The Role And Mechanism Of MicroRNA-127-5p And Circular RNAs In Premature Ovarian Insufficiency

Part ⅠMicroRNA-127-5p impairs function of granulosa cells via HMGB2 gene in premature ovarian insufficiencyObjective:MicroRNAs(miRNAs)regulate gene expression posttranscriptionally and are associated with female reproductive function.A lot of evidences have uncovered that miRNAs contributes to follicle atresia through dysregulation of granulose cell proliferation,progression,and apoptosis.Distinct microRNA profiles have been revealed in premature ovarian insufficiency(POI),but their function in POI is not yet clearly reported.Herein,this study was aimed to determine whether miRNA-127-5p is involved in the ovarian dysfunction of biochemical POI and the mechanism of DNA repair deficiency of granulosa cells.Methods:Aberrant expressions of miR-127-5p and high mobility group box 2(HMGB2)were observed by microarrays in granulosa cells(GCs)from biochemical POI(bPOI)women and further confirmed by quantitative reverse transcription(qRT)-PCR.Immortalized human granulosa cell line was used for functional validation.Additionally,the expression of miR-127-5p was measured in the plasma of bPOI women.The receiver operating characteristic(ROC)curve analysis was performed to determine the indicative role of miR-127-5p for ovarian reserve.Results:The up-regulation of miR-127-5p was identified in GCs from bPOI patients.It inhibited GCs proliferation and impaired DNA damage repair capacity through targeting HMGB2,which was significantly down-regulated in GCs from the same cohort of cases.Intriguingly,the up-expression of miR-127-5p was also detected in plasma of bPOI individuals,suggesting that miR-127-5p could be a promising indicator for the diminished ovarian function.Conclusion:Our results discovered the deleterious effects of miR-127-5p on GCs function and its predictive value in POI process.The target gene HMGB2 could be considered as a new candidate for POI.This study highlights the importance of DNA repair capacity for ovarian function and sheds light on the epigenetic mechanism in the pathogenicity of POI.Part Ⅱ MicroRNA-127-5p aggravates GCs dysfunction and contributes to ovarian insufficiency in mouse in vivoObjective:The up-regulation of miR-127-5p was identified in GCs from bPOI patients.It inhibited GCs proliferation and impaired DNA damage repair capacity through targeting HMGB2.However,whether miR-127-5p could influence ovarian function in vivo is still known.Hence in this study,mouse primary GCs and orthotopic mouse model were used to explore the role of miR-127-5p.Methods:As the seed sequences of miR-127-5p are highly conserved among orthologues,mouse primary GCs was used in vitro to explore the function of miR-127-5p on cell proliferation and DNA repair.Then the 21-day-old female C57BL/6J mice were purchased and maintained in a temperature-and humidity-controlled room on a 12-hour light/12-hour dark cycle.The orthotopic ovarian mouse model was established.The miR-127-5p mimics or controls was injected into the left and right ovarian bursa by insulin syringes,respectively.Forty-eight hours after injection,mice were treated with ETO intraperitoneally to induce DNA damage.After 6 hours,the ovaries were excised and ruptured for yH2AX detection.Results:High expression of miR-127-5p inhibited the proliferation of mouse GCs.Furthermore,miR-127-5p delayed DNA repair process indicated by yH2AX detection in mouse GCs.Additionally,orthotopic mouse model was established by ovarian bursa injection with miR-127-5p mimics.After 48 hour,miR-127-5p was significantly increased and HMGB2 was remarkably decreased in ovaries injected with the miR-127-5p mimics,indicating the regulation of miR-127-5p on Hmgb2 expression in vivo.Furthermore,after intraperitoneal injection of ETO for 6 hours,more yH2AX was detected in the ovaries injected with miR-127-5p mimics,implying a severe damage was existed in vivo.No significant differences in the appearance and weight of the ovaries and the number of follicles were observed after miR-127-5p mimics injection.Conclusion:MicroRNA-127-5p aggravates GCs dysfunction via Hmgb2 and contributes to ovarian insufficiency in mouse in vivo.Part Ⅲ The role and mechanism of differentially expressed CircTRIM24 and circEMSY in POIObjective:Circular RNAs(circRNAs)are covalently closed,endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns,that have been implicated in many biological processes and diseases such as diabetes mellitus,neurological disorders and cancer.However,the contribution of circRNAs in the pathogenesis of POI remains unclear.In this chapter,we planned to delineate the circRNAs expression profile in granulosa cells and to determine the deferentially expressed circRNAs in biochemical POI patients.Methods:A total of 34 individuals with biochemical POI and 32 age-and body mass index-matched control subjects of Han Chinese ancestry were recruited.Granulosa cells of 8 patients and 9 matched controls were collected and profiled by the RNA-seq(RNA-sequencing)to identified differentially expressed circRNAs,quantification of selected circRNAs was replicated by quantitative RT-PCR.Immortalized human granulosa cell line was used for functional validation.Results:The RNA-seq was performed and identified that circTRIM24 and circEMSY were up-regulated in the GCs from bPOI patients.Both of circTRIM24 and circEMSY were revealed mainly located in the cytoplasm of GCs.Then forced expression of circTRIM24 and circEMSY suppressed GCs steroidogenesis,impaired DNA damage repair function and promoted apoptosis of GCs.Conclusion:Up-regulated circTRIM24 and circEMSY attenuate the steroidogenesis and enhanced damage-induced apoptosis of GCs,suggesting the plausible role of circRNAs involved in POI etiology.