The Mechanism Of Granulosa Cells Mir-186 And Mir-135a Targeting ESR2 In Polycystic Ovary Syndrome

Background and purposePolycystic ovary syndrome(PCOS)is a complex and heterogeneous endocrine disease,with a prevalence of 10%and an increased risk of long-term complications.PCOS is mainly manifested in hyperandrogenemia(HA),ovulation dysfunction and polycystic ovary,which can lead to infertility and multiple metabolic diseases.However,the cause of PCOS is still largely unknown.MicroRNA(miRNA)is a small noncoding RNA(nearly 22 nucleotides long),and it can regulate gene expression at the post transcriptional level.Abnormal miRNA expression levels are closely related to the occurrence of diseases,including diabetes,cancer and atherosclerosis,and miRNA can be used as a predictor of cancer and a diagnostic biomarker.Interestingly,the role of miRNA in the PCOS pathological process has attracted extensive attention in recent years.Research showed that the change of miRNA expression in PCOS women may be a noninvasive biomarker and a new therapeutic target compared with healthy women.Granulosa cells(GCs)which are located on the surface of the follicle,can maintain the basic function of ovary by secreting estrogen.Ovarian GCs provide nutrition for the development and maturation of follicles.It was found that the proliferation and apoptosis of GCs in PCOS patients were abnormal compared with normal ovulation women.Hence,it is important to understand the pathogenesis of GCs dysfunction in PCOS patients,which may provide clues for identifying new biomarkers for diagnosis and treatment.Studies have shown that miRNA plays an important role in the development and maturation of follicles and embryo growth.So,miRNA plays a key role in maintaining the normal reproductive function of women.Since the pathogenesis of PCOS is related to the development and maturation of follicles,and miRNAs are involved in regulating the formation of follicles,which suggests that miRNAs may be related to the pathogenesis of PCOS.At present,most of the related researches focus on the abnormal expression of miRNAs in the plasma and follicular fluid of PCOS patients,but few in the GCs of PCOS patients and its role in the pathogenesis of PCOS.Therefore,studying the biological functions and regulatory mechanisms of abnormally expressed miRNAs in PCOS GCs can provide new strategies for the diagnosis and treatment of PCOS.In this study,we will focus on the role of miRNAs in the pathogenesis of PCOS.This study aims to further deepen our understanding of the pathogenesis of PCOS by regulating the relationship between abnormally expressed miRNAs in GCs and their target proteins and provide new ideas and theoretical basis for the clinical diagnosis and treatment of PCOS.This research mainly includes the following two parts:Part 1 Study on the expression and biological function of miRNAs in granulosa cells of PCOS patientsPurpose:At present,a large number of studies have shown that the abnormal expression of miRNAs in plasma and follicular fluid of PCOS patients is closely related to the occurrence and development of PCOS.In the follicular fluid of PCOS patients,177 miRNAs were up-regulated and 86 miRNAs were down-regulated.The alteration of GCs in follicles may be an intrinsic mechanism of PCOS.The abnormal expression of miRNA in GCs is closely related to the occurrence of PCOS.In this study,we selected 6 kinds of miRNAs based on literature review,which were abnormal expressed in serum,plasma and follicular fluid of PCOS patients,as the research objects from a variety of miRNAs with different expressions.Then the expression levels of these 6 candidate miRNAs in GCs of follicular fluid of PCOS patients and healthy controls were detected to determine the abnormally expressed miRNAs(miR-186 and miR-135a)in the PCOS patients and the effect of FSH on the expression levels of E2,miR-186 and miR-135a.To explore the biological functions of miR-186 and miR-135a in GCs in PCOS patients,we analyzed the effects of miR-186 and miR-135a on GCs proliferation,apoptosis and cell cycle;the correlation between the serum E2 level and the expression levels of miR-186 and miR-135a in GCs of PCOS patients to determine whether E2 has a direct regulatory role in the expression of miR-186 and miR-135a,so as to further deepen the understanding of the pathogenesis of PCOS and provide new ideas for the diagnosis and treatment of PCOS.Methods:1.The study included 63 PCOS patients and 58 healthy women with normal menstrual cycles recruited in the Department of Reproductive Medicine of the Second Affiliated Hospital of Zhengzhou University from December 2015 to February 2018.Follicular fluid and GCs were collected from PCOS patients and healthy controls.RT-qPCR was used to detect the expression differences of 6 candidate miRNAs(miR-154-5p,miR-186,miR-26a,miR-224-3p,miR-135a and miR-103a)in GCs of PCOS patients and healthy controls.2.GCs were extracted from PCOS patients and healthy controls and treated with FSH respectively.The E2 level was detected by ELISA,and the expression