The Role Of Long Non-coding RNAs In The Pathogenesis Of Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome(PCOS),one of the most common metabolic and endocrine disorders in premenopausal women.PCOS is characterised by signs and symptoms of hyperandrogenaemia,chronic oligo/anovulation,and polycystic ovarian morphology on exclusion of other specific diagnoses.PCOS is commonly associated not only with ovarian dysfunction such as abnormal folliculogenesis,anovulation,and female infertility,but also frequently with metabolic disorders such as abdominal adiposity,hirsutism,insulin resistance,obesity and type 2 diabetes.The pathology of PCOS is complex.As a heterogeneous syndrome,PCOS aetiology remains largely unknown.Both genetic and environmental factors including diets and lifestyle might contribute to the pathogenesis of this multigenic disorder.Ovarian follicles are formed from oocytes,surrounding granulosa cells,and theca internal cells.Follicular atresia,a common process in female mammalian ovaries,constitutes the degeneration of follicles at any stage of growth and development.In women with PCOS,more subsequent and ripening atretic follicles were observed indicating abnormal follicular development.Granulosa cell apoptosis is the major cause of follicular atresia,inducing DNA degradation and caspase activation,contributing to aberrant folliculogenesis.Thus,the abnormality of granulosa cell apoptosis might participate in the pathogenesis of PCOS follicular development,leading to anovulation and infertility.Insulin resistance,one of the typical clinical features of PCOS,is defined as an impaired ability for insulin to regulate metabolic actions such as glucose production,glucose uptake,and/or lipolysis,resulting in a requirement for increased amounts of insulin to achieve metabolic actions.Insulin resistance and compensatory hyperinsulinism contribute to hyperandrogenemia and ovulatory disturbances leading to the arrest of cell proliferation and follicle growth in PCOS.In women with PCOS,insulin resistance tends to aggravate over time and is associated with the development of obesity and type 2 diabetes.Moreover,the associated metabolic disorders,obesity,and type 2 diabetes have recently become among the most important long term concerns in PCOS and warrant increased attention.Long non-coding RNAs(lncRNAs)are a type of noncoding RNAs(nc RNAs)defined as transcripts longer than 200 nucleotides.LncRNAs constitute vital factors in a variety of biological processes such as cell growth,proliferation,apoptosis,development,and differentiation.These lncRNAs exert their functions via transcriptional and post-transcriptional regulation,chromatin modification,recruitment of epigenetic modifiers,ce RNA networks,and control of m RNA decay.LncRNAs have been reported to being involved in folliculogenesis including ovarian cumulus expansion,luteinisation,and oocyte development and maturation.In the past decades,emerging evidences have suggested that lncRNAs are participated in pathological process of PCOS.There are general three aspects of lncRNAs in PCOS: the first deals with cell growth,proliferation,and apoptosis(e.g.LINC-01572:28 and lncRNA HCG26);the second aspect relates to steroid-genesis,-secretion,and receptor function(e.g.lncRNA SRA and CTBP1-AS);and the third aspect is involved in metabolic processes(e.g.lncRNA RP11-151A6.4 and lncRNA GAS5).Despite those findings,the potential roles of lncRNAs and underlying mechanism in PCOS still remains poorly understood and needs to be further clarified.Part 1.The role of Lnc-CCNL1-3:1 in pathology of follicular atresia and insulin resistance in women with PCOSAims: In our previously work,the lncRNAs expression profile microarray analysis of granulosa cells in women with PCOS demonstrated that Lnc-CCNL1-3:1(CCNL)expression was significantly increased.We aim to identify the differently expressd CCNL and explore its regulatory function and underlying mechanisms in PCOS.Methods: 1.We recruited patients diagnosed as PCOS basing on the Revised Rotterdam Diagnostic Criteria.Non-PCOS patients were women with regular menstrual cycles(25 to 35 days),no endocrine abnormalities,and normal ovarian morphology confirmed by ultrasound.Ovarian granulosa cell samples were collected from patients who underwent in vitro fertilisation(IVF)or intracytoplasmic sperm injection(ICSI)treatment.2.Clinical variables,such as age,weight,body mass index(BMI),and select biochemical parameters such as follicle-Stimulating hormone(FSH),luteinising hormone(LH),testosterone(T),and anti Müllerian hormone(AMH)were recorded and analysed.3.The CCNL expression levels in human luteinised granulosa cells(h LGCs)derived from women with and without PCOS were analyzed via quantitative real-time polymerase chain reaction(q RT-PCR).4.The immortalised human granulosa KCN cell line and h LGCs were cultured for 72 hours following transfection with plasmid DNA(Enter or CCNL)and/or short interfering RNAs(si RNAs)(si-Ctrl or si-FOXO1).The successfully infected cells were then therefore used for futher experiments.5.The CCNL roles in cell apoptosis were examined through annexin V/propidium iodide(PI)staining followed by flow cytometry,Terminal deoxynucleotidyl transferase d UTP nick end labelling(TUNEL)staining,and immunoblotting.Western blotting was used for measurement of relatived proteins expression including cleaved and total poly-ADP-ribose polymerase(PARP),cleaved and total caspase3,B cell lymphoma-2(Bcl-2),and BCL2-associated X(Bax).6.The CCNL functions in glucose transport ablity were verified by glucose uptake assay and immunoblotting.Western blotting was applied for measurement of relatived proteins expression such as insulin receptor substrate-1(IRS-1)and glucose transporter 4(GLUT4).7.The CCNL effects on cell mitochondrial function were evaluated through the detection of reactive oxygen species(ROS)generation levels and adenosine triphosphate(ATP)levels.8.The abundant of FOXO1 was analyzed