Study On The Regulatory Role And Mechanism Of Long Non-coding RNA In The Pathogenesis Of Premature Ovarian Insufficiency

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Study on the role and mechanism of lncRNA DDGC in Premature Ovarian InsufficiencyBackground:Premature ovarian insufficiency(POI)affects about 1%women of reproductive age.Decline of ovarian function in patients with POI progresses rapidly and is irreversible in most cases,and there is a lack of effective treatment in clinical practice.POI not only leads to infertility but also increases the risk of a variety of chronic diseases,leading to increased all-cause mortality.Therefore,the study of etiology is of great significance for early warning,diagnosis and intervention of POI.Previous studies on the pathogenesis of POI mainly focused on the protein-coding region,but transcriptome studies have confirmed that more than 98%of the transcripts of the human genome are non-coding RNAs,more than half of which are long noncoding RNAs(lncRNAs).However,the regulatory role of lncRNA in follicular development and the pathogenesis of POI is still poorly understood.Exploring the role of lncRNA in the epigenetic regulation is one of the new directions in the etiology study of POI.Objective:To clarify the expression profile of lncRNA in granulosa cells(GCs)from patients with POI,and further explore the regulatory role and mechanism of differentially expressed lncRNA in follicular development and POI pathogenesis,and provide new epigenetic regulation evidence for the exploration of the pathogenesis of POI and provide a research basis for the clinical diagnosis and treatment of POI.Methods:First,GCs from 8 patients with biochemical POI(bPOI)and 9 age/BMI matched healthy women were collected,and transcriptome sequencing analysis was performed to identify differentially expressed lncRNAs in GCs from patients with POI.The RNA-seq results were verified by qRT-PCR validation.After silencing of lncRNA DDGC in human GC-derived KGN cell lines,the RNA-seq was performed to observe the effect of DDGC down-regulation on the overall function of GCs.The regulatory roles of lncRNA DDGC on GC proliferation,cell cycle progression,DNA damage repair,apoptosis,and hormone synthesis were investigated by functional experiments in KGN and SVOG cell lines.The regulatory mechanism of lncRNA DDGC in follicular development and POI was clarified by nucleoplasmic separation,fluorescence in situ hybridization,RNA binding protein immunoprecipitation(RIP),RNA pulldown,and other experimental methods.Finally,DDGC was over-expressed by adenovirus in mouse ovaries to observe the regulatory effect of DDGC on ovarian function in vitro and in vivo.Results:1.LncRNA DDGC is significantly down-regulated in bPOI GCs.RNA-seq found that 78 lncRNAs were significantly differentially expressed.qRT-PCR confirmed that lncRNAs AC112721.1,ZNF674-AS1,and DDGC were significantly downregulated,and lncRNAs GS1-358P8.4,SNAI3-AS1,and RP11-3D4.3 were significantly up-regulated.Among them,lncRNA DDGC had the largest foldchange and showed a relatively high expression in ovary.2.LncRNA DDGC binds with miR-589-5p to regulate the expression of RAD51.KGN RNA-seq analysis combined with qRT-PCR validation suggested that DDGC down-regulation resulted in an abnormal DNA damage repair process in GCs.Functional experiments showed that DDGC silencing significantly prolonged the time to repair DNA damage and promoted the apoptosis.Further studies showed that DDGC silencing resulted in decreased RAD51 expression and impaired homologous recombination repair process.RAD51 over-expression can rescue the delay of DNA damage repair caused by DDGC silencing,suggesting that RAD 51 is a key target gene of DDGC in regulating DNA damage repair of GCs.DDGC was mainly distributed in the cytoplasm.DDGC silencing accelerated RAD51 mRNA degradation.RIP assay showed that DDGC was specifically bound to AGO2 protein,and silencing RAD51 also down-regulated the expression of DDGC,suggesting that DDGC regulates the degradation of RAD51 mRNA by adsorbing miRNA.Bioinformatics prediction combined with functional experiments showed that overexpression of miR-589-5p down-regulated DDGC and RAD51.Functional experiments confirmed that miR-589-5p binds with DDGC and RAD51 mRNA 3'UTR.In