Bioinformatics Studies On The Signaling Pathways And Genes Involved In The Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Testicular Mesenchymal Cells

Objective:RNA-seq technology and bioinformatics analysis were used to investigate the differences in the signaling pathway and gene expression of bone marrow mesenchymal stem cells(BMSCs)in their differentiation into testicular mesenchymal cells(LC),and to explore the differentiation mechanism.Methods:The purified and identified bone marrow mesenchymal stem cells(BMSCs)were inoculated in A petri dish,with group A as the control group and group B as the experimental group.The addition of bFGF+PDGF+hCG in the experimental group induced BMSCs to differentiate into LC.RNA of group A(BMSCs)at day 0(BMSCs),group B(BMSCs)at day 7(BMSCs),group B(Leydig cell)at day 7(BMSCs),group B(Leydig cell)at day 7(BMSCs),group B(Leydig cell)at day 7(BMSCs),group B(Leydig cell)at day 14(Leydig cell)at day 21(Leydig cell)were extracted for transcriptome sequencing and analyzed according to mRNA sequencing results:1.Differential screening criteria.Reads were assembled using the latest transcriptional assembly software String Tie,and the transcriptional splicing was annotated.The difference analysis of StringTie assembled and quantified mrnas was carried out with the Ballgown package of R language and the screening conditions were set as |log2FC|>2.2.Screening differentially expressed genes.Function and pathway enrichment of differentially expressed genes were analyzed based on Gene ontology database and KEGG pathway database.3.Differential expression analysis.We used the R package Ballgown for differential expression analysis and visualization.RNA expression level was mainly measured by RPKM or FPKM.Ballgown used the normalized FPKM value in the calculation of RNA expression to avoid some false positive results and the influence of high abundance expressed RNA on the expression of medium and low abundance RNA.Results:1.The testicular Leydig cell(Leydig cell)was obtained through experiments,and the morphology of each cell at 7d and 21 d in the differentiation process was consistent with the differentiation process,and the obtained Leydig cell could secrete testosterone.2.By comparing the expression data of control group A(BMSCs),experimental group(7d,21d)and LC,A total of 7546 genes were obtained,among which 356 genes were continuously up-regulated and 960 genes were continuously down-regulated.3.By further GO enrichment analysis of continuously up-regulated and down-regulated genes,these differential genes are involved in biological processes including signaling pathway,regulation of signaling pathway,macromolecular metabolism,cell cycle or cell division,etc.KEGG enrichment analysis mainly correlated with 244 key signaling pathways,among which the signals with more enrichment were: Wnt signaling pathway,fibroblast growth factor signaling pathway,BMP signaling pathway,Notch signaling pathway,etc.Among all the signal paths,the Wnt signaling pathway has the highest number of enrichment.4.By screening all genes related to the Wnt signaling pathway(130),a total of 22 genes were found to conform to the up-regulation and down-regulation trend.The genes with continuously increasing expression were Dact3,DKK3,Gli3,Ddit3,etc.,while the genes with continuously decreasing expression were Lrp1,Wnt5 a,Depdc1b,Smad3,etc.Genes with expression difference of more than 10 times were: Wnt5 a,Depdc1b,DKK3,Gli3.Conclusion:Wnt signaling pathway exists in the signaling pathway of BMSCs differentiation to LC,and the possible key genes are Wnt5 a,Depdc1b,DKK3,Gli3.