via q RT-PCR and western blotting in CCNL-overexpressed KGN cells and h LGCs.9.Then,CCNL function was further confirmed by the rescue assays to silence FOXO1.In addition,the subcellular fractionation was used to detect the intracellular localization of CCNL and relative proteins.RNA immunoprecipitation(RIP),and immunofluorescence were used to further verify the CCNL interact with FOXO1.Findings: 1.There is no significant difference in regard to age and BMI between women with PCOS and the control group.Women with PCOS exhibited significantly higher serum levels of LH,testosterone,and AMH compared with healthy women.2.We validated that CCNL is highly expressed in the granulosa cells of women with PCOS via r RT-PCR.Moreover,the CCNL expression was significantly positively associated with human serum testosterone levels in PCOS.The expression of CCNL was increased concomitant with higher BMI levels and homeostasis model assessment of insulin resistance(HOMA-IR)values in women with PCOS.3.In vitro,the overexpression of CCNL in KGN cells and h LGCs up-regulated FOXO1 expression,promoted cell apoptosis,reduced glucose uptake,and impaired mitochondrial function.4.The study of underlying mechanism revealed that elevated CCNL bind with FOXO1 to regulate its transcriptional activity by preventing FOXO1 exclusion from the nucleus.5.In addition,the rescue experiements were conducted.The silence of FOXO1 can partially abolished CCNL overexpression-mediated effects on cell apoptosis and glucose transport ablity.Conclusions: In Part 1,We determined that CCNL might serve as a facilitator in the processes of PCOS.The dysregulation of CCNL impaired mitochondria in granulosa cells therefore induce cell apoptosis,and reduce glucose uptake.In conclusion,we suggest that CCNL was elevated in PCOS and might thereby participate in PCOS pathologies such as follicular atresia and insulin resistance.Part 2.The construction of insulin resistance-associated lncRNA-m RNA network in women with PCOSAims: We plan to explore the potential functional lncRNAs in women with PCOS accompanied by insulin resistance and indentify the therapeutic target and noval biomakers for PCOS.Methods: 1.Associated gene expression profiles of PCOS with insulin resistance were searched in Gene Expression Omnibus(GEO)database and normalized via “affy” package in R software.BLASTn program were used to re-annotate HG-U133 probe.RNA-binding protein(RBP)-lncRNA and RBP-m RNA interactions data were obtained from star Base v3.0(http://starbase.sysu.edu.cn/).2.Significance analysis of microarrays(SAM)was applied to discover differentially expressed genes(DEGs)between the PCOS and healthy groups,accomplished via “siggenes” package in R software.The R software package “Venn Diagram” were used to describe the relationship between DEGs.3.By performing a hypergeometric test,a Global triple network including RNA-binding protein,m RNA,and lncRNAs,an insulin resistance-associated lncRNA-m RNA network(IRLMN),was constructed from star Base and GEO.To investigate the cross-talks between m RNAs and lncRNAs on IRLMN,R package“pheatmap” was applied to performed bidirectional hierarchical clustering.4.To identify the potential function and biological meaning of those genes,DAVID 6.7 provided a comprehensive set of tools for gene ontology(GO)analysis and “subpathway Miner” package in R was used to analyze the potential genes enriched in the the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.5.Combined with former weighted gene co-expression network analysis(WGCNA)results,we.indentify several hub lncRNAs,The expressions of hub lncRNAs were further validated in another independent GEO dataset.6.We recruited patients with and without PCOS followed the same criteria in Part 1,methods 1.Clinical variables,such as age,weight and biochemical parameters such as LH,FSH were also collected and analayzed.Ovarian granulosa cell samples were collected from patients who underwent IVF or ICSI treatment.Finally,the expression level of hub lncRNAs were examined and analyzed in clinical samples of PCOS group and control group.Findings: 1.We chose GSE8157 for further analysis considering the quality of data and sample size.We compared the DEGs in the skeletal muscles between 10 patients with PCOS and 13 healthy women.181 lncRNAs and 1322 m RNAs were identified.2.We constructed a novel IRLMN,containing 47 lncRNAs nodes,852 m RNA nodes,and 5705 edges.Through bidirectional hierarchical clustering on the IRLMN,we identified a highly related insulin resistance-associated module including 6 hub lncRNAs.3.We analyzed and merged those hub lncRNAs obtained from IRLMN and WGCNA.GO enrichment analysis and subpathway analysis were conducted for the hub genes in this module.RP11-151A6.4 was identified as the most possible functional lncRNA.4.The abundance of RP11-151A6.4 was examined in an independent GEO dataset(GSE20950)and ovarian granulosa cells samples.Results showed that RP11-151A6.4 expression was elevated in granulosa cells in women with PCOS compared to that in control.Moreover,the expression levels of RP11-151A6.4 were increased in PCOS patients with hyperinsulinemia,higher BMI and HOMA-IR values.Besides,RP11-151A6.4 was highly expressed and subcutaneous and omental adipose tissues of patients with insulin resistance.Conclusions: In Part 2,We provided the differently expressed lncRNA and m RNA profiles in women with PCOS associated insulin resistance compared to healthy controls.We indentified the RP11-151A6.4 as a hub lncRNA basing on IRLMN and WGCNA.RP11-151A6.4 was highly expressed in skeletal muscle,ovarian granulosa cells,and subcutaneous and omental adipose tissues of patients with insulin resistance.In conclusion,we revealed that RP11-151A6.4 play a important role in insulin resistance,androgen excess and adipose dysfunction in women with PCOS.To further explore RP11-151A6.4 function and underlying mechanisms of insulin resistance in PCOS,more experiment remains to conduct.