Expression,Function And Mechanism Of MicroRNA In Human Sertoli Cells And Germ Cells

Background and objective: Human spermatogenesis comprises three distinctive phases,including the mitosis of spermatogonia,meiosis of spermatocytes,and spermiogenesis of haploid spermatids.Human Sertoli cell,the only somatic cell within the seminiferous tubule,is a vital component of the blood-testis barrier,and it can also generate numerous growth factors required for normal human spermatogenesis.Spermatogenesis is an intricate process that can be regulated by many factors,e.g.,genetic factors,epigenetic factors,and the niche or the microenvironment.In rodents,it has been reported that non-coding small RNA(microRNA)plays essential roles in mediating spermatogenesis and abnormality of microRNA leads male infertility.Nevertheless,it remains unknown about differential microRNA profiles in human germ cells and Sertoli cells between normal men and non-obstructive azoospermia(NOA)patients.In addition,the roles and mechanisms of microRNA in regulating human Sertoli cells need to be clarified.Therefore,the objective of this study was as follows: i)we have for the first time compared the global microRNA profiles in human Sertoli cells between normal men and NOA patients;ii)we explored the function and target of miRNA-133b in regulating the proliferation of human Sertoli cells;and iii)furthermore,we identified the differential microRNA patterns of human spermatogonia,pachytene spermatocytes,and round spermatids between normal men and NOA patients,and we verified the differentially expressed microRNA of human spermatogonia between normal men and NOA patients and predicted the targets of these miRNA.Methods: PART Ⅰ: Differential microRNA profiles in human Sertoli cells between normal men and NOA patients as well as the function and mechanism of miRNA-133b in the regulation of human Sertoli cells.Micro RNA microarray and real-time PCR were used to identify and verify differentially expressed microRNA in human Sertoli cells between normal men and Sertoli-cell-only syndrome(SCOS)patients.MiRNA-133b specific mimics and inhibitors,CCK-8 assays and Western blots were employed to probe its effect on the proliferation of human Sertoli cells.Immunohistochemistry was utilized to compare the proliferation state of the testis of OA patients and SCOS patients.Bioinformatics was employed to predict that GLI3 was a target of miRNA-133b in human Sertoli cells,which was confirmed by real-time PCR and Western blots.Finally,using the RNA interference and CCK-8 assay,we explored the influence of GLI3 on controlling the proliferation of human Sertoli cells.PART Ⅱ: Differential microRNA profiles in human spermatogonia,spermatocytes and spermatids between normal men and NOA patients.Human spermatogonia,pachytene spermatocytes,and round spermatids were isolated from the testes of OA patients and NOA patients using the two-step enzymatic digestion,differential plating and STA-PUT.Total RNA was extracted from human spermatogonia,pachytene spermatocytes,and round spermatids of the testis of OA patients and NOA patients.RNA amplification and RNA deep-sequencing were performed to identify the differentially expressed microRNA in human spermatogonia,pachytene spermatocytes,and round spermatids between normal men and NOA patients.Real-time PCR was used to verify 11 differentially expressed microRNA by RNA deep-sequencing.Using the bioinformatics,we predicted the targeting genes of the these microRNAs,and real-time PCR was used to confirm these targets.Results:PART Ⅰ: Human Sertoli cells were isolated from human testis by a two-step enzymatic digestion and followed by differential plating.The fresh isolated cells were positive for GATA4,WT1,SOX9,BMP4,SCF,and GDNF,suggesting that the isolated cells were human Sertoli cells in phenotype.Microarray analysis revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between SCOS patients and OA patients with normal spermatogenesis.In OA testis,88 microRNA were upregulated,whereas 86 microRNAs were downregulated,and real time PCR verified these data.We further found that microRNA-133b could promote the proliferation of human Sertoli cells using miRNA-133b specific mimics and inhibitors,CCK-8 assays,and Western blots.Immunohistochemistry showed that more human Sertoli cells proliferated in SCOS patients than OA patients.Notably,GLI3 was identified to be a binding target of miRNA-133b in human Sertoli cells,and miRNA-133b stimulated the proliferation of human Sertoli cells by the activation of Cyclin B1 and Cyclin D1.RNA interference and CCK-8 assay showed that GLI3 knockdown promoted the growth of human Sertoli cells.PART Ⅱ: Human spermatogonia,pachytene spermatocytes,and round spermatids with a high purity and viability were isolated from the testis of OA patients and NOA patients using the two-step enzymatic digestion and STA-PUT.RNA deep-sequencing assay revealed that the expression of microRNA in NOA testis is much higher than that in OA patients.In contrary,the expression of piRNA in NOA testis is relatively lower than that in OA testis.In this study,we focused on differentially expressed microRNA in human spermatogonia between OA and NOA patients.In total,76 miRNAs were present in OA spermatogonia but absent in NOA spermatogonia,and 88 miRNAs had higher expression in OA spermatogonia compared to NOA spermatogonia.Inversely,91 miRNAs were expressed in NOA spermatogonia but not in OA spermatogonia,whereas 308 miRNAs showed higher expression in NOA spermatogonia compared with OA spermatogonia.Next,11 miRNAs were randomly chosen to confirm their expression differences in human spermatogonia between OA and NOA patients,the targeting genes of these miRNAs were predicted by bioinformatics and verified by real-time PCR.Conclusions:We have for the first time uncovered a number of miRNAs that are differentially expressed in human Sertoli cells and male germ cells between OA patients and NOA patients.We predict targeting genes of these miRNAs using bioinformatics and verify these targets utilizing real-time PCR.Significantly,we have demonstrated that miRNA-133b promotes the proliferation of human Sertoli cells by targeting GLI3.This study thus sheds novel insights into epigenetic regulation of human spermatogenesis and the etiology of azoospermia,and it offers new targets for the diagnosis and treatment of male infertility.