Screening,Verification Of Differential Expression And Ovarian Specific LncRNA And Correlation Signal Pathway Analysisbefore And After Initiation Of Primordial Follicles In Mice

Background:The size of the primordial follicle pool is the decisive factor in the female reproductive lifespan,and the main factors affecting the size of the primordial follicle pool and the consumption rate of primordial follicle initiation,so in-depth study of mechanisms of primordial follicles initiation and development is important to slow ovarian aging and extend the female reproductive life.In twenty-first Century,with the completion of the human genome project,the regulation and control of genes for the process of life and the occurrence and development of diseases has been recognized and valued by more and more people.RNA,as the intermediate bridge between DNA and protein,is rich in content and complex in function.It is a very large group.RNA can be divided into 2 major categories,encoding genes and non coding genes.In recent years,many studies have shown that the content of non coding RNA accounts for about 98% of the whole transcriptome.It plays a very important role in regulating the process of organism’s life.LncRNA is a nucleotide sequence of more than 200 long chain non encoding RNA,which participates lots of physiological and pathological processes in body,many researchers have confirmed that lncRNA can influence human cumulus cell expansion and oocyte maturation and early embryo development.However,the role of lncRNA in the initiation of primordial follicles has not yet been reported,nor the relationship between ovarian specific lncRNAs and primordial follicle activation related signaling pathways is well studied.Objective:1.Constructed lncRNA expression profiles before and after primordial follicles activation.2.Screened and verified the ovarian specific lncRNA that differentially expressed and differentially expressed before and after primordial follicle initiation.3.Analyzed the relationship between lncRNAs and primordial follicle initiation related signaling pathways.Methods:1.Establish the culture model of primordial follicles in vitro,and high throughput sequencing was used to detect the expression of lncRNAs before and after initiation of primordial follicles,and the expression profiles of lncRNAs were constructed.2.Analyze the differentially expressed lncRNAs by bioinformatics.(1)GO enrichment analysis: the difference of lncRNAs target genes was analyzed by Functional enrichment;(2)KEGG enrichment analysis: pathway analysis of differential lncRNAs target genes.3.Real-time fluorescent quantitative PCR technology was used to verify the differential expression of lncRNAs in the sequencing results(random selection of 10),so as to further verify the reliability of the sequencing results.4.Construction of LncRNA-mRNA co-expression network: Based on cis-trans principle,we screened out differentially expressed and ovarian specific mRNA and lncRNA located in initiation of primordial follicles related signaling pathways of primordial follicles,and constructed mRNA-lncRNA co-expression network with targeted relationship.5.Real-time fluorescence quantitative PCR and conventional PCR technology were used to detect differentially expressed and ovarian specific lncRNAs in primordial follicle related signaling pathways,and select the key lncRNAs.Results:1.Compared with the control group,there were 8676 differentially expressed lncRNAs and 5966 differentially expressed mRNAs in the experimental group.In the differential expression of lncRNAs,there were 6541 up-regulated and 2135 down-regulated expressions.Of the differentially expressed mRNAs,there were 4171 up-regulated and 1795 downgrades.2.The results of biological information analysis;(1)Through GO enrichment analysis,5336 items were obtained,including: immune system process,cell differentiation,cell activation,cell proliferation regulation,angiogenesis,gene expression regulation,cell cycle regulation,cell division,signal transduction and other functional items.(2)Through pathway analysis,290 signal transduction pathways were obtained,including TNF signaling pathway,MAPK signaling pathway,Notch signaling pathway,PI3K-Akt signaling pathway,Wnt signaling pathway,p53 signaling pathway,Hippo signaling pathway,TGF-beta signaling pathway and so on.3.Real time fluorescence quantitative PCR validation was performed on 10 randomly selected lncRNAs expression.The results showed that q-PCR verification results were consistent with the expression results of sequencing results.4.In the KEGG database,10 with the original follicle initiation pathway,were related to PI3K-Akt signaling pathway,MAPK signaling pathway,cAMP signaling pathway,Wnt signaling pathway,TNF signaling pathway,Hippo signaling pathway,AMPK signaling pathway,Notch signaling pathway,TGF-beta signaling pathway and p53 signaling pathway.The 10 signal paths are enriched with 375 mRNA.According to the cis-trans principle,there are 854 mRNA corresponding lncRNA in target.Among them,there were 67 differentially expressed and ovarian specific lncRNA,corresponding 69 mRNA,and Cytoscape software was used to construct the co expression network of differentially expressed primordial follicle related signal pathway and ovarian specific lncRNA and corresponding mRNA.5.Real time fluorescence quantitative PCR and ordinary PCR showed that 6 differential expression and relative ovarian specific lncRNAs in Hippo signaling pathway,PI3K-AKT signaling pathway,TGF-beta signaling pathway and P53 signaling pathway.Conclusions:1.A large number of lncRNAs expressed differently before and after the initiation of primordial follicles in mice.Some of these differentially expressed lncRNAs were ovarian specific.2.Of the lncRNAs,which had differential expression and ovarian specificity before and after priming of the primordial follicles,some of them were closely related to the signal pathway related to the priming of the original follicle.