The Regulation Of Let-7g In Ovarian Granulosa Cell Apoptosis And Autophagy And Regulatory Mechanisms During Follicular Development

During mammalian ovarian development,follicles progress from the primordial stage to the primary,secondary,and tertiary stage;and ovulation occurs once mature follicles are formed.However,more than 99%of ovarian follicles undergo degeneration and atresia,leading to a tremendous waste of resources.Previous studies demonstrated that follicular atresia is triggered by GC apoptosis;however,the molecular mechanisms involved in this process remain elusive.MicroRNAs(miRNAs)are 20-24-nucleotides-long small non-coding RNAs that regulate gene expression in the posttranscriptional level by directly binding to the 3’untranslated region(UTR).Followed by Lin-4,the let-7 miRNA family is the second discovered miRNA,which has been shown extensively involved in development,proliferation,diffentitation,apoptosis and disease.In ovary,dysregulation of let-7 negatively affectes the germ cell pool,subsequently leads to female infertility.let-7g,an important member of the let-7 family,is the only one miRNA that significantly upregulated during ovarian follicular atresia according to previous study.However,the underlying regulatory mechanisms in granulosa cell apoptosis and follicular atresia remian elusive.Autophagy,a highly conserved degradation/recycling system,plays an important role in maintaining cellular homestasis by degradation and turnover of protein during cell metabolism in eukaryotic cells.Recently reports investigate autophagy regulation is also involved in granulosa cell apoptosis during follicular atresia.Studies indicate autophagy is closely related with miRNA let-7g,which suggests the possibility that autophagy is responsable for granulosa cell apoptosis and follicular atresia regulated by let-7g.Thus,it is nesscessary to investigate the intereaction of autophagy and apoptosis mediated by let-7g,and the molecular mechanism of autophagy regulated by let-7g.Follicle-stimulating Hormone(FSH),the major reproductive hormone factor,plays an important role in promoting granulosa cell proliferation,follicular development and mammalian reproduction.It keeps cellular homestasis and subsequently promotes granulosa cell survival by upregulating autophagy.Our results suggest FSH enhances autophagy level in antral follicles(FSH-sensitive follicles)significantly,which indicating autophagy is upregulated during follicular develop mediated by FSH.In addition,there has a strong molecular crosstalk among cell autophagy,cell apoptosis and downstream signaling cascades.Therefore,FSH may act as the primary survival factor during granulosa cell apoptosis by activating autophagy.By conducting most of the experiments in pig and mouse ovaries,the current research focused on the following aspects:(1)Explore the apoptosis rate after overexpression of let-7g in primary cultured porcine granulosa cell and identify the downstream target gene of let-7g.(2)Detect the autophagy level after overexpression of let-7g in primary cultured mouse granulosa cell and confirm the downstream target gene of let-7g.(3)Clarify the interplay between granulosa cell autophagy and apoptosis by blocking the autophagy and then analyzing the cell apoptotic level.(4)Screen the possible downstream signaling pathway in the process of autophagy activation in response to FSH injection in vivo.(5)Validate the reliability of the signaling pathway selected based on vivo assay through knockdown the gene expression in simulated vivo hypoxic environment cultured granulosa cell in vitro.(6)Explore the function of autophagy in granulosa cell of follicular development upon FSH signaling by detecting apoptosis,proliferation,and functionally-related genes expression in autophagy suppressed granulosa cell.According to the above results,the following findings are thereby presented:(1)let-7g promotes follicular granulosa cell apoptosis by targeting transforming growth factor-β type 1 receptorOverexpression of let-7g in cultured porcine granulosa cell results in apoptosis,as well as increased proapoptotic gene Bax expression,decreased anti-apoptosis gene Bcl-2 expression and enhanced Caspase-3 activity.Bioinformatics analyses showed that transforming growth factor-β type 1 receptor(TGFBR1)is an important let-7g target.Luciferase activity analysis indicates that let-7g inhibits TGFBR1-3’UTR luciferase activity,but has no effect on the mutant TGFBR1-3’UTR luciferase activity,which suggests let-7g can directly bind to TGFBR1 3’UTR and inhibits the expression.Overexpression of let-7g induced apoptosis of porcine granulosa cells in vitro and repressed the mRNA and protein levels of TGFBR1,as well as the level of phosphorylated