The Molecular Regulatory Mechanism Of Let-7 MiRNA Family During Porcine Follicle Atresia

In female mammal,ovary is the major reproductive organ;however,most of the follicles(-99%)undergo atresia.Although,the development of follicles is an ongoing process,atresia affects all stages of follicular growth and development by preventing follicles from ovulation to control reproductive activities.While,the apoptosis of granulosa cells is defined as a hallmark during follicle atresia based on numerous studies.Regulation by programmed death of granulosa cells has impacts on follicular growth and atresia in mammalian ovaries,furthermore,affects mammalian breeding and reproduction.MircoRNAs(miRNAs)are a class of small endogenous noncoding-RNAs,containing 18-23 nt in length,which have emerged as pivotal post-transcriptional regulators resulting in gene silencing.MiRNAs participate in widespread regulation of biological and cellular processes,including cell development,proliferation,and apoptosis.However,more investigation on the molecular regulatory mechanism of granulosa cell apoptosis mediated by miRNA is still required.To elucidate the regulatory mechanisms of follicle atresia mediated by granulosa cells apoptosis,we discriminated healthy(H),early atretic(EA)and progressively atretic(PA)porcine ovary follicles by morphology and the ratio of Progesterone(P4)/17b-estradiol(E2).Furthermore,stem-loop RT-PCR followed by SYBR-Green I-based real-time quantitative PCR was used to confirm the expression patterns of let-7 miRNA family identified by miRNA microarray.Next,we used cell culture,flow cytometry,real-time quantitative PCR,immunofluorescence and westernblot to analyze granulosa cells transfected with let-7g mimic and the expression of apoptosis-associated genes.Our results showed that:1)To investigate whether microarray reflected the correct changes in response to atresia,we used stem-loop real-time PCR to detect the relative expression of let-7 miRNA family during follicle atresia.MiRNA let-7a,let-7b,let-7c and let-7i were exhibiting a decrease in abundance of EA and PA compared to H;however,let-7g was significantly increased in EA and PA relative to its expression in H.It suggested that the expression pattern of let-7 family matched with our miRNA microarray assay and let-7 miRNA family is essential for the development of reproductive system.2)Flow cytometry analysis confirmed that the cell apoptosis rate increased significantly in granulosa cells transfected with let-7g mimic.It indicated that let-7g may promote apoptosis in porcine granulosa cells.To further confirm microRNA let-7g would have effects on granulosa cell apoptosis in vitro,real-time quantitative PCR was used to detest the expression of apoptosis-associated genes,such as FasL,Bax,Bcl-2 Mcl-1 and Caspase-3.It could be seen that increased levels of the pro-apoptotic gene Bax and Caspase-3,meanwhile,decreased levels of the anti-apoptotic gene Bcl-2 and Mcl-1.Meanwhile,dephosphorylated FoxO1 translocated to the nucleus of granulosa cells transfected with let-7g mimic responsing for its transcriptional regulatory functions.3)We demonstrated that MAP3K1 is the direct target gene binding by let-7g.siRNA knockdown of MAP3K1 increased the apoptosis rate of porcine granulosa cells,meanwhile,FoxO1 was found to translocate to the nucleus.It suggested that let-7g inhibits the post-transcrpition of MAP3K1,attenuated MAP3K1 supresses FoxO1 export from the nucleus,thereby promoting its capacity to upregulate proapoptotic genes.In this study,we profiled the expression pattern of let-7 miRNA family during porcine ovary follicle atresia and indicated that let-7 miRNA family participated in the regulation of follicular astesia.At the same time,we illustrated the the molecular regulatory mechanism of let-7g inducing granulosa cell apoptosis.We explored and replenished the mechanism of miRNA-mediated ovarian follicle atresia contributing to animal genetic breeding and reproduction.