MicroRNA Regulation Mechanism Of PI3K Signaling Pathway In Granulosa Cells Of Patients With Polycystic Ovary Syndrome

Part I Comprehensive assessment the expression of core elements related to IGF1R/PI3K pathway in granulosa cells from women with polycystic ovary syndromeBackground:Polycystic ovary syndrome(PCOS)is a relatively common endocrine disorder of women in reproductive age,characterized by hyperandrogenism,polycystic ovaries and anovulation.A plethora of information suggests phosphatidylinositol-3-kinase(PI3K)pathway is the key to the pathogenesis of PCOS,but little is known about the expression pattern and possible role of insulin like growth factor 1 receptor(IGFIR)/PI3K pathway in PCOS.Therefore,the goal of this study was to detect the core elements of the IGF1R/PI3K pathway in PCOS ovarian granulosa cells(GCs)and to speculate its role in the occurrence and development of PCOS.Study design:Western blot and quantitative real-time PCR(qRT-PCR)for IGF1R,insulin receptor substrate 1(IRS1),insulin receptor substrate 2(IRS2),phosphatase and tensin homolog(PTEN)and forkead box protein Ol(FOXO1)related to IGF1R/PI3K pathway were performed in GCs isolated from 60 PCOS patients(PCOS group)and 60 controls(control group),and their clinical significance was analyzed.Results:Compared to controls,body mass index(BMI),the levels of fasting plasma glucose(FPG),fasting insulin(FINS),anti-Mullerian hormone(AMH),testosterone(T)3 luteotropic hormone(LH),homeostasis model assessment of insulin resistance(HOMA-IR),antral follicle count(AFC)were markedly elevated,while follicle stimulating hormone(FSH)decreased(P<0.05).Furthermore,at both mRNA and protein levels,the expression of IGF1R,IRSI,IRS2 were significantly increased,whereas PTEN and FOXO1 were dramatically decreased in PCOS patients(P<0.05).Conclusion:Our findings indicate that IGF1R/PI3K pathway is up-regulated in PCOS GCs compared with controls,with IGF1R,IRS1,IRS2 significantly increased,while PTEN and FOXO1 decreased.Thus,our study probably provides new evidences about the pathogenesis of PCOS in term of molecular mechanism.Part II MicroRNA-183 suppresses granulosa cells proliferation by targeting FOXO1Background:MicroRNAs(miRNAs)perform their functions in multiple biological and pathology processes,the alteration of which is closely related to the pathogenesis of PCOS.The purpose of this study is to explore whether miR-183 is involved in the abnormal proliferation of PCOS GCs and its underlying mechanisms.Study design:The expression of miR-183 in GCs isolated from 115 patients(55 PCOS vs 60 controls)was detected by qRT-PCR.Granulosa-like tumor cell line cells were used to alter the levels of miR-183 and target gene by transfection,and cell proliferation was detected by cell counting kit-8(CCK-8)assay.The target gene of miR-183 was first predicted by Targetscan bioinformatics analysis and then verified using qRT-PCR,western blot and luciferase reporter assay.Granulosa cells were treating with insulin to further confirm the relationship between miR-183 and target gene.Results:MiR-183 was significantly increased in PCOS GCs,and over-expression of miR-183 led to an inhibition in granulosa cells proliferation.In addition,FOXO1 was a direct target of miR-183,and down-regulation of it could significantly reduce the proliferation of granulosa cells.Furthermore,insulin increased the expression of miR-183,while FOXO1 was suppressed in both mRNA and protein levels.Conclusion:Our Results revealed that miR-183 was significantly increased in PCOS and targeted FOXO1 to suppress granulosa cells proliferation.Insulin could up-regulate the expression of miR-183 and down-regulate of FOXO1.Taken together,this study provides new evidence for the function role of miR-183 in the dysfunction proliferation of GCs,which might contribute a lot to the pathogenesis of PCOS.Part III MicroRNA-200c inhibits granulosa cells proliferation by targeting PTENBackground:PCOS is one of the most common endocrine metabolic disorders,characterized by hyperandrogenism,polycystic ovaries and ovulatory dysfunction.A large number of studies have reported tha abnormal expression of miRNAs plays an important role in the pathogenesis of PCOS,though the expression and biological function of miR-200c in PCOS GCs are still unclear.Study design:The expression of miR-200c in GCs derived from 90 PCOS patients and 70 controls was analyzed by qRT-PCR.TargetScan was used to predict the target gene of miR-200c,which was further verified by qRT-PCR,western blot and luciferase assays.Granulosa cells were cultured for CCK-8 after over-expression of miR-200c or knockdown PTEN,as well as after insulin treatment.Results:Significantly increased expression of miR-200c was observed in PCOS patients compared with the controls.Moreover,over-expression of miR-200c inhibited the proliferation of granulosa cells.In addition,our results verified that miR-200c directly targeted PTEN,which could be reduced by insulin and then inhibited proliferation in KGN cells.Conclusion:Our findings,therefore,demonstrate that miR-200c suppresses the proliferation of granulosa cells by targeting PTEN,providing new evidence for abnormal proliferation of GCs in PCOS.