Function And Mechanism Study Of New Candidate Risk Gene HMGA2 In Polycystic Ovary Syndrome

Chapter Ⅰ Expression and function of HMGA2 in polycystic ovary syndrome granulosa cellObjective:Polycystic ovary syndrome(PCOS)is a complex reproductive endocrine and metabolic disorder characterized by hyperandrogenism,irregular ovulation and polycystic ovarian morphology,which may affect a woman’s lifelong health and the quality of her offspring.According to the epidemiological investigation,which was conducted by the research group led by professor Qiao Jie from the Peking University Third Hospital,the incidence of PCOS in China was 5.6%.PCOS is characterized by genetic heterogeneity,phenotypic complexity,and obvious family aggregation,which may be affected by multiple micro-effect genes and environmental factors.Chen Zi-Jiang’s research group has reported 11 susceptibility genes related to PCOS disease in the journal Nature Genetics,which have been widely verified in European and American populations.Through genome-wide association analysis(GWAS),we found that HMGA2(high-mobility group at-hook 2)is a PCOS susceptibility gene for the first time.HMGA2 is a structural transcription factor with three DNA binding domains that regulate transcription by assembling and stabilizing the structure and function of the nucleo-protein transcription complex.The follicles development depends on the regular proliferation and differentiation of granulosa cells.Recent studies have found that granulosa cells undergo abnormal proliferation and differentiation in PCOS patients.Therefore,from the perspective of proliferation and differentiation of granulosa cells,we can explore the regulatory mechanisms of ovulation abnormalities in PCOS patients to provide new etiological,prevention and treatment targets for PCOS patients.We collected granulosa cells from 96 patients with PCOS and 58 control patients to detect the expression level and function of HMGA2 in granulosa cells.Methods:Immunohistochemistry staining was used to determine the localization of HMGA2 in ovaries.Real-time quantitative PCR(qRT-PCR)and Western Blot were used to detect the expression level of HMGA2 in PCOS patient granulosa cells.The subcellular localization of HMGA2 granulosa cell was determined by immunofluorescence staining.Adenoviruse expressing HMGA2 was used to overexpress HMGA2 in human granulosa cell lines KGN and SVOG.CCK8 and flow cytometry were used to detect the effect of overexpression of HMGA2 on granulosa cell proliferation.Interfering RNA was used to down-regulate HMGA2 in KGN and SVOG cells,and detect granulosa cell proliferation.Results:HMGA2 is mainly located in granulosa cells in the ovary.It is also expressed in oocytes,but the expression level is low in theca cells.Compared with the control group,HMGA2 mRNA and protein levels were significantly increased in PCOS patient granulosa cells.The immunofluorescence staining showed that the expression of HMGA2 in granulosa cells was mainly in the nucleus,which was consistent with its function.After overexpression of HMGA2,the proliferation activity of granulosa cells was increased.After the down-regulation of HMGA2,the proliferation activity of granulosa cells was significantly reduced,and the cell cycle G1-S phase was prolongedConclusion:HMGA2 is mainly located in granulosa cells,suggesting that it affects the function of granulosa cells.HMGA2 expression level was significantly increased in PCOS granulosa cells,indicating that HMGA2 may be involved in the pathogenesis of PCOS.HMGA2 is mainly located in the nucleus of granulosa cells,which is adapted to the function of transcription factors.HMGA2 promotes the proliferation of granulosa cells and promotes the G1-S transition.Chapter Ⅱ HMGA2-IGF2BP2 pathway is activated in granulosa cells and promotes granulosa cell proliferation Objective:Through what mechanism HMGA2 participated in the proliferation of granulosa cells is unknown.It has been found that insulin like growth factor mRNA binding protein Ⅱ(IGF2BP2/IMP2)is a direct target gene downstream of HMGA2 in embryonic development and various cancers.IGF2BP2 is an mRNA binding protein that regulates downstream gene expression.GWAS found IGF2BP2 was associated with the onset of type 2 diabetes mellitus(T2DM),and Igf2bp2-/-mice were resistant to obesity induced by high-fat diet,suggesting its potential function in metabolism diseases.The HMGA2-IGF2BP2 pathway is conserved and activated in a variety of tumors to promote tumor cell proliferation and invasion,but the function of HMGA2-IGF2BP2 pathway