Bioinformatics Analysis Of Potential Regulatory Mechanisms About Diabetic Nephropathy

Objective:To explore the potential biomarkers and therapeutic targets for diabetic nephropathy by bioinformatics methods.Methods:1.GSE1009 microarray data was downloaded from Gene Expression Omnibus(GEO)database.The differentially expressed genes(DEGs)between DN patients and normal individuals were analyzed by GEO2 R.2.STRING database was utilized to build proteins encoded by DEGs interaction network,and visual protein-protein interaction(PPI)network diagram were constructed through Cytoscape software.Besides,the key genes of DN were calculated using cyto Hubba plugin based on cytoscape.3.GO functional annotation and KEGG pathway analysis of DEGs by using DAVID online analytical database.Meanwhile,we screened the main signaling pathways that hub genes involved.4.The regulatory miRNAs for DEGs were screened by Web-based Gene Set Enrichment Analysis Toolkit(Web Gestalt)system.Then we constructed the DEGs-miRNA visualized network in Cytoscape software,and calculated topological features of nodes in the network.At last,key miRNAs were filtered out according to the node degree of network.5.The regulatory relationship between key miRNAs and target genes in DN were builded by Cytoscape software,and verified through five kinds of online database for miRNA targets prediction.Finally,we established the potential regulatory mechanisms of diabetic nephropathy combining with the results of pathway analysis.Results:1.A total of 600 DEGs were screened out,including 257 down-regulated genes and343 up-regulated ones.2.The 15 down-and 15 up-regulated hub genes were filtered out,of which 7 were the most critical differential genes,namely VEGFA,EGFR,ITGA3,ITGAV,COL4A3,COL4A4 and COL4A5.3.GO analysis: Most of the down-regulated DEGs had molecular functions such as protein binding,integrin binding and calcium ion binding;and significantly enriched in extracellular exosome,cell surface and plasma membrane;and mainly participated in cell migration,heart development,lung development,glomerular development,extracellular matrix organization and angiogenesis.The up-regulated DEGs mainly had molecular functions including growth factor and cytokine activity,protease binding,and calcium ion binding;chiefly concentrated in extracellular region,plasma membranes,cell junction;mostly involved in cell death and proliferation,protein autophosphorylation,GTPase activity,protein import into nucleus,translocation and transcription,etc.4.KEGG analysis: The down-regulated DEGs were mainly involved in a total of 14 pathways,including ECM-receptor interaction,focal adhesion,PI3K-Akt signaling pathway,calcium signaling pathway,Rap1 signaling pathway,Erb B signaling pathway.The up-regulated DEGs were markedly enriched in 6pathways such as PI3K-Akt signaling pathway,renin secretion,neuroactive ligand-receptor interaction,cytokine-cytokine receptor interaction,etc.Among them,seven key genes mainly involved in ECM-receptor interaction,PI3K-Akt signaling pathway,focal adhesion,and Rap1 signaling pathway.5.The down-regulated DEGs were mainly regulated by 9 miRNAs clusters,respectively,mi R-506,mi R-124 a,mi R-200 family(mi R-200b/c,mi R-429),mi R-23a/b,mi R-181 family(mi R-181a/b/c/d),mi R-25/32/92/363/367 clusters,mi R-17 family(mi R-17-5p,mi R-20a/b,mi R-106a/b,mi R-519d),mi R-27a/b,mi R-29 family(mi R-29a/b/c).And the up-regulated DEGs were significantly regulated by 2 miRNAs clusters,namely,mi R-17 family and mi R-25/32/92/363/367 clusters.6.The regulatory relationship between key miRNAs and target genes showed that COL4 A and VEGFA were target genes of mi R-29,mi R-200 targeted VEGFA expression,mi R-25 targeted ITGAV expression,mi R-27 targeted EGFR expression.Finally,Combining with the KEGG pathway enrichment analysis,these key miRNA-target genes were mainly involved in signaling pathways such as ECM-receptor interaction and PI3K-Akt signaling pathway.Conclusions:This study was based on the microarray data about diabetic nephropathy from GEO database.The differentially expressed genes of DN were downloaded and screened.The potential molecular mechanisms and theoretical targets for DN were analyzed using bioinformatics methods.Eventually,we drawed the following conclusions:1.A total of 600 DEGs were successfully screened for diabetic nephropathy,of which VEGFA,EGFR,ITGA3,ITGAV,COL4A3,COL4A4,COL4A5 were expected to be biomarkers for the early detection or diagnosis of DN.2.The key miRNAs closely related to the development of diabetic nephropathy were mi R-200b/c,mi R-29a/b/c,mi R-25 and mi R-27,which mainly involved in podocyte apoptosis,extracellular matrix accumulation and kidney fibrosis.And we highlighted the significance of these miRNAs as novel therapeutic targets to prevent progression of DN.3.Mi R-29 targeted COL4 A and VEGFA expression,mi R-200 targeted VEGFA expression,mi R-25 targeted ITGAV expression,mi R-27 targeted EGFR expression,which might further regulate the development of diabetic nephropathy through ECM receptor interaction pathway and PI3K-Akt signaling pathway.