprod Sci 2020; 27: 301–308.
[28] Liu WM, Pang RT, Chiu PC, Wong BP, Lao K, Lee KF, et al. Sperm-borne microRNA-34c is required for the first cleavage division in mouse. Proc Natl Acad Sci 2012; 109: 490–494.
[29] Comazzetto S, Di Giacomo M, Rasmussen KD, Much Ch, Azzi Ch, Perlas E, et al. Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci. PLoS Genet 2014; 10: e1004597: 1–11.
[30] Nissan T, Parker R. Computational analysis of miRNAmediated repression of translation: Implications for models of translation initiation inhibition. RNA 2008; 14: 1480–1491.
[31] Noonan EJ, Place RF, Pookot D, Basak S, Whitson JM, Hirata H, et al. miR-449a targets HDAC-1 and induces growth arrest in prostate cancer. Oncogene 2009; 28: 1714–1724.
[2] Freitas e Silva KS. Molecular genetics of male infertility: A mini-review. Trends in Res 2018; 1: 1–3.
[3] Kim SY, Kim HJ, Lee BY, Park SY, Lee HS, Seo JT. Y Chromosome microdeletions in infertile men with nonobstructive azoospermia and severe oligozoospermia. J Reprod Infert 2017; 18: 307–315.
[4] O’Brien J, Hayder H, Zayed Y, Peng Ch. Overview of microRNA biogenesis, mechanisms of actions, and circulation. Frontiers in Endocrinology 2018; 9: 402–413.
[5] Plotnikova O, Baranova A, Skoblov M. Comprehensive analysis of human microRNA–mRNA interactome. Frontiers in Genetics 2019; 10: 933–401.
[6] Si W, Shen J, Zheng H, Fan W. The role and mechanisms of action of microRNAs in cancer drug resistance. Clinical Epigenetics 2019; 11: 25-48.
[7] Gebert LFR, MacRae IJ. Regulation of microRNA function in animals. Nature Reviews Molecular Cell Biology 2019; 20: 21–37.
[8] Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RHA, Cuppen E. Phylogenetic shadowing and computational identification of human microRNA genes. Cell 2005; 120: 21–24.
[9] Pasquinelli AE, Hunter Sh, Bracht J. MicroRNAs: a developing story. Curr Opin Genet Dev 2005; 15: 200– 205.
[10] Kotaja N. Micro RNA and spermatogenesis. Fertility and Sterility 2014; 101: 1552–1562.
[11] Harton GL, Tempest HG. Chromosomal disorders and male infertility. Asian J Androl 2012; 14: 32–39.
[12] Urdinguio RG, Bayon GF, Dmitrijeva M, Torano EG, Bravo C, Fraga MF, et al. Aberrant DNA methylation patterns of spermatozoa in men with nexplained infertility. Hum Reprod 2015; 30: 1014–1028.
[13] Tuttelmann F, Simoni M, Kliesch S, Ledig S, Dworniczak B, Wieacker P, et al. Copy number variants in patients with severe oligozoospermia and Sertoli-cell-only syndrome. PLoS One 2011; 6: e19426.
[14] Gunes S, Asci R, Okten G, Atac F, Onar OE, Ogur G, et al. Two males with SRY-positive 46, XX testicular disorder of sex development. Syst Biol Reprod Med 2013; 59: 42–47.
[15] Pomper N, Liu Y, Hoye ML, Dougherty JD. CNS MicroRNA profiles: a database for cell type enriched microRNA expression across the mouse central nervous system. Sci Rep 2020; 20: 4914–4921.
[16] Zhang Zh, Zhuang L, Lin ChH. Roles of microRNAs in establishing and modulating stem cell potential. Int J Mol Sci 2019; 20: 3643–3673.
[17] Xie K, Liu J, Chen J, Dong J, Ma H, Liu Y, et al. Methylationassociated silencing of microRNA-34b in hepatocellular carcinoma cancer. Gene 2014; 543: 101–107.
[18] Yang X, Feng M, Jiang X, Wu Zh, Li Zh, Aau M, et al. miR- 449a and miR-449b are direct transcriptional targets of E2F1 and negatively regulate pRb-E2F1 activity through a feedback loop by targeting CDK6 and CDC25A. Genes Dev 2009; 23: 2388–2393.
[19] Czech B, Hannon GJ. Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 2011; 12: 19–31.
[20] Boeva V. Analysis of genomic sequence motifs for deciphering transcription factor binding and transcriptional regulation in eukaryotic cells. Front Genet 2016; 7: 1–16.
[21] Bao J, Li D, Wang L, Wu J, Hu Y, Wang Zh, et al. MicroRNA- 449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway. J Biol Chem 2012; 287: 21686–21698.
[22] Wu Q, Song R, Ortogero N, Zheng H, Evanoff R, Small CL, et al. The RNase III enzyme DROSHA is essential for MicroRNA production and spermatogenesis. J Biol Chem 2012; 287: 25173–25190.
[23] Li Q, LI H, Zhao X, Wang B, Zhang L, Zhang C, et al. DNA methylation mediated downregulation of miR-449c controls osteosarcoma cell cycle progression by directly targeting oncogene c-Myc. Int J Biol Sci 2017; 13: 1038–1050.
[24] Zhang Q, Yang Zh, Shan J, Liu L, Liu Ch, Shen J, et al. MicroRNA-449a maintains self-renewal in liver cancer stem-like cells by targeting Tcf3. Oncotarget 2017; 8: 110187–110200.
[25] Wu J, Bao J, Kim M, Yuan Sh, Tang Ch, Zheng H, et al. Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis. Proc Natl Acad Sci USA 2014; 111: E2851–E2857.
[26] World Health Organization Do RHaR. WHO Laboratory Manual for the Examination and Processing of Human Semen. Geneva: WHO Press; 2013.
[27] Momeni AM, Najafiour R, Hamta A, Jahani S, Moghbelinejad S. Experssion and methylation pattern of has_miR_34 family in sperm samples of infertile men. Reprod Sci 2020; 27: 301–308.
[28] Liu WM, Pang RT, Chiu PC, Wong BP, Lao K, Lee KF, et al. Sperm-borne microRNA-34c is required for the first cleavage division in mouse. Proc Natl Acad Sci 2012; 109: 490–494.
[29] Comazzetto S, Di Giacomo M, Rasmussen KD, Much Ch, Azzi Ch, Perlas E, et al. Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci. PLoS Genet 2014; 10: e1004597: 1–11.
[30] Nissan T, Parker R. Computational analysis of miRNAmediated repression of translation: Implications for models of translation initiation inhibition. RNA 2008; 14: 1480–1491.
[31] Noonan EJ, Place RF, Pookot D, Basak S, Whitson JM, Hirata H, et al. miR-449a targets HDAC-1 and induces growth arrest in prostate cancer. Oncogene 2009; 28: 1714–1724.