levels of miR-186 and miR-135a were detected by RT-qPCR.3.In order to study the relationship between E2 level and the expression of miR-186 and miR-135a,we divided PCOS patients into normal E2 group(n=24)and high E2 group(n=39).To determine whether E2 can regulate the expression of miR-186 and miR-135a in GCs,we first treated KGN GCs with 10 nM E2 for 24 h,and detected the expression levels of miR-186 and miR-135a by RT-qPCR.Then,primary GCs were extracted from healthy participants,treated with 10nM E2 for 24 hours,and the expression changes of miR-186 and miR-13 5a were detected by RT-qPCR.4.KGN cells were treated with miR-186 mimic or miR-135a mimic for 48 h,and then MTT assay was used to detect cell viability,FITC-annexin V/PI double staining was used to detect cell apoptosis and flow cytometry was used to detect cell cycle.Results:1.The expression of miR-186 and miR-135a in GCs of patients with PCOS was significantly up-regulated(p<0.05),and the other four miRNAs was not significant difference between the two groups.2.FSH treatment could increase E2 level and the expression of miR-186 and miR-135a in GCs of PCOS patients and healthy controls(p<0.01).There was no significant difference in E2 level between the two groups,but FSH treatment could increase the expression levels of miR-186(p<0.05)and miR-135a(p<0.01)in GCs of PCOS group.3.The serum E2 level was positively correlated with the levels of miR-186 and miR-135ain GCs.4.After E2 treatment,the expression levels of miR-186 and miR-135a in KGN cells and primary granulosa cells were significantly increased(p<0.01).5.After treatment with miR-186 mimic or miR-135a mimic,the cell viability of KGN cells significantly increased compared with the mismatched control group(p<0.01).6.After treatment with miR-186 mimic or miR-135a mimic,the apoptosis rate of KGN cells was significantly lower than that of the control group(p<0.05).7.After treatment with miR-186 mimic or miR-135a mimic,the proportion of KGN cells in G1 phase was significantly decreased,while the proportion of KGN cells in S phase was significantly increased(p<0.05),but no significant change in G2/M phase.Part 2 Validation of(miR-186 and miR-135a)/ESR2 signaling pathway in granulosa cells of PCOS patientsPurpose:The expressions of ESR2 and CDKN1A in GCs of PCOS patients were detected;Then the effect of abnormal miRNAs on ESR2 expression was analyzed,Online target prediction and dual luciferase analysis were used to determine the target of miRNAs,which provided the basis for further exploring the biological function of miRNAs in GCs.Methods:1.Western blotting was used to detect the protein expression levels of ESR2 and CDKN1A in granulosa cells from PCOS patients and healthy controls.2.KGN GCs were transfected with miR-186 mimic and miR-135a mimic,respectively.The effects of differentially expressed miRNAs on the expression of ESR2 pathway related mRNA and protein(ESR2 and CDKN1A)were detected by RT-qPCR and western blotting.3.Based on the principle of bioinformatics,the target genes of miRNAs were predicted by TargetScan,miRDB and miRanda online prediction website.4.KGN cells were co-transfected with miR-186 and miR-135a mimic/inhibitor and the reporter vector of ESR2,respectively.The expression levels of miR-186 and miR-135a were detected by RT-qPCR.5.KGN cells were co-transfected with miR-186 and miR-135a mimic/inhibitor and vector with ESR2-3’UTR wildtype or mutant,respectively.The change of luciferase activity was detected by dual luciferase reporter to verify the targeting relationship between miR-186/ESR2 and miR-135a/ESR2.Results:1.The protein contents of ESR2 and CDKN1A in GCs of PCOS patients were significantly decreased.2.When KGN cells were transfected with miR-186 mimic or miR-135a mimic,the mRNA and protein expression levels of ESR2 and CDKN1A were significantly decreased(p<0.05).3.The target genes of miRNAs were predicted by TargetScan,miRDB and miRanda online prediction website.It was found that the target genes of miR-186 and miR-13 5a were ESR2.4.After KGN cells were transfected with miR-1 86 or miR-135a mimic,the levels of intracellular miR-186 and miR-135a were significantly increased,while after transfection with miR-186 or miR-135a inhibitor,the levels of intracellular miR-186 and miR-135a were significantly reduced.5.KGN cells were transfected with miR-186 or miR-135a mimic/inhibitor,the luciferase activity decreased in miR-186 or miR-135a mimic transfected cells.However,luciferase activity increased in cells transfected with miR-186 or miR-135a inhibitor.And when the binding sites of miR-186 and miR-135a are mutated,both miR-186 and miR-135a mimic/inhibitor lost their ability to regulate the luciferase activity of ESR2-3’UTR.Conclusions:1.The expression of miR-186 and miR-135a is increased in granulosa cells of PCOS patients,and E2 can directly regulate the expression of miR-186 and miR-135a in granulosa cells.2.MiR-186 and miR-135a affect the proliferation,apoptosis and cell cycle of granulosa cells by targeting ESR2,which further reveals the pathogenesis of PCOS.