Mechanism Of MiR-194 Promoting Apoptosis Of Granulosa Cells In Polycystic Ovary Syndrome By Targeting HB-EGF

BackgroundPolycystic ovarian syndrome(PCOS)is a common and complex endocrine disorder that affects 7-15% of women of reproductive age.It is characterized by clinical or laboratory hyperandrogenemia,sporadic ovulation,polycystic ovarian changes,with long-term metabolic complications such as insulin resistance and cardiovascular disease,and is one of the main causes of ovulatory disorders in women.It is one of the main causes of dysfunctional infertility in women.Due to the highly heterogeneous phenotype of PCOS,the etiology is unclear and relies on exclusionary diagnostic criteria,clinical treatment is mainly symptomatic.In the reproductive cycle of follicular development,granulosa cell proliferation and apoptosis are necessary to maintain the normality of oocyte development,follicular growth and differentiation,and follicular atresia.Granulosa cells nutritionally support oocytes in an autocrine and paracrine manner through gap junctions,and bi-directional intercellular signal transduction ultimately promotes normal follicular development and maturation.Among them,excessive granulosa cell apoptosis is one of the important manifestations of polycystic ovary syndrome.An increasing number of studies have found that micro RNAs(miRNAs)are tiny non-coding single-stranded RNAs produced endogenously that regulate the expression levels of post-transcriptional genes and can regulate various key biological functions such as cell growth and differentiation and apoptosis,and their altered expression levels are closely associated with various diseases.A large number of studies have shown that miRNAs play an important role in the proliferation and apoptosis of PCOS granulosa cells.It has been reported that miR-194 plays a role as a tumor suppressor and also regulates a variety of diseases by targeting genes.overexpression of miR-194 inhibits cell proliferation,delays disease progression and improves prognosis.However,the specific mechanism regarding the preventive and curative effects of miR-194 on PCOS is still unclear.Therefore,we propose to investigate the effect of miR-194 on proliferation and apoptosis of granulosa cells in patients with polycystic ovary syndrome by using relevant indicators of cell proliferation and apoptosis,such as flow cytometry(FC),and to explore the molecular biological mechanism of miR-194 by targeting heparin-binding epidermal growth factor-like growth factor(HB-EGF).The present study investigated the possible mechanisms by which miR-194 regulates the development of PCOS,and provided a theoretical basis for exploring new ways of miR-194 treatment for PCOS.Part Ⅰ: Differential expression and correlation of miR-194 and HB-EGF in granulosa cells of PCOS patients and PCOS mouse modelsPurpose1.to investigate whether there is a difference in the expression levels of miR-194 and HB-EGF in ovarian granulosa cells in PCOS and non-PCOS groups and whether there is a correlation;2.To investigate whether the levels of miR-194 and HB-EGF are differentially expressed in the ovaries of PCOS mouse models versus non-PCOS mice.MethodOvarian granulosa cells were extracted from 10 cases of follicular fluid from the PCOS group and 10 cases from the control group(non-PCOS group),and the expression levels of miR-194 and HB-EGF were measured by real-time quantitative reverse transcription-polymerase chain reaction(RT-q PCR)and protein immunoblotting(Western blot,Wb).The m RNA and/or protein expression levels of miR-194 and HB-EGF in the granulosa cells were detected by western blot(Wb)and linear correlation analysis of the m RNA expression of miR-194 and HB-EG in the granulosa cells using Pearson correlation coefficient.21-day-old C57 female mice were selected and subcutaneously injected with dehydroepiandrosterone(6mg/100g)(DHEA)for 20 consecutive days on the back to establish PCOS model mice;enzyme-linked immunosorbent assay(ELISA)was used to Serum estrogen(E2),follicle stimulating hormone(FSH),luteinizing hormone(LH)and testosterone(T)levels were measured by ELISA.The m RNA expression levels of miR-194 and HB-EGF were measured by RT-q PCR.All participating patients signed the informed consent form and the animal experiments were performed under the approval of the ethics committee of the relevant units.Results7.Significantly higher m RNA expression levels of miR-194 in ovarian granulosa cells in PCOS versus non-PCOS groups;8.Significantly