| Previous studies found that kisspeptin treatment promoted steroid hormone secretion,proliferation and inhibited granulosa cell apoptosis in vitro cultured Tan sheep ovarian granulosa cells,but the regulatory mechanism is unclear.Numerous studies have shown that miRNAs can significantly regulate ovarian granulosa cell function.To this end,in this study,miRNA-mRNA sequencing was performed on kisspeptin-treated primary Tan sheep ovarian granulosa cells to screen for differential miRNAs and candidate target genes regulating granulosa cell function.Based on the results of the previous analysis,the most significant difference let-7b was further validated to regulate the function of Tan sheep ovarian granulosa cells.Secondly,the target gene of let-7b was identified as ITGB7,and the effects of its target gene ITGB7 on steroid hormone secretion,proliferation and apoptosis of Tan sheep ovarian granulosa cells were elucidated.The main results of the experiment were as follows:1.In vitro isolation and culture of Tan sheep ovarian granulosa cells,bioinformatics analysis after transcriptome sequencing of 500 nM kisspeptin-treated granulosa cells,preliminary screening of key miRNAs regulating ovarian granulosa cell function(let-7b,miR-10a)and candidate mRNAs with potential targeting relationship with key miRNAs(EGR1,HNRNPD,ITGB7).2.In vitro cultured granulosa cells were transfected with let-7b mimics and inhibitors.The results showed that let-7b inhibitor highly significantly promoted the secretion of progesterone in granulosa cells(P<0.001),and the secretion of estradiol was consistent with the trend of progesterone but not significantly different(P>0.05).let-7b downregulated CYP19A1,CYP11A1,LHR,ERβ,INHBA and other genes(P<0.05).let-7b inhibited granulosa cell proliferation(P<0.05),while let-7b significantly inhibited the rate of granulosa cells in S phase(P<0.05);in addition,let-7b highly significantly downregulated the expression of proliferation-related genes CDK4,CDK2,and PCNA(P<0.001).In addition,let-7b highly significantly promoted ovarian granulosa cell apoptosis(P<0.001).Meanwhile,the expression of apoptosis-related gene Bax was up-regulated(P<0.001).3.ITGB7 was found to be a potential target gene of let-7b by miRNA target gene prediction;the analysis of the results of dual luciferase reporter system assay further demonstrated that let-7b targeted ITGB7.In addition,the expression of ITGB7 was further confirmed to be negatively regulated by let-7b in primary Tan sheep ovarian granulosa cells.4.In vitro cultured granulosa cells were transfected with pcDNA3.1-ITGB7 overexpression vector and siRNA-ITGB7 interference fragment,and the results showed that ITGB7 promoted the secretion of estrogen and progesterone in beach sheep ovarian granulosa cells(P<0.05),and ITGB7 up-regulated the expression of STAR,CYP19A1,CYP11A1,3β-HSD,FSHR,ERβ,and PGR expression(P<0.05).ITGB7 promoted granulosa cell proliferation(P<0.01),ITGB7 upregulated CDK4,CDK2,and PCNA expression in granulosa cells(P<0.05),while ITGB7 increased the percentage of granulosa cells in S-phase cells(P<0.05)and accelerated cell cycle progression;in addition,ITGB7 inhibited granulosa cell apoptosis(P<0.001),upregulated bcl-2 expression,and downregulated Bax,p53,and Caspase-3 expression(P<0.05).In conclusion,key miRNAs(miR-10a,let-7b)and key mRNAs(EGR1,HNRNPD,ITGB7)regulating ovarian granulosa cell function after kisspeptin treatment were screened.let-7b inhibited steroid hormone secretion,proliferative cycle and promoted granulosa cell apoptosis in Tan sheep ovarian granulosa cells through ITGB7 binding.In summary,the key miRNAs(miR-10a,let-7b)and key mRNAs(EGR1,HNRNPD,ITGB7)that regulate ovarian granulosa cell function after kisspeptin treatment were screened,revealing that let-7b inhibits steroid hormone secretion,proliferative cycle and promotes granulosa cell through ITGB7 binding in Tan sheep ovarian granulosa cells The mechanism of action was revealed. |
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