MiR-101-5p Regulates The Biological Characteristics Of Goat Spermatogonial Stem Cells Via Targeting EZH2

Spermatogonial stem cells(SSCs)are regulated by epigenetic modification.Micro RNA(miRNA)and histone methylation exert a critical role of gene regulation.miR-101 regulated cellular proliferation,apoptosis via targeted genes.Enhancer of zeste homolog 2(EZH2)catalyzed methylation of lysine-27 of histone H3(H3K27)and regulated the differentiation and aging of mouse spermatogenic cells and embryonic stem cells.However,miR-101-5p and EZH2 expression in goat testis and its effect on goat SSCs have rarely been studied.Therefore,the experiment detected the expression of EZH2 and miR-101-5p in the testes of postpubertal goats,and explored its role in goat SSCs,which revealed miR-101-5p regulated the biological characteristics of goat SSCs via targeting EZH2.The results are as follows:1.miR-101-5p expression and its effect on cell proliferation and apoptosis of goat SSCsThe study was to detect the expression of miR-101-5p in goat testes and its effect on proliferation and apoptosis in goat SSCs.The results showed that miR-101-5p expression increased in the testes of post pubertal goats(P < 0.01).Compared with negative control(NC),miR-101-5p decreased cell viability(P < 0.01),the expression of anti-apoptotic factor BCL-2(P < 0.01),G2/M phase(P < 0.05),pluripotency genes such as NANOG(P <0.01),CDX2(P < 0.01),and spermatogenesis-related genes PLZF(P < 0.05),GDNF(P <0.01),and DAZL(P < 0.01),but increased the expression level of pro-apoptotic genes CASP3(P < 0.01),and CASP9(P < 0.01).After miR-101-5p inhibitor treatment,it had reverse results with the miR-101-5p mimics compared to the NC inhibitor.Above results indicates that miR-101-5p stimulated apoptosis,inhibited the proliferation,blocked the cycle and comprised the pluripotency of goat SSCs.2.miR-101-5p regulated proliferation and apoptosis of goat SSCs via EZH2The experiment verified target of miR-101-5p,investigated the expression pattern of EZH2 and its role of self-renewal in goat SSCs.The results of the dual luciferase reporter assay showed that the luciferase activity of co-transfection with EZH2-WT and miR-101-5p mimics was significantly reduced in goat SSCs compared with the co-transfection with EZH2-WT and NC mimics(P < 0.01),and there were no significant differences in other groups(P > 0.05).Compared with NC,miR-101-5p mimics significantly decreased the protein and m RNA levels of EZH2(P < 0.01),while the level of EZH2 had no differences(P > 0.05)after miR-101-5p inhibitor transfection in goat SSCs.EZH2 protein(P < 0.05)and m RNA(P < 0.01)expression were significantly higher(P < 0.01)in the testes of postpubertal goats.Moreover,the expression of EZH2 in goat SSCs was higher(P < 0.01)than in Leydig cells and Sertoli cells,as determined by q PCR,western blot,and immunofluorescence.Compared to the NC,cell proliferation and viability were decreased in SSCs which EZH2 was knocked down(P < 0.01),and the G2/M phase of the cell cycle was blocked(P < 0.01),by using Edu staining,CCK-8 assay,and flow cytometry analysis.Additionally,the expression of pro-apoptotic factors CASP3,BAX,and CASP9 was significantly increased(P < 0.01),while BCL-2 expression was decreased in EZH2 knockdown SSCs(P < 0.01).Notably,the expression of GDNF,a SSCs marker gene,and DAZL,were significantly decreased(P < 0.01)while GFRA1 expression was significantly up-regulated in EZH2 knockdown SSCs(P < 0.01).Our data suggested that miR-101-5p might impair self-renewal of goat SSCs,and spermatogenesis in goats via targeting EZH2.3.Molecular mechanism of EZH2 on regulating the pluripotency in goat SSCsThe experiment explored the effects of EZH2 on the pluripotency of goat SSCs,and analyzed the changes of pluripotency genes,imprinted gene and its DNA methylation status of goat SSCs by BSP,Western blot and q PCR.The results indicated that the protein and m RNA level of EZH2 significantly decreased(P < 0.01)after EZH2 knockdown goat SSCs.Compared with NC,the m RNA expression of pluripotency genes OCT4,NANOG was reduced in EZH2 knockdown goat SSCs(P < 0.01),and the fourth dot of NANOG gene in methylation levels decreased.In addition,EZH2 knockdown increased the expression levels of Dnmt3a(P < 0.01)and Dnmt3b(P < 0.01),but decreased Tet1,Tet2,and Tet3expression(P < 0.01).Expression of the imprint gene H19 decreased significantly(P <0.01),but methylation levels of H19(P < 0.05)and IGF2(P < 0.01)significantly increased.The results suggested that EZH2 influenced the pluripotency of goat SSCs independent of DNA methylation,but affected the gene imprinting through DNA methylation.