lower m RNA and protein expression levels of HB-EGF in ovarian granulosa cells in PCOS versus non-PCOS groups;9.A negative correlation between the m RNA expression levels of miR-194 and HB-EGF in ovarian granulosa cells in the PCOS and non-PCOS groups;10.Serum levels of sex hormones E2,T and LH were increased and FSH levels were decreased in the PCOS mouse model,all with statistical differences(P < 0.05),and then the LH/FSH ratio was increased,consistent with the endocrine function of the ovaries in PCOS;11.HE staining of the ovaries of PCOS mice showed enlarged ovarian volume and increased mass,follicles in the ovaries were cystically dilated,and changes in the radial crown,granulosa cells,corpus luteum and atretic follicles were consistent with histomorphological changes in the ovaries of PCOS mice;12.In PCOS mice ovaries with non-PCOS,miR-194 with m RNA expression levels were significantly increased,while m RNA expression levels of HB-EGF were significantly downregulated,consistent with the trend in the PCOS group in the aforementioned patients.Conclusion3.miR-194 expression levels were significantly upregulated in both granulosa cells of PCOS patients and PCOS mouse models,while HB-EGF expression levels were significantly downregulated in both;4.The m RNA expression levels of miR-194 and HB-EGF in ovarian granulosa cells of PCOS patients were negatively correlated.Part Ⅱ: Possible molecular mechanisms by which miR-194 regulates proliferation and apoptosis of human ovarian granulosa cell line KGN cellsPurpose1.To investigate the effect of miR-194 on the growth and apoptosis of human ovarian granulosa cells(KGN);2.To investigate whether there is a targeting relationship between miR-194 and HB-EGF molecules and to further explore the potential regulatory mechanism.MethodsHuman ovarian granulosa cell line KGN cells were cultured in vitro and transfected with miR-NC-inhibitor,miR-194-inhibitor,miR-NC mimic,miR-194 mimic,HB-EGF and miR-194 mimic+HB-EGF plasmids according to the specific experimental requirements by cell transfection technique.RT-q PCR to detect the transfection efficiency;using cell proliferation and activity assay kits(CCK-8,Ki-67)and flow cytometry to detect cell viability,proliferation and apoptosis;dual luciferase reporter genes were used to determine the corresponding fluorescence values to determine the presence of target correlation;Wb was also used to detect the expression levels of apoptosis-related proteins P53,P21 and P16.Results1.in KGN cells,the expression level of miR-194 m RNA level was significantly decreased in the group transfected with miR-194-inhibitor,while the expression level was significantly increased in the miR-194-mimic group,and the other groups were not significantly different from the blank control(P < 0.05);2.detecting significantly higher KGN cell viability in the transfected miR-194-inhibitor group and significantly lower KGN cell viability in the transfected miR-194-mimic group using CCK-8(P < 0.05);3.Cell proliferation ability and apoptosis level were detected using Ki-67 and flow cytometry.In the miR-194-inhibitor group,cell proliferation ability was increased and apoptosis level was decreased;after transfection with miR-194-mimic,KGN cells proliferation ability was decreased and apoptosis level was significantly upregulated,and their expression of apoptosis-related proteins such as P53,P21 and P16 were significantly upregulated(P < 0.05);4.In KGN cell double luciferase reporter gene assay,after cotransfection with HB-EGF-WT and overexpression of miR-194 vector,luciferase activity was reduced compared with HB-EGF-WT + miR-NC blank control;in contrast,after cotransfection with HB-EGF-Mut and overexpression of miR-194 vector,luciferase activity was reduced compared with HB-EGF-Mut + miR-NC control did not produce significant changes(P < 0.05);5.the expression level of HB-EGF was significantly reduced in KGN cells overexpressing miR-194;however,there was some upregulation of HB-EGF expression level in cotransfected miR-194 + HB-EGF,and in flow cytometry,compared to the miR-194 overexpression only group,after cotransfection of miR-194 mimic + HB-EGF the apoptosis rate of the cells was decreased(P < 0.05).Conclusion1.miR-194 can inhibit the ability of KGN cells to grow and proliferate and promote the effect of apoptosis level.2.There is a direct targeting relationship between miR-194 and HB-EGF,and upregulation of HB-EGF can reverse the role of overexpressed miR-194 in KGN cell growth and apoptosis.