Study On The Role And Mechanism Of ANGPTL4 Gene In Polycystic Ovary Syndrome

Part I Expression and clinical analysis of ANGPTL4 in women with polycystic ovary syndromeObjective:Polycystic ovary syndrome(PCOS)is one of the most complicated and common endocrine and metabolic disorders,affecting about 6%-20%women of reproductive age.In addition to reproductive dysfunction,PCOS can also manifest insulin resistance(IR),obesity,dyslipidemia,metabolic syndrome(MetS)and cardiovascular disease(CVD).Throughout the process of oocyte development,there is an interdependence between oocytes and its surrounding granulosa cells(GC)providing growth regulators and nutrients.For patients with PCOS,the associated endocrine and glycolipid metabolic disorder can disrupt the interaction between them,interfere with the development and ovulation of oocytes by affecting the function of GCs,and eventually lead to reproductive dysfunction.A series of evidence have been provided that the glycolipid metabolism in GCs plays an important role in maintaining ovarian follicle development.However,the mechanisms underlying these associations have not been fully investigated,especially the origins of the metabolic alterations for PCOS as well as new biomarkers.Angiopoietin-like 4(ANGPTL4),as a secreted glycoprotein,is mainly involved in regulating glycolipid metabolism,and also plays an important role in angiogenesis,inflammatory response and tumorigenesis.However,the research of ANGPTL4 in PCOS is limited.This part of the study aims to characterize the expression of ANGPTL4 in ovarian GCs and its association with polycystic ovary syndrome(PCOS).Methods:Three independent cohorts in this study involved a total of 160 PCOS patients and 164 control women to detect the level of ANGPTL4 in serum and follicular fluid,and its expression level in ovarian GCs,while Pearson correlation and binary logistic regression analysis were performed with clinical baseline data,hormone levels and glycolipid metabolism indicators.The independent predictive ability of ANGPTL4 was analyzed using ROC curve.Results:1.The level of serum ANGPTL4 in PCOS patients was significantly higher than that in the control group(5.86±3.04 vs.3.90±1.39 ng/ml,P<0.01);The level of ANGPTL4 in serum was positively correlated with BMI,T,LH/FSH and AMH,and negatively correlated with FSH.The area under curve(AUC)of serum ANGPTL4 was 0.727(P<0.01)with sensitivity(98%)and specificity(41.3%).After adjusting for age,BMI,AMH,T and LH/FSH,serum ANGPTL4 level could predict the risk of PCOS patients independently.2.The level of ANGPTL4 in follicular fluid of PCOS patients was significantly higher than that of the control group(9.43±1.54 vs.12.5±5.12 ng/ml,P=0.01);The AUC of ANGPTL4 in follicular fluid was 0.792(P<0.01)with sensitivity(83.3%)and specificity(68.7%).The level of ANGPTL4 in follicular fluid was positively correlated with the relative expression of ANGPTL4 mRNA in ovarian GCs(r=0.757,P<0.001).3.In ovarian GCs of PCOS patients,the mRNA level and protein level of ANGPTL4 were significantly higher than that of the control group;The relative expression of ANGPTL4 mRNA in ovarian GCs was positively correlated with AMH,HOMR-IR,LDL/HDL,ApoB/ApoAI and TC/HDL.The AUC of ANGPTL4 mRNA level in GCs was 0.704(P<0.01)with sensitivity(67.3%)and specificity(70.5%).After adjusting for BMI,AMH,T and LH/FSH,the relative expression of ANGPTL4 mRNA in GCs was an independent factor affecting the occurrence of PCOS.Conclusion:The level of ANGPTL4 in serum,follicular fluid,as well as its expression level in ovarian GCs were significantly increased in PCOS patients,and its association with PCOS and glycolipid metabolism showed that the high expression of ANGPTL4 might be an independent factor in predicting the incidence of PCOS,which contributes to illustrate the etiology and pathogenesis of PCOS,Part Ⅱ High expression of ANGPTL4 inhibits the proliferation of ovarian granulosa cellsObjective:The development of follicle and ovulation process are related to female fertility.There is a dynamic balance between the proliferation and apoptosis of granulosa cells(GCs)surrounding oocytes.It has been reported that when the apoptosis rate of ovarian GCs in the late stage reached more than 10%,the apoptosis of oocytes would occur and include follicular atresia.PCOS is one of the main causes of ovulatory dysfunctional infertility with a large number of follicular atresia during follicular development.Therefore,exploring the mechanism regulating proliferation and apoptosis of ovarian GCs can improve the awareness of pathogenesis in PCOS.In previous studies,ANGPTL4 not only participated in the regulation of glycolipid metabolism,but also contributed to cell proliferation.Nevertheless,it is still unknown whether ANGPTL4 is involved in regulating the proliferation of ovarian GCs in PCOS.Based on the results of our case-control study in chapter 1,we conducted functional experiments to explore the effect of ANGPTL4 on the proliferation of ovarian GCs.Methods:To identify the role of ANGPTL4 in the function of ovarian GCs,we overexpressed ANGPTL4 in KGN and SVOG cell lines using adenoviruses.CCK-8 and EdU staining assay were used to detect cell proliferation,while cell cycle analysis was performed by flow cytometry.Results:Overexpression of ANGPTL4 could inhibit cell activity and proliferation,and block the G1/S phase of the cell cycle in KGN and SVOG cells.Conclusion:Overexpression of ANGPTL4 inhibited the proliferation of ovarian GCs by blocking the G1/S phase of cell cycle,thereby participating in the development of PCOS.Part III ANGPTL4 inhibits granulosa cell proliferation in polycystic ovary syndrome by EGFR/JAK1/STAT3-mediated induction of p21Objective:The results of the previous parts of the study showed that ANGPTL4 level in serum,follicular fluid,as well as its expression level in ovarian GCs were significantly increased in PCOS patients.Overexpression of ANGPTL4 inhibited the proliferation of ovarian GCs by blocking the G1/S phase of cell cycle,thereby participating in the development of PCOS.However,the mechanism by which ANGPTL4 regulates the proliferation of ovarian GCs remains unknown.This part of study aims to find the downstream pathway of ANGPTL4 and explore its mechanism of regulating the downstream pathway.Methods:We overexpressed ANGPTL4 in KGN cell using adenoviruses and high-throughput sequencing techniques were used to verify expression changes of downstream genes.Further Co-IP,ChIP and antagonist treatment were performed to reveal the molecular mechanism in detail.Results:CDKN1A(p21),CCNA2(CyclinA2)and CDK2 were detected by high throughput sequencing as downstream genes of ANGPTL4 to participate in the regulation of cell cycle.ANGPTL4 protein could bind to EGFR/JAK1 and activate the phosphorylation of EGFR/JAK1/STAT3 pathway to mediate p21 transcription,thereby down regulating the expression of CyclinA2 and CDK2 and blocking G1/S cell cycle progression and cell proliferation.In ovarian GCs of PCOS patients,the expression of CDKN1A(p21)was also up-regulated,while the expression of CCNA2(CyclinA2)and CDK2 was down-regulated,suggesting that this mechanism was involved in the incidence and development of PCOS.Conclusion:Overexpression of ANGPTL4 inhibited the proliferation of GCs through EGFR/JAK1/STAT3-mediated induction of p21,thus providing a novel mechanism for the pathogenesis of PCOS.