conclusion,DDGC regulates the stability of RAD51 mRNA and the process of homologous recombination repair of GCs through direct binding with miR-589-5p.3.LncRNA DDGC regulates the stability of WT1 protein and the differentiation of GCs by interaction with HSP90.KGN RNA-seq analysis identified the differential expression of genes related to estrogen synthesis in GCs,suggesting that DDGC silencing led to abnormal differentiation of GCs.Functional experiments showed that DDGC silencing resulted in elevated estradiol synthesis and up-regulated expression of FSHR and CYP19A1 in GCs.Furthermore,results showed that DDGC silencing led to down-regulation of WT1 expression,and the over-expression of WT1 could reverse the effect of DDGC down-regulation on GC differentiation,suggesting that DDGC regulates the GCs differentiation through down-regulating WT1.Further mechanism studies showed that DDGC silencing resulted in decreased stability and increased ubiquitination of WT1 protein.RNA pulldown and RIP experiments showed that DDGC directly bound with HSP90 in GCs,and inhibition of HSP90 activity also resulted in increased ubiquitination of WT1 protein.Immunofluorescence showed that HSP90 co-localized with WT1 in GCs.DDGC silencing had no impact on the expression of HSP90,but significantly reduced the binding efficiency between HSP90 and WT1 in GCs.In summary,DDGC regulates the stability of WT1 protein and differentiation of GCs through directly binding with HSP90.4.Over-expression of DDGC promoted the DNA damage repair of mice ovaries by up-regulating Rad51.DDGC over-expression ameliorated the etoposide-induced DNA damage and apoptosis,however,had no impact on the WT1 ubiquitination of in vitro cultured mice ovaries.Over-expression of DDGC by adenovirus upregulated the expression of Rad51 but did not change the expression of WT1,FSHR,and CYP19A1 in vivo.Furthermore,DNA damage in mice ovaries was induced by intraperitoneal injection of etoposide,and it was found that over-expression of DDGC significantly reduced the DNA double-strand breaks and apoptosis in mice ovaries.These results suggested that DDGC promotes the DNA damage repair of mice ovaries by up-regulating Rad51.Conclusion:LncRNA DDGC plays an important role in DNA damage repair and differentiation of GCs.Low-expressed DDGC leads to decreased stability of RAD51 mRNA and WT1 protein in GCs,resulting in impaired homologous recombination repair,increased apoptosis and abnormal differentiation of GCs,which may lead to follicular atresia and POI eventually.Chapter ? Study on the role and mechanism of lncRNA ZNF674-AS1 in Premature Ovarian InsufficiencyBackground:Ovarian follicle is the basic female reproductive unit which consist of oocyte and two types of somatic cells,granulosa cells(GCs)and theca cells.During the process of follicular development,GCs mediates the ordered activation,growth,and maturation of follicles through cellular junctions and paracrine pathways,thus maintaining the normal reproductive function under physiological conditions.By providing amino acids,cholesterol,glucose,and glycolytic products,GCs can improve the efficiency of oocyte protein synthesis,promote the acquisition of embryonic development potential of oocytes,and meet the high energy metabolism demand of oocytes during the development process.Restricted proliferation,abnormal differentiation,and defective DNA damage repair of GCs have been shown to contribute to follicular atresia and POI.Therefore,more and more attention has been paid to the relationship between GC status and POI development.As transcripts with diverse functions and regulatory mechanisms,lncRNAs are involved in the regulation of physical process from early cellular development to final senescence.Recent studies have shown that certain lncRNAs are involved in the maintenance of GCs proliferation,hormone synthesis,but the regulatory role of lncRNAs on GCs status and POI development is still lacking in detailed functional and mechanistic studies.Objective:Based on the study in the previous chapter,continuous exploration was conducted to clarify the regulatory role and mechanism of differentially expressed lncRNAs in follicular development and POI pathogenesis,and to provide new research evidence for the involvement of lncRNAs in the occurrence