The Role Of Long Noncoding RNA In The Dysfunctional Folliculogenesis And Insulin Resistance Of Polycystic Ovary Syndrome

BackgroundPolycystic ovary syndrome(PCOS),a common endocrine disease among reproductive-aged women,is characterized by hyperandrogenemia,chronic oligo/anovulation,and polycystic ovarian morphology.PCOS is a leading cause of female infertility.Although women with PCOS exhibit more follicles than women without PCOS,none of these follicles develop into a dominant follicle,leading to abnormal ovulation.The granulosa cell layers surrounding these follicles show signs of atresia,degradation,and hypertrophy,indicating abnormal proliferation and/or apoptosis.The granulosa cells are essential for providing the oocyte with nutrients and growth regulators during oocyte development.Their dysfunction,therefore,may contribute to the aberrant folliculogenesis observed in PCOS.In addition,PCOS is also recognized as an important metabolic as well as reproductive disorder conferring substantially increased risk for type 2 diabetes.A high percentage of women with PCOS have marked insulin resistance(IR).Several clinical trials indicated that insulin sensitizer could benefit for the outcome of controlled ovarian stimulation treatment of PCOS patients.Furthermore,androgens contribute to insulin resistance in PCOS,but the underlying mechanism still remained obscure.Microarray analysis of tissue from women with and without PCOS identifies a significant proportion of the differentially expressed transcripts in PCOS as non-coding RNAs.Non-coding RNA,especially long non-coding RNA(lnc RNA),have important potential regulatory effects on gene expression.lnc RNAs,which are defined as transcripts longer than 200 nucleotides without coding potential,play a crucial role in cell development,differentiation,proliferation,and apoptosis via interactions with RNA-binding proteins,chromatin modification,and ce RNA networks.Previous studies have demonstrated that lnc RNAs may be involved in the etiology of PCOS.For example,Huang et al.demonstrated different microarray expression patterns of lnc RNAs and m RNAs in cumulus cells isolated from patients with and without PCOS.Liu et al.also found differential expression of lnc RNA-HCG26 in women with PCOS,which may influence the proliferation and steroidogenesis of the granulosa cells.The lnc RNAs that involved in the metabolism disorder such as SRA were also aberrantly expressed in the tissue of PCOS.However,the molecular mechanism underlying the involvement of lnc RNAs in disordered folliculogenesis and insulin resistance in PCOS remains unclear.Part 1 Microarray analysis of granulosa cells from women with and without PCOSAims:1.We conducted microarray analysis to identify differentially expressed protein-coding genes and lnc RNAs expression profiles in luteinized granulosa cells obtained from women with and without PCOS.2.We planned to identified aberrantly expressed lnc RNAs and explore the underlying mechanism around candidate genes.Methods:Long non-coding RNA(lnc RNA)expression in luteinized granulosa cells(h LGCs)derived from women with and without PCOS were analyzed using microarray and q RT-PCR.Immortalized human granulosa cell lines were cultured for proliferation assays after transfection with the LINC-01572:28 over-expression vector in the presence or absence of p27 si RNA.Protein expression analysis,rescue assays,and RNA immunoprecipitation(RIP)were used to confirm the LINC-01572:28 substrate.Results:LINC-01572:28 and p27 protein were elevated whereas proliferating cell nuclear antigen protein was decreased in the h LGCs of women with PCOS.LINC-01572:28 expression was positively correlated with basal testosterone levels.Over-expression of LINC-01572:28 inhibited cell proliferation and impeded G1/S transition,which were partially reversed by si RNA-mediated p27 knockdown.Conclusion:Our findings,therefore,suggest that LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via SKP2 binding.Part 2 Data mining of insulin resistance related lnc RNA invovled in the pathogenesis of PCOSAims:This study aimed to screen critical long non-coding RNAs(lnc RNAs)that might play pivotal roles in insulin resistance therefore to provide candidate biomarkers potential treatment target of PCOS.Methods:Gene expression profiles in skeletal muscle of PCOS patients accompanying insulin resistance and the healthy control were obtained from the publicly available Gene Expression Omnibus(GEO).After re-annotation,we performed WGCNA analysis on the differential expressed genes(DEGs)between the women with and without PCOS to identified hub lnc RNAs therefore validated the former result.Results:lnc RNA LINC02239:2 was identified as the hub lnc RNA in the insulin resistance related module and was significantly elevated in the skeletal muscle of women with PCOS.Besides,the expression of LINC02239:2 in the skeletal muscle was decreased after the regular treatment of pioglitazone in PCOS patients.Conclusion:Our results showed that lnc RNA LINC02239:2 might play a vital role in the insulin resistance and further study concerning LINC02239:2 could shed new light to the understanding underlying mechanisms of insulin resistance.Part 3 Long non-coding RNA LINC02239:2 suppress Hepatic glucose transport and fat oxidation in PCOS patients with insulin resistanceAims:This study aimed to explore the underlying mechanism of long non-coding RNA LINC02239:2 involved in the insulin resistance of PCOS patients.Methods:Hep G2 cell lines were cultured for glucose uptake assays,oil red O and nile red staining after transfection with the LINC02239:2 over-expression vector.Protein expression analysis,q RT-PCR and chromatin immunoprecipitation(Ch IP)were used to confirm the molecular mechanism of LINC02239:2.Results:The ability of glucose uptake was damaged after transfection with LINC02239:2 over-expression vector in Hep G2 cells,as well as elevated lipid accumulation.Furthermore,GLUT4(the insulin-regulated glucose transporter gene)and CPT1B(a mitochondrial enzyme involved in fatty acid metabolism)is down-regulated in LINC02239:2 over-expressed Hep G2 cells.Interestingly,this non-coding gene can be downregulated by PPAR-? agonist.Further research revealed that not only rosiglitazone but also metformin could reduce the expression of LINC02239:2 due to regulation on androgen receptor activity or expression respectively.Conclusion:LINC02239:2 suppress hepatic glucose transport and fat oxidation and can be regulated by androgen receptor in PCOS patients with insulin resistance.