Study On The Mechanism Of MiR-204-5p/lnc-GULP1-2:1/COL3A1 In Synergistic Regulation Of Premature Ovarian Failure

Premature ovarian failure(POF)refers to the premature failure of female ovarian reserve function.The etiology of POF remains elusive,among which genetic factor is one of the most important factors in the occurrence of POF.Previous researches on the genetic etiology of POF were mainly focused on the protein-coding genes.However,protein-coding genes could only explain the etiology of POF in less than 1% of those patients.In the human genome,97% of the deoxyribonucleotide sequences are not encoded into protein.Most of these deoxyribonucleotides sequences are transcribed into non-coding RNA(ncRNA).Recent studies have shown that these ncRNAs are widely involved in the occurrence and progression of the diverse diseases,and they are more important for the early diagnosis and treatment of related diseases than the coding genes.However,the roles of ncRNA including microRNA(miRNA)and long non-coding(lncRNA)in the co-regulation of ovarian follicular development and the occurance of POF are unclear.Competitive endogenous RNA(ceRNA)is not a new kind of RNA molecules,but rather a class of RNA transcripts competitively binding to mi RNA,including lncRNA,messenger RNA(mRNA),circular RNA(circRNA),transcripts of pseudogenes.Those RNA transcripts bind to miRNA by miRNA response element(MREs),thereby relieving the "gene silencing" effect of miRNA on mRNA.In addition,when these transcripts bind to miRNA,they also occupy the binding sites where other mi RNA transcripts with the same MREs bind,so the expressions of these transcripts are interdependent.The ceRNA hypothesis provides a new perspective to understand the etiology of POF.In this study,lncRNA,miRNA and mRNA expression profiles in ovarian tissues of POF patients and the remaining normal ovarian tissues of patients with benign ovarian tumors after surgical removal and pathological examination of the tumors were detected by transcriptome sequencing technology.The CeRNA regulatory pathway was constructed by bioinformatics method and molecular biology method.The CeRNA regulatory pathway was studied in mutilple tissues and granulosa cells to explore the potential role of miRNA-lncRNA-mRNA co-regulatory network in the pathogenesis of POF.Objective:To investigate the mechanisim of lncRNA as ce RNA involved in the regulation of mRAN by miRNA by comparing the differentially expressed lncRNAs,miRNAs,mRNAs between ovarian tissues of POF patients and the remaining normal ovarian tissue of patients with benign ovarian tumors after surgical removal and pathological examination of the tumors using the transcriptome sequencing technology and,and to preliminarily explore the role of the RNA regulation network in the occurrence of POF.Methods:The differentially expressed lncRNAs,miRNAs,mRNAs were screened in the ovarian tissues from 5 POF patients and 5 control patients.Based on the sequencing results and data from bioinformatics database,GeneOntology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to investigated the functions and signal pathwaya of the differentially expressed RNA.The regulation network of the differentially expressed RNA and regulation pathway in which lncRNA as ceRNA invovled in the regulation of mRNA by miRNA were investigated.By using real-time PCR(real time PCR),cell proliferation and hormone detection,the mechanism of lnc-GULP1-2: 1 involved in the regulation of COL3A1 by miR-204-5p and the pathway in which miR-204-5p / lnc-GULP1-2: 1 / COL3A1 involved in the occurrence of POF were preliminarily studied.Results:1.RNA transcriptome analysis showed that there were 20 differentially expressed lncRNAs(12 lncRNAs up-regulated,8 lncRNAs down-regulated),95 miRNAs(39 miRNAs up-regulated,56 miRNAs down-regulated)and 56 mRNA(35 mRNA up-regulated,21 mRNA down-regulated).2.GO analysis showed that the differentially expressed RNA may be involved in hormone response,extracellular matrix,cellular aging and proliferation and other biological processes which are closely related to ovarian function.KEGG analysis revealed that the differentially expressed RNAs were involved multiple signaling pathways,such as the TNF signaling pathway,the PI3K-Akt signaling pathway,the MAPK signaling pathway,and the ovarian steroid production,which are closely related to ovarian follicular development3.Expression correlation analysis showed that multiple miRNA-lncRNA-mRNAs had a strong expression correlation.4.In a variety of cells and tissues,there was a positive correlation between the expresion level of lnc-GULP1-2:1 and COL3A1,whereas the expresion level of miR-204-5p was negatively correlated with that of lnc-GULP1-2:1 and COL3A1.5.The results of real time PCR,cell proliferation and hormone detection showed that miR-204-5p could inhibite the expression of lnc-GULP1-2:1 and COL3A1;miR-204-5p could inhibite the proliferation of ovarian granulosa cancer cell line KGN;miR-204-5p could inhibite both CYP19A1 expression and estrogen production.Conclusion:There are a large number of differentially expressed RNA between the ovarian tissue of POF patients and normal fertility women,including 20 lncRNAs,95 miRNAs and 56 mRNA.Bioinformatics analysis showed that those differentially expressed RNA are mutual regulated,which coluld regulate the developmental process of ovarian follicles,thus involving in the occurrence of POF.Among which,lnc-GULP1-2:1 may affect the regulation of COL3A1 by miR-204-5p,thus participating in the occurrence of POF.