Study On The Role Of Long Noncoding RNA-dependent Epigenetic Regulation In Premature Ovarian Insufficiency

Chapter I Study on the role and mechanism of lncRNA HCP5 in Premature Ovarian InsufficiencyObjective:Premature ovarian insufficiency(POI),defined as a cessation of menstruation prior to the expected age of menopause at 40,mainly manifested as abnormal menstrual,elevated follicle-stimulating hormone,and decreased estrogen levels.POI is one of the most ordinary reproductive endocrine disorders,which affects approximately 1%?5%of women of childbearing age.To our knowledge,POI is believed to be an intricate and multistep process involving both genetic and epigenetic alterations during the formation of primordial follicles pool to premature follicle depletion.Of all the possible pathogenic reasons,genetic causes account for approximately 20%?25%of patients.Moreover,chromosomal abnormalities,autoimmune,infectious,and iatrogenic factors have also been recognized as major risk factors.Nevertheless,the majority of etiology remains unknown.Thus,detailed elucidation of the molecular mechanisms involved in POI is imperative.Long non-coding RNAs(lncRNAs)are a group of non-protein-coding RNAs that are longer than 200 nucleotides and poorly conserved identified by the accumulating data from large-scale transcriptome studies.Increasingly,studies suggest that lncRNAs modulate the expression of protein-coding genes in a variety of tissues and organs by altering chromatin modification,transcription,mRNA decay,protein subcellular localization or other key processes like that.Beyond the well-established etiologic mechanism,lncRNAs may have a functional role and provide an additional layer of complexity on the etiology of POI.Recently,dysregulated expression of IncRNAs was discovered in ovarian cortical tissues from POI patients which might be critical for the pathogenesis of POI whereas a better knowledge of this class of regulatory factors in the etiology of human POI has yet to be determined.Methods:The differential expression of lncRNAs in granulosa cells(GCs)derived from women with and without POI were analyzed using microarray and further verified by qRT-PCR.Functionally,KGN,COV434,and SVOG cell lines were cultured for DNA damage repair assays and apoptosis detection in the presence or absence of lncRNA HCP5 shRNA in vitro.Next,we examined the expression of HCP5 neighboring protein-coding gene MSH5 in both mRNA and protein levels by means of qRT-PCR and Western Blot after knockdown of HCP5.Mechanically,subcellular localization of HCP5 was confirmed by Fluorescent in situ Hybridization(FISH)and nuclear/cytoplasmic RNA fractionation.RNA pull-down assay combined with mass spectrometry to seek the protein partner of HCP5.And RNA-binding protein immunoprecipitation using specific antibody followed by qRT-PCR confirmed HCP5-protein binding in KGN cells.Chromatin immunoprecipitation followed by qRT-PCR was used to study the occupancy on the promoter region of MSH5 gene.Results:In this study,we identified a down expressed lncRNA HCP5 in GCs from biochemical POI patients,which impaired DNA damage repair and promoted apoptosis of GCs.Mechanistically,we discovered that HCP5 stabilized the interaction between YB1 and its partner ILF2,which could mediate YB1 transferring into the nucleus of GCs.HCP5 silencing affected the transporting of YB1 into the nucleus and reduced the binding of YB1 to the promoter of MSH5 gene,thereby diminishing MSH5 transcription.Conclusion:Collectively,we identified that the decreased expression of HCP5 in bPOI contributed to dysfunctional GCs by regulating MSH5 transcription and DNA damage repair via the interaction with YB1,providing a novel epigenetic mechanism for POI pathogenesis.Chapter ? Study on the role and mechanism of lncRNA GCAT1 in Premature Ovarian InsufficiencyObjective:Premature ovarian insufficiency(POI),defined as a cessation of menstruation prior to the age of 40 years,mainly manifests with irregular menstruation,elevated follicle-stimulating hormone(FSH>25 U/L),and estrogen deficiency.POI is one of the most common reproductive endocrine disorders,which affects approximately 1%?5%of women of childbearing age.POI encompasses three stages in clinic,i.e.,occult,biochemical and overt(formerly called premature ovarian failure)stage.Patients with biochemical POI(bPOI)typically have regular menstruation,but elevated FSH levels and reduced fertility.The etiology of POI is clinically challenging involving genetic,autoimmune,metabolic,and infectious factors.However,the pathogenesis remains to be elucidated in most cases.Of all the causes,genetic defects account for about quarter of patients,including chromosomal abnormalities and gene mutations.Causative genes of POI have been extensively studied to date in coding regions with alterations to disrupt protein function.However,the protein-coding region only accounts for 1.5%of the whole human genome.The noncoding RNAs,including microRNAs,long noncoding RNAs(IncRNAs),and circRNAs,have recently begun to be explored in ovaries and human diseases.Recently,twenty differentially expressed lncRNAs have been discovered in the ovarian cortex from POI patients,which suggested that lncRNAs may be involved in the maintenance of ovarian function.However,the mechanism of lncRNAs contributing to human POI has yet to be determined.In this chapter,we planned to explore aberrant expressed lncRNAs in granulosa cells of bPOI patients and clarified the role and mechanism of these molecules.Methods:Two independent cohorts in this study included a total of 34 bPOI patients and 34 age-and body mass index-matched control women who underwent in vitro fertilization or intracytoplasmic sperm injection and embryo transfer.Ovarian GCs samples were collected from all participants and processed according to standard procedure.The differential expression of lncRNAs in granulosa cells(GCs)derived from women with and without POI were analyzed using microarray and further verified by qRT-PCR.Functionally,the granulosa-like tumor cell line,KGN cells were cultured for cell proliferation assays and cell cycle analysis in the presence or absence of lncRNA GCAT1 in vitro.Further FISH,RNA pull-down and RIP assays were performed to reveal the mechanism of GCAT1 in detail.Results:We previously explored the global expression profile of lncRNAs in Chapter I.After initial screening,the top seven most differentially expressed IncRNAs were confirmed by qRT-PCR.The Pearson Correlation Analysis showed that only LINC02690 was significantly correlated with FSH and AMH.Therefore,due to its potential pathological and clinical value,we hence named it as Granulosa Cell-Associated Transcript 1(GCAT1).Moreover,the functional study established an outcome of silencing GCAT1 in inhibiting GCs proliferation accomplished by G1/S cell cycle arrest.Mechanically,we revealed that GCAT1 competes with p27 mRNA for binding to PTBP1,therefore regulating p27 translation and cell proliferation.Conclusion:Study in this chapter on functional lncRNA GCAT1,in women with bPOI,suggested that the influence of GCAT1-deficiency on granulosa cell proliferation and G1/S cell cycle progression is mediated by p27 greatly.These data indicated the pivotal role of lncRNA in granulosa cells,therefore folliculogenesis and provide a novel contribution to the etiology of POI.