A Study On The Role Of MicroRNA-181a In Ovarian Granulosa Cell Apoptosis

Background:Normal ovarian follicle development is the basis for maintaining the secretion of female steroid hormones and the formation of mature oocytes.Increased ovarian follicular atresia leads to accelerated ovarian dysfunction,such as premature ovarian failure(POF).Previous studies demonstrate that granulosa cell apoptosis is the main cause of follicular atresia.Many factors are involved in the process of granulosa cell apoptosis.FoxO1,a member of the transcriptional factor FoxOs,is one of the most important factors regulating granulosa cell apoptosis.FoxO1 binds to the promoter of target gene FasL,up-regulates FasL expression,and triggers granulosa cell apoptosis.Oxidative stress induces granulosa cell apoptosis and follicular atresia by promoting FoxOl nuclear accumulation and activation.However,the mechanism by which the oxidative stress regulates FoxO1 nuclear translocation remains unknown.microRNAs(miRNAs),small noncoding RNAs,are involved in many biological and pathological processes,such as proliferation,differentiation,and apoptosis.We previously demonstrated that miR-181a level increases in the blood of POF patients compared with that in the normal controls and miR-181a expression decreases in the growing follicles compared with that in primary ones.Moreover,miR-181a overexpression attenuates activin A-induced granulosa cell proliferation by down-regulating activin receptor 2A expression.To date,whether miR-181a is associated with granulosa cell apoptosis is unclear.Objective:In the present study,we aimed to investigate the role of miR-181a in the process of oxidative stress-induced granulosa cell apoptosis.Methods:(1)miRNAs were extracted from the blood of POF patients(n=40)and of normal fertility females(n=20)using the mirVanaTM PARISTTM Kit and quantitative real time PCR(qRT-PCR)assay was performed to examine miR-181a level.(2)Human ovarian granulosa-like tumor cell line KGN cells and mouse primary granulosa cells(mGC)were cultured in vitro and treated with H2O2 using different concentration or for different time.miR-181a expression was detected using qRT-PCR.(3)Then adenovirus-mediated miR-181a overexpression(MOI=0,25,50)and anti-sense oligonucleotides-mediated miR-181a inhibition(100nM),combined with qRT-PCR,cell death detection assay,and Western blot assay,were performed to investigate the role of miR-181a in regulating granulosa cell apoptosis.(4)Cytoplasmic and nuclear protein was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents.Western blotting and immunofluorence assay were also used to reveal the effect of miR-181a on FoxO1 cytoplasm-to-nuclear translocation and post-translational modification.(5)Luciferase reporter assay,qRT-PCR,and Western blot assay were performed to elucidate the inhibitory role of miR-181a in SIRT1 gene expression.(6)Ad-h-SIRT1 was used to test whether SIRT1 was involved in the process of H2O2-and miR-181a-regulated granulosa cell apoptosis.Results:(1)qRT-PCR results showed that miR-181a level increased～3.68-fold in the blood of POF patients compared with that in normal fertility females(P<0.01).miR-181a expression was also enhanced by H2O2 in a dose-and time-dependent manner in both KGN cells and mGC.After Ad-miR-181a infection for 48 h,KGN cell and mGC apoptosis was promoted,and the pro-apoptotic marker gene caspase 3 was also activated.H2O2-induced cell apoptosis was also inhibited after knockdown of endogenous miR-181a using an miR-181a inhibitor in KGN cells and mGC.(2)Both miR-181a and H2O2 promoted the pro-apoptotic factor FoxO1 nuclear accumulation in KGN cells and mGC.Moreover,FasL,the target gene of FoxO1,was also up-regulated after miR-181a overexpression or H2O2 treatment,combined with activation of the apoptotic pathway.Knockdown of endogenous FoxO1 using siRNA which specifically targeting FoxO1 blocked the promoting effect of miR-181a on apoptosis in both KGN cells and mGC.Interestingly,miR-181a overexpression had no effect on FoxO1 phosphorylation.Instead,FoxO1 acetylation was apparently enhanced by miR-181a overexpression and H2O2 treatment.miR-181a inhibitor weakened H2O2-induced FoxO1 acetylation.(3)Luciferase reporter assay results confirmed that miR-181a inhibited the luciferase activity by binding to the seed sequence "TGAATGT" of SIRT1 3’UTR.The results of Western blotting and qRT-PCR further confirmed that miR-181a overexpression inhibited SIRT1 protein and mRNA expression in both KGN cells and mGC.Furthermore,adenovirus-mediated overexpression of human SIRT1 gene attenuated H2O2-and miR-181a-induced FoxO1 acetylation and cell apoptosis in KGN cells.Treatment of SIRT1 activator 3(SA3)also showed an inhibitory effect on miR-181a-induced FoxO1 acetylation and apoptosis in both KGN cells and mGC.Conclusion:miR-181a level is increased in the blood of POF patients.H2O2 enhances miR-181a expression in granulosa cells.The overexpression of miR-181a induces FoxO1 acetylation and nuclear accumulation,triggering granulosa cell apoptosis by suppressing SIRT1 gene expression.Our study reveals the role and mechanism of miR-181a in regulating granulosa cell apoptosis.Our study not only implies the important role of miR-181a in follicular development and atresia,but also provides a new direction for research in the area of POF diagnosis and treatment.