SMAD3(p-SMAD3)protein,Indicating let-7g regulates TGFBR1 expression and modulates the phosphorylation level of downstream molecule in granulosa cell.In addition,RNA interference-mediated knockdown of TGFBR1 and blocking of TGFBR1 significantly increased the rate of apoptosis of granulosa cell and Caspase-3 activity.In contrast,let-7g inhibition by let-7g inhibitor decreases the apoptosis rate;however,cotreated let-7g inhibitor with TGFBR1 siRNA recovers the apoptosis rate.The result suggests that knockdown of TGFBR1 can significantly suppress the inhibition function of let-7g inhibitor on GC apoptosis,further indicates let-7g promotes granulosa cell apoptosis through TGFBR1.Treatment of granulosa cell with TGF-β1 reduces the level of let-7g and increases the expression,which indicates let-7g can inhibit TGF-β1-induced TGFBR1 expression in granulosa cell.(2)let-7g regulates mouse granulosa cell autophagy by targeting insulin-like growth factor 1 receptorlet-7g regulates the mouse granulosa cell autophagy signaling pathway by inhibiting IGF1R expression,increasing the accumulation of GFP-LC3 puncta,accelerating the conversion of LC3-1 to LC3-2 and p62 degradation.By using bioinformatics analyses,IGF1R has two potential let-7g response elements in 3’UTR.Luciferase activity analysis further indicates let-7g can directly bind to IGF1R 3’UTR and inhibit the expression.In granulosa cell,let-7g suppresses IGF1R expression and affects the phosphorylation of PKB/mTOR.Small interference-mediated knockdown of IGF1R significantly promotes autophagy signaling of mouse granulosa cell.Conversely,overexpression of IGF1R attenuates the let-7g dependent autophagy.This result further suggests let-7g regulates granulosa cell autophagy by targeting IGF 1R.(3)Autophagy induced by let-7g contributes to apoptosis in granulosa cellOverexpression of miRNA let-7g results in granulosa cell apoptosis;however,blocking of let-7g-dependent autophagy decreases cell apoptosis rate,as well as increases the cell viability.In normal cell,autophagy is remained in a lower level to protect cell from stress and maintain cellular homeostasis.However,excessive autophagy could cause autophagic cell death;the type Ⅱ programmed cell death.let-7g regulates cell autophagy by targeting IGF1R,which in turn modulates the activation of Akt and mTOR.It is thought that let-7g-dependent autophagy in granulosa cell leads to the increasing of apoptosis rate.(4)Autophagy is induced in FSH dependent follicular developmentIn mouse granulosa cells of antral follicles,FSH promotes autophagy marker LC3 and lysotracker staining,induces the autophagy mainly occurs in antral follicles(or FSH-sensitive follicles)during follicular development responsible for FSH.In line with the activated autophagy response to FSH,study investigates the Akt-mTOR signaling pathway is inhibited in a time dependent manner.Further experiments investigate FSH could lead to oxygen stress and metabolism disorder problems in mouse granulosa cell,which may in turn lead to cell autophagy.(5)Hypoxia plays a dominate role in FSH induced cell autophagyBy using hypoxia inhibitor,autophagy signaling is suppressed in granulosa cell.Likewise,HIF-la and its downstream molecule Bnip3,autophagy key gene Beclinl play an essential role in Cocl2 treated granulosa cell.Knockdown these genes expression decrese autophagy in vitro,which suggests hypoxia,which subsequently regulates HIF-1α,is a major factor in regulating granulosa cell autophagy.(6)Inhibiting autophagy induced by FSH inhibits granulosa cell proliferationInhibiting FSH induced autophagy signaling by blocking autophagy has no effect on apoptosis rated gene expression,which indicates FSH inhibits granulosa cell apoptosis is independent of autophagy.Conversely,the expressions of CyclinA2,CyclinD2 are significantly inhibited,indicates a relationship between autophagy and proliferation in FSH induced follicular development.In addition,the cell cycle result reveals autophagy inhibition has a negative effect on cell proliferation mediated by FSH,further leads to a cell cycle delay in S phase.The cell viability also shows a similar result.In summary,this study shows the regulation of let-7g and the related-signaling pathway in ovarian granulosa cell apoptosis and autophagy,as well as the relationship between autophagy and apoptosis.We propose a mechanism of FSH regulates cell autophagy through HIF-la-Bnip3 pathway during follicular development and the autophagy induced by FSH is related to granulosa cell proliferation.This study provides some evidence for molecular mechanism of granulosa cell apoptosis and autophagy,also helps to develop novel strategies that reduce the number primordial follicles that undergo degeneration and atresia.