The Mechanism Of MiR-495-3p Regulating The Function Of Goat Ovarian Granulosa Cells

The reproductive performance of goats affects the development of the goat industry.Dazu black goats are characterized by early sexual maturity and high fecundity,and are an excellent germplasm resource to improve the economic benefit of goat husbandry.However,the regulation mechanism behind its high fecundity is still unclear.Granulosa cells in the ovary play a very important role in regulating follicle development and sex hormone synthesis and thus affect the reproductive performance of goats.miRNAs have a wide range of gene regulatory functions,and they play an important role in follicular growth and development,oocyte maturation,ovulation and luteinsation.Although miR-495-3p determines cell fate by targeting the regulation of gene expression,its function in goat ovarian granulosa cells is unclear.Therefore,elucidating the mechanism of miR-495-3p in regulating the function of goat ovarian granulosa cells will provide a theoretical basis for improving the reproductive performance of goats.In this study,Dazu black goat ovarian granulosa cells were used as the research object.Through immunohistochemistry,miRNA overexpression or interference,RT-PCR,Western blot,flow cytometry,transcriptome sequencing,and dual luciferase reporting,the influence mechanism of miR-495-3p on the apoptosis,proliferation and steroid secretion of goat ovarian granulosa cells was explored,and the following results were obtained:1.miR-495-3p promoted the apoptosis of ovarian granulosa cells and inhibited their proliferationIn this study,mimics and inhibitor were transfected into ovarian granulosa cells to construct the overexpression and inhibition model of miR-495-3p.The results showed that miR-495-3p overexpression significantly promoted the apoptosis of granulosa cells,whilst miR-495-3p inhibition inhibited the apoptosis of granulosa cells(p < 0.05).Further detection showed that miR-495-3p overexpression significantly increased the mRNA and protein expression of pro-apoptotic gene BAX(p < 0.01),and inhibited the mRNA and protein expression of anti-apoptotic gene BCL2(p < 0.01).The inhibition of miR-495-3p decreased the mRNA and protein expression of BAX(p < 0.01),but had no significant effect on the expression of BCL2.The results showed that miR-495-3p promoted the apoptosis of ovarian granulosa cells.CCK-8 was used to detect cell proliferation activity at 24,36 and 48 h.The results showed that miR-495-3p inhibited cell proliferation in a time-dependent manner.Flow cytometry showed that miR-495-3p overexpression blocked the cell cycle in the G2/M phase(p < 0.05).The overexpression and inhibition of miR-495-3p had no significant effect on the mRNA level of PCNA,but miR-495-3p overexpression significantly inhibited the protein level of PCNA(p < 0.05).Further detection showed that miR-495-3p overexpression significantly inhibited the mRNA levels of CDK4 and CCND1(p < 0.05),but had no significant effect on CCNE1.The inhibition of miR-495-3p significantly decreased the mRNA level of CCNE1(p < 0.01),but had no significant effect on CDK4 and CCND1.The results showed that miR-495-3p inhibited the proliferation of granulosa cells.2.miR-495-3p regulates steroid secretion in ovarian granulosa cellsThe overexpression or inhibition of miR-495-3p promoted the secretion of estradiol(E2)and progesterone(P4).Further analysis showed that miR-495-3p overexpression significantly promoted the mRNA and protein expression of 3β-HSD,CYP11A1 and CYP19A1,although it had no significant effect on the expression of St AR(p < 0.05).Interfering with miR-495-3p inhibited the expression of 3β-HSD(p < 0.01)and promoted the expression of CYP11A1(p < 0.001).The results suggested that miR-495-3p may regulate the secretion of E2 and P4 by promoting the expression of key genes in the steroid hormone synthesis pathway.3.Verification of the targeting relationship of miR-495-3pmiR-495-3p was overexpressed in granulosa cells,and the candidate target genes of miR-495-3p were screened by transcriptome sequencing technology.GO and KEGG enrichment analysis showed that the differentially expressed mRNAs were mainly concentrated in the pathways related to cell proliferation,differentiation,apoptosis and inflammation.Through differential expression analysis of the mimics group and mimics NC group,12 differentially expressed genes were screened,eight of which were down-regulated and four were up regulated.The eight down regulated genes were verified by RT-PCR.Seven genes were significantly down-regulated except for HBEGF(p < 0.05).Go and KEGG enrichment analysis was performed to obtain signaling pathways related to cell proliferation and apoptosis.For example,regulation of biological process,cellular process,catalytic activity and transporter activity,molecular transformation,cell part,cell,etc.Analysis of the binding sites between the 3’UTR region of the above seven differentially expressed genes(HMOX1、STC1、PLAU、STRA6、F2RL1、GDPD5、C4BPA)and miR-495-3p revealed that there were binding sites between F2RL1,HOMX1 and STC1 and miR-495-3p.Further verification by dual luciferase reports showed that miR-495-3p had a targeting relationship with STC1.In conclusion,this study demonstrated that(1)miR-495-3p promoted the apoptosis and inhibited the proliferation of goat ovarian granulosa cells,(2)may promote the secretion of E2 and P4 by regulating the expression of genes related to steroid hormone synthesis and(3)targeted the expression of STC1.These results provide theoretical support for further revealing the mechanism of miR-495-3p’s targeted regulation of goat follicular development and atresia.