Mir-183-5p Regulates The Self-renewal Process Of Spermatogonial Stem Cells By Targeting ZEB2 To Regulate FGF5 Ubiquitination

Objective(s):Infertility in present-day men is a common reproductive disorder,but we lack a comprehensive understanding of its etiology,as well as proper treatments.Spermatogonial stem cells(SSCs)with differentiation and genetic capabilities are considered a promising approach to treat male infertility.Therefore,the regulatory mechanisms of SSCs were deeply investigated at the molecular level in this study.Methods: The expression of mir-183-5p in SSCs was detected by QRT PCR.SSCs with mir-183-5p overexpression and inhibition were applied to identify the effects of mir-183-5p on cell growth and differentiation of SSCs.Cell viability was detected by cell counting kit-8(cell counting kit-8,CCK-8)assay.Thymidine analog(5-ethynyl-2 deoxyuridine,EDU)staining and PCNA Western blot(WB)were used for cell proliferation assays.The level of apoptosis was detected by flow cytometry.The targeted relationship between mir-183-5p and E box zinc finger binding protein 2(zinc finger E-box binding homeobox 2,ZEB2)was predicted by targetscan and then validated by dual luciferase reporter assay.Regulation of fibroblast growth factor 5(FGF5)ubiquitination and degradation by ZEB2 was also detected.Results: Upregulation of mir-183-5p in SSCs enhances cell viability and proliferation while suppressing apoptosis,which upregulates SSC promoting factors including gfra1,Etv5 and PLZF.Through bioinformatics data and subsequent validation,mir-183-5p was demonstrated to directly target ZEB2 and exert the above effects by downregulating the expression of ZEB2 in SSCs.In addition,this study confirmed that ZEB2 negatively regulates FGF5 by regulating ubiquitination and degradation,which in turn affects the phosphoinositide 3-kinase / protein kinase B(PI3K / Akt)signaling pathway in SSCs.Conclusion(s): Mir-183-5p inhibits the expression of ZEB2 and promotes the expression of FGF5,thereby triggering SSCs activity and promoting cell growth and differentiation.Therefore,the mir-183-5p / ZEB2 / FGF5 axis may be a potential target for SSC treatment of male infertility.