Effects Of Follicular Fluid-Derived Exosomal MiRNA-143-3p And MiRNA-155-5p On Glycolysis In Granulosa Cells Of Patients With Polycystic Ovary Syndrome

Insufficient glycolytic energy supply of follicular granulosa cells(GCs)is an important cause of follicular dysplasia in patients with polycystic ovary syndrome(PCOS).Follicular fluid(FF)-derived exosomal miRNAs have been shown to broadly regulate the function of GCs.To explore whether FF-derived exosomal miRNAs are involved in the regulation of GCs glycolysis.This study used clinical patient FF-derived exosome transcriptome sequencing(RNA-seq)and in vitro cell simulation experiments.Screening found that human FF-derived exosomal miRNA-143-3p and miRNA-155-5p antagonistically regulate glycolysis in granulosa cells of PCOS patients.The specific results are as follows:1.Isolation and characterization of FF-derived exosomes:This study recruited infertility patients with PCOS factors(PCOS group,n=6)and non-PCOS factors infertility patients(Control group,n=6)based on the Rotterdam Consensus(2003).By measuring the baseline hormone levels of the two groups of patients,the results showed that luteinizing hormone(LH),anti-Mullerian hormone(AMH),testosterone(T),and antral follicle count were clinical features that were significantly up-regulated in the PCOS group.Ovulation induction was performed with a short-acting and long-term protocol and bilateral follicular fluid was collected by puncture.The exosomes were collected by ultracentrifugation.The exosomes were identified by transmission electron microscopy(TEM)and nano-tracking(NTA).There are cup-mouthed vesicle exosomes with a diameter of 100-150 nm.2.RNA sequencing analysis of differentially expressed exosomal RNAs :Through RNA-seq data analysis,this study screened 83 differential miRNAs(44 up-regulated,39 down-regulated)and 337 differential m RNAs(120 up-regulated,117 down-regulated)in the PCOS group.Random RNAs real-time quantitative PCR(RT-q PCR)results showed a consistent discrepancy trend that was highly correlated with RNA-seq.The results of KEGG pathway enrichment analysis of differential RNAs showed that there were significant differences in the down-regulation of glycolysis-related pathways and the up-regulation of apoptosis-related pathways in FF-derived exosomes in the PCOS group.3.Screening of miRNA-m RNA targeting relationships regulating glycolysis in GCs : The differentially expressed miRNA-143-3p and miRNA-155-5p in RNA-seq were screened by miRBD,Target Scan and RNAhybrid to find potential targets of glycolysis key genes(HK2,PKM2 and LDHA).After verification by dual luciferase reporter gene experiments,the results showed that HK2 is a key gene of miRNA-143-3p and miRNA-155-5p regulating glycolytic pathway.4.Effects of PCOS environment on glycolysis and apoptosis in Human ovarian granulosa tumor cell line(KGN cells): KGN cells was cultured by using high glucose medium and testosterone-containing high glucose medium to simulate the in vivo environment of patients in Control group and PCOS group.This study found that compared with the Control group,the expression of miRNA-143-3p was significantly up-regulated in the PCOS group,the expression of miRNA-155-5p was down-regulated but not significantly,the glycolysis intensity of KGN cells was significantly reduced,and the apoptosis rate was significantly increased.5.Effects of miRNA-143-3p and miRNA-155-5p differential expression systems on glycolysis and apoptosis of KGN cells in different environments : By constructing differential expression systems of miRNA-143-3p and miRNA-155-5p in Control and PCOS environments,this study found that up-regulated miRNA-143-3p inhibited the glycolysis of KGN cells and knocking down miRNA-143-3p can significantly alleviate the down-regulation of glycolysis in KGN cells of PCOS.Down-regulated miRNA-155-5p attenuated the activation of glycolysis in KGN cells,and overexpression of miRNA-155-5p could significantly promote glycolysis in KGN cells of PCOS.Taken together,FF-derived exosomal miRNAs significantly inhibited GCs glycolysis and accelerated apoptosis in PCOS patients.FF-derived exosomal miRNA-143-3p and miRNA-155-5p regulate glycolysis in GCs of PCOS patients via HK2 antagonism.