The Mechanisms Of MicroRNA 9-5p/SIRT1 Participate In The Pathogenesis Of PCOS

BackgroundPolycystic ovary syndrome(PCOS)is the most common endocrine and metabolic dysfunction disease in women of reproductive age,mainly affecting reproductive and metabolic functions,manifested as hyperandrogenism,chronic anovulation and polycystic ovaries.This syndrome is heterogeneous associated with genetics,it positively associated with the prevalence of subclinical cardiovascular disease,insulin resistance(IR),endometrial cancer and ovarian cancer.IR and granulosa cell apoptosis play important role in the development of PCOS.And the pathogenesis is also closely affected by environmental factors,current research has shown that epigenetics is involved in the development.MicroRNA(miRNA)is a short-chain non-coding RNA that inhibits the expression of downstream target genes at the post-transcriptional level,participates in the gene expression regulation and plays key role in many biological processes,such as cell growth,differentiation,metabolism,apoptosis,hormone synthesis and angiogenesis.Many miRNAs(miR-22,miR-132,miR-141,miR-21,etc.)are differentially expressed in ovarian tissue from PCOS,thus abnormal miRNA profiles may be associated with epigenetic pathophysiological processes of PCOS.SIRT1 is a member of the Sirtuin family of histone deacetylases and plays an important regulatory role in regulating cell proliferation/apoptosis,immune response,insulin recreation and glucose metabolism.In order to improve the clinical individualized precision treatment level,studying the miRNA and its target genes has become a hotspot research.ObjectiveIn the previous study,we have shown that miRNA 9-5p is up-regulated in PCOS patients.This time we hope to find new targets for regulating granulosa cell insulin resistance and apoptosis in PCOS by studying the regulation mechanism of miR-9-5p on SIRT1.And by changing the expression of miR-9-5p,SIRT1-mediated intervention of granulosa cell insulin resistance and apoptosis can be achieved,which provides a theoretical basis for PCOS future drug intervention.Methods1?38 cases of human follicular fluid were collected for granulosa cells,including 25 cases of PCOS groups(13 cases of insulin resistance group and 12 cases of non-insulin resistance group)and 13 control group.Clinical data were retrospectively analyzed.2?The four data chips GSE34526,GSE98595,GSE10946,and GSE102293 published in the GEO database(https://www.ncbi.nlm.nih.gov/geo/)were integrated to analyze and screen differentially expressed genes.The expression levels of SIRT1 were analyzed,GO and KEGG analyses were performed.3?The differential expression of miR-9-5p in the three groups of granulosa cells was verified by real-time quantitative PCR.The differential expression of SIRT1 in the three groups of granulosa cells was confirmed by western blotting.4?The bioinformatics was used to verify that one of the downstream target genes of miR-9-5p is SIRT1.5?Western blotting and immunofluorescence experiments were used to detect the effect of miRNA 9-5p/SIRT1 pathway on granulosa cell insulin resistance,and the effects on AKT and PI3 K phosphorylation levels were observed.6?Cell proliferation assay and Tunel fluorescence staining were used to detect the effect of miRNA 9-5p/SIRT1 on granulosa cell proliferation and apoptosis.Result1?According to the HOMA-IR index,PCOS patients were divided into insulin resistance group(PCOS-IR group,HOMA-IR?2.57)and non-insulin resistance group(PCOS-NIR group,HOMA-IR<2.57).Compared with the control group,the BMI,T,LH/FSH,FPG,FINS,and HOMA-IR values were significantly higher in the PCOS group(P<0.05).The PCOS-IR group and the PCOS-NIR group were compared.The ratios of BMI,T,FPG and INS have increased and the differences were statistically significant(P<0.05).Compared with the PCOS-NIR group,the BMI,T,FPG,and INS values were significantly higher in the PCOS-IR group(P<0.05).2?After integration and analysis of four data chips,included 26 PCOS granulosa cell samples and 16 non-PCOS control granulosa cell samples,and 970 differentially expressed genes were screened.The expression of SIRT1 gene in the PCOS groups were down-regulated in each data chip(P<0.05).The KEGG pathway was enriched and screened to obtain a highly expressed PI3K/AKT cell pathway for further experimental verification.3?The expression of miR-9-5p in PCOS-IR group was higher than that in PCOS-NIR group and control group(P<0.05).The expression of SIRT1 in PCOS-IR group was lower than that in PCOS-NIR group and control group(P<0.05).4?There is a complementary pairing between miR-9-5p and the 3'-UTR of SIRT1 mRNA.Bioinformatics analysis suggests that SIRT1 is the target gene of miR-9-5p.5 ? After successful transfection of cells,it was found that miR-9-5p down-regulated SIRT1 expression at the post-transcriptional level,miR-9-5p/SIRT1 can induce insulin resistance by inhibiting PI3K/AKT cell pathway and reducing the expression of GLUT4 molecules in granulosa cells.6?miR-9-5p/SIRT1 can promote the apoptosis of granulosa cells.Conclusions1?This study demonstrates that miR-9-5p targets SIRT1 and induces granulosa cell insulin resistance by inhibiting the PI3K/AKT signaling pathway.2?Up-regulated miR-9-5p in the PCOS-IR group can promote the apoptosis of granulosa cells by inhibiting the expression of SIRT1,thereby promoting follicular atresia and leading to maturation disorders.