in granulosa cells has rarely been studied.Therefore,we detect the expression level of IGF2BP2 in PCOS patient granulosa cells,so as to verify whether HMGA2-IGF2BP2 pathway is activated in patients.And we investigate the localization and function of IGF2BP2 in granulosa cells.And we detect whether or not HMGA2 regulates IGF2BP2 in granulosa cells.Methods:qRT-PCR and Western Blot were used to detect the expression level of IGF2BP2 in PCOS patient granulosa cells and analyze its relationship with HMGA2 expression.Immunofluorescence staining was used to determine the subcellular localization of IGF2BP2 in granulosa cells.The IGF2BP2-lentivirus was transfected into human granulosa cell lines KGN and SVOG to overexpress IGF2BP2.And then we detect granulosa cell proliferation by CCK8 and flow cytometry.IGF2BP2 was down-regulated in KGN and SVOG cells using interfering RNA technology.We investigate IGF2BP2 expression when HMGA2 was overexpressed or knocked down.Results:mRNA and protein levels of IGF2BP2 were significantly increased in PCOS granulosa cells and positively correlated with HMGA2 expression level,suggesting that HMGA2-IGF2BP2 pathway was activated in PCOS granulosa cells.IGF2BP2 is mainly located in the cytoplasm of granulosa cells,which is adapted to the function of RNA binding protein.After overexpression of IGF2BP2,the proliferation activity of granulosa cells increased.After the down-regulation of IGF2BP2,the proliferation activity of granulosa cells decreased significantly,and the cell cycle G1-S phase was prolonged.The expression level of IGF2BP2 increased significantly after overexpression of HMGA2,and decreased significantly after down-regulation of HMGA2,suggesting that HMGA2 positively regulated the expression of IGF2BP2 in granulosa cells.Conclusion:HMGA2-IGF2BP2 pathway is activated in PCOS granulosa cells,and the expression levels of two genes are positively correlated.IGF2BP2 was localized in the cytoplasm and overexpression of IGF2BP2 significantly promoted the proliferation of granulosa cells.HMGA2 positively regulates the expression of IGF2BP2 in granulosa cells.Chapter Ⅲ Study on the mechanism of HMGA2-IGF2BP2 in granulosa cell proliferation Objective:HMGA2-IGF2BP2 pathway is activated in granulosa cells and promotes granulosa cell proliferation,so the mechanism of HMGA2-IGF2BP2 pathway mediating granulosa cell proliferation is the aim of the following research.IGF2BP2 is an mRNA binding protein,and Hafner M et al.reported that the downstream target genes of IGF2BP2 in HEK293 cells were found through the PAR-CLIP technology(photoactively-enhanced ribonucleoside cross-linking and immunoprecipitation)in the international journal Cell.Among which,the cell cycle factor CCND2(Cyclin D2)and the RNA-binding protein SERBP1(SERPINE1 mRNA binding protein 1)were the targets which we got interested in.CCND2 is a cyclin protein that affects the transition of Gi/S transition and promotes the granulosa cells proliferation induced by FSH(follicle-stimulating hormone).SERBP1 inhibits granulosa cell apoptosis by binding to PGRMC1(Progesterone receptor membrane complex 1).In this chapter,we will seek the downstream target genes of HMGA2-IGF2BP2 pathway to promote granulosa cell proliferation.And we will explore the mechanism how HMGA2-IGF2BP2 regulates the gene expression.Methods:To find the downstream target genes of HMGA2-IGF2BP2 pathway in granulosa cells by RBP-IP(RNA binding protein immunoprecipitation)assay.We overexpressed and knocked down HMGA2-IGF2BP2 pathway in granulosa cells to detect the downstream genes expression level.We use double luciferase reporter gene assay to explore the regulation relationship between IGF2BP2 and downstream genes.CCND2/SERBP1 expression was detected in clinical samples to study the predictive value of HMGA2-IGF2BP2 pathway in PCOS.Results:RIP-Seq data analysis revealed that CCND2 and SERBP1 were the downstream genes of IGF2BP2.Sequence analysis showed that IGF2BP2 could bind to the 3 ’-UTR of CCND2/SERBP1 mRNA.Luciferase reporter assay confirmed that IGF2BP2 can positively regulate CCND2/SERBP1 expression.HMGA2-IGF2BP2 positively regulates CCND2/SERBP1 expression in granulosa cells.CCND2/SERBP1 expression level is increased in PCOS patients,indicating its predictive value in disease diagnosis.Conclusion:IGF2BP2 can bind to the 3’-UTR of CCND2/SERBP1 mRNA and promote their expression in granulosa cells.HMGA2-IGF2BP2 pathway can positively regulate the expression of CCND2/SERBP1 and promote granulosa cell proliferation,thus mediate the pathogenesis of PCOS.