The Mechanism Of MiR-335-5p Regulating Proliferation Of Granulose Cells By Targeting SGK3 In Polycystic Ovary Syndrome

Background Polycystic ovary syndrome(PCOS)is a clinical syndrome with metabolic abnormalities and reproductive disorders in reproductive-aged women.It is the most common endocrine disorder with an approximate incidence of 5% to 10% in women.The etiology of PCOS is not yet clear.Many studies suggested that the etiology of PCOS is related to genetic and environmental factors in past decades,whereas little is known about its exact etiology.PCOS is characterized by the persistent presence of abnormal folliculogenesis in ovary.PCOS has heterogeneity and its pathological changes are very complicated,including abnormal follicular development,proliferation and apoptosis of granulosa cells,hypertrophy of theca cells,hyperandrogenism and chronic ovulation disorders. Ovarian follicular development mainly includes the growth of dominant follicle,the division of granulosa cells and the expansion of follicular basal layer.Finally,follicular fluid is formed in the follicular sinus.Human follicular fluid can provide an important microenvironment for oocyte development.It contains many substances including proteins,metabolites and ionic compounds,which are closely related to the maturation and quality of the oocyte.The latest study showed that human follicular fluid contains not only these substances,but also contains microRNAs(miRNAs). miRNAs are single-stranded,small non-coding RNAs approximately 19-26 nucleotides in length,which participate in transcriptional and post-transcriptional regulation of genes and bind to the 3’ untranslated region(3’UTR)region of target mRNAs to negatively regulate their expression.They are involved in a variety of regulatory pathways,and associated with many diseases such as cancer,metabolic diseases and so on.Furthermore,miRNAs are also widely involved in the process of follicle maturation,fertilization,embryo implantation and early embryonic development,which play an irreplaceable role in maintaining the normal fertility function of female.At present,there is little research on miRNA in human follicular fluid of PCOS patients.Therefore,we aimed to explore the abnormal expression of miRNAs in human follicular fluid of patients with PCOS,and what are the important roles of these abnormal miRNAs in the pathogenesis of PCOS? Aims:1.We aim to explore the differential expression of miRNAs in follicular fluid of women with PCOS.2.Our study also aims to investigate the role of differential miRNAs played in pathological defects in PCOS,such as dysfunction of granulosa cells or abnormal folliculogenesis.Methods:Firstly,we collected follicular fluid of IVF / ICSI assisted pregnancy in PCOS patients and control group at Reproductive Center of Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and found the difference by miRNA high-throughput sequencing The expressed miRNAs were verified by quantitative real-time polymerase chain reaction(qRT-PCR).Bioinformatics analysis software was then used to identify the differentially expressed miRNA biological functions,related signaling pathways and possible target genes.Finally,the effects of miR-335-5p and its target genes on the proliferation of granulosa cells were verified by Western blot,in vitro cell culture and transfection,dual luciferase reporter system and cell counting kit-8(CCK-8).Results:A total of 19 miRNAs were differentially expressed in both groups.Three miRNAs were upregulated and 16 miRNAs were downregulated in PCOS group.MiR-335-5p expression was significantly decreased in follicular and granulosa cells of PCOS group(P <0.01)by RT-PCR.Follicular fluid The expression level of miR-335-5p in follicular fluid was significantly negatively correlated with AFC,AMH and TT(ρ =-0.623,P = 0.001;ρ = 0.570,P = 0.01,p =-0.708,P = 0.001).Serum / glucocorticoid-inducible kinase family member 3(SGK3)is the target gene of miR-335-5p;the mRNA and protein levels of SGK3 in PCOS group were significantly higher than those in control group(P <0.05);miR-335-5p mimics can inhibit KGN cell proliferation,and reduce the phosphorylation of Akt and mTOR.Conclusions:In PCOS patients,low expression of miR-335-5p in follicular fluid is related to its endocrine abnormality;low expression of miR-335-5p in granulosa cells can promote the proliferation of granulosa cells by up-regulating the expression of SGK3,and its mechanism of action is related to PI3K-Akt-mTOR signaling pathway,which may lead to over-recruitment of PCOS follicles and maturation disorders.This study provides a new molecular target for dysfunction of granulosa cells in PCOS.