and development of POI.Methods:Based on the transcriptome sequencing in the previous chapter and the qRTPCR validation,the correlation analysis was conducted between the expression levels of lncRNAs and the commonly used evaluation indicators of ovarian function in clinical practice.LncRNA ZNF674-AS1 was silenced in human GC-derived KGN and COV434 cell lines by antisense nucleotide probe,and the effect of ZNF674-AS1 on GCs proliferation was observed by CCK-8 and EdU staining.The glycolytic aldolase activity,glycolytic products,and ATP levels of GCs were detected by the commercial kits.The expression of ZNF674-AS1 were detected after cell metabolic pressure induced by glucose-free medium,and the regulatory effect of lncRNA ZNF674-AS1 on GCs glucose metabolism was also observed.Finally,the specific regulatory mechanism of lncRNA ZNF674-AS1 was elucidated by nucleoplasmic separation,RNA pulldown,mass spectrometry,RIP,and immunofluorescence techniques.Results:1.LncRNA ZNF674-AS1 is significantly down-regulated in GCs from patients with bPOI and is associated with ovarian reserve.qRT-PCR detection was performed in GCs from 33 patients with bPOI and 41 age/BMI matched healthy women,and the results showed that lncRNA ZNF674-AS1 was significantly down-regulated in GCs from patients with bPOI.And the expression levels of ZNF674-AS1 was positively correlated with the basal estradiol and AMH,and negatively correlated with the basal FSH,suggesting that the expression level of ZNF674-AS1 is correlated with ovarian reserve.2.The regulatory role and mechanism of lncRNA ZNF674-AS1 in proliferation and glycolysis of GCs.Functional experiments showed that ZNF674AS1 silencing significantly reduced the cell viability and proliferation of KGN and COV434 cells,but did not affect the ability of estrogen synthesis.Nucleoplasmic isolation combined with qRT-PCR revealed that ZNF674-AS1 was mainly distributed in the cytoplasm,suggesting the post-transcriptional regulation of ZNF674-AS1 function.RNA pulldown combined with mass spectrometry indicated that ZNF674-AS1 was bound to ALDOA protein.Further RIP assays showed that ZNF674-AS1 was significantly enriched by ALDOA antibodies compared with IgG,confirmed the direct binding of ALDOA protein to ZNF674-AS1.ZNF674-AS1 silencing had no impact on the expression and subcellular localization of ALDOA,however,significantly reduced the cleavage activity of ALDOA in KGN and COV434 cells.Silence of ZNF674-AS1 significantly reduced the concentrations of fructose 1,6-bisphosphate,and lactic acid,indicating that insufficient expression of ZNF674-AS1 inhibited the glycolysis process of GCs.Silence of ZNF674-AS1 resulted in decreased ATP production and significantly increased ADP/ATP ratio,indicating that insufficient expression of ZNF674-AS1 resulted in the defective energy metabolism of GCs.In addition,metabolic stress induced by glucose deficiency significantly up-regulated the expression of ZNF674AS1 in GCs.These results suggest that ZNF674-AS1 is necessary for the glycolysis of GCs.3.LncRNA ZNF674-AS1 regulates GC proliferation through the ALDOA/vATPase-dependent AMPK activation pathway.Silence of ZNF674-AS1 significantly increased the phosphorylation level of AMPK and its downstream ACC proteins in GCs,suggesting that insufficient expression of ZNF674-AS1 promoted the activation of AMPK.Abnormal activation of AMPK also inhibited the viability and proliferation of GCs,suggesting that down-regulation of ZNF674-AS1 may inhibit GC proliferation by activating AMPK.Immunoprecipitation experiments showed that silencing of ZNF674AS1 promoted the binding of ALDOA to ATP6V1B2,and silencing of ATP6V1B2 significantly inhibited the abnormal activation of AMPK caused by insufficient expression of ZNF674-AS1.In addition,functional experiments also showed that ATP6V1B2 silencing could reverse the inhibitory effect of ZNF674-AS1 deficiency on GC viability and proliferation.In summary,the low-expressed ZNF674-AS1 inhibited GCs proliferation by promoting ALDOA/v-ATPase-dependent AMPK activation.Conclusion:LncRNA ZNF674-AS1 is necessary for GCs proliferation and glycolysis.The direct binding of ZNF674-AS1 to ALDOA maintains the process of glycolysis and normal proliferation of GCs,and ensures the normal development of ovarian follicles.