Protein Interaction Network Associated With Diabetic Nephropathy And Rack1 Promotes The Expressions Of Inflammatory Cytokines In Diabetic Nephropathy

Diabetic nephropathy(DN)is a severe diabetic microvascular complications and the major cause of end-stage renal disease.It'spathogenesis is so complicated thatit is differentto clarify that by the study of a single gene.In recent years,with the rise of systems biologyand the accumulation of enormous high throughput data,applying molecular network to study genetic relationship is possible and the method from the perspective of complex network to understand the development of the disease arises at the historic moment.How to search for the gene that may play a key role in the oneset and development of a diseases and find the drug targetthrough molecular network is the key problem about the study complex diseases.In this study,the protein interaction network is the core and some key problems about the selection of key genes in DN are studied.Firstlybased onRNA-seq results and protein database constructed the protein interaction networks(PPIN),selected nine genes may play an important role in the network by systems biology approach.Finally by comparing the expression level and conservative of predicted genes,we chooseRack1 andstudy it's role in the regulation of inflammatory cytokines expression in DN.Through decades of research,more and more evidence indicates that DN involves the mutation of many genes,and its pathogenesis involves the comprehensive change of cellular molecular networks.From the perspective of complex network,the screening of key genes is the essential problem of network analysis.The rapid construction of DN related PPIN(DNPPIN)can be achieved by integrating the differences of the gene expression associated with DN and the information of protein interaction.The first part of the paper proposes a method for identifying key genes based on gene expression differences and correlation.Based on this method and the network topological correlation index,nine genes that maybe signifisant in the development of DN were found.Recent studies have found that continued high glucose environment can induce the occurrence of inflammation,cause the excessive secretionof immune cells and inflammatory factors,and play a key role in promoting development of the occurrence of DN.The activation of PKC is closely related to the occurrence of DN,and the role of Rack1 which as the activation receptor of PKC in the process of DN has attracted our attention.The second part of the thesis mainly discusses the regulation process of Rack to the expression of DN inflammatory factors.To sum up,through the analysis of protein interaction networks,we find some genes participate in the development of DN and studied the effect of Rack1 on the expression of inflammatory factors.Part ? The construction analysis and verification of the protein interaction network of diabetic nephropathyObjective:To detect the differential expression gene of diabetic nephropathy and construct a protein interaction network associated with diabetic nephropathy.To Screen for genes that may play a key role in the development of diabetic nephropathy.Methods: Next-Generation Sequencing(NGS)was used to identify the different expression of mRNA in DNmice;mouse protein interactions was downloaded fromSTRING database;BioMart was employed to transform informations between different databases;"R" and Cytoscapen were applied to Constructed and visualized DNPPIN;Network Analyzer and CytoNCA were used to Calculated the centrality parameters of Network(Degree,Betweenness,Closeness,Eigenvector);"R" was emplied toAnalyzed the Robustness of network;MCODE was used to recognized the module of the network;KOBAS was applied to completed the GO and KEGG pathway analysis of the first-ranked module in DNPPIN;q RT-PCR was used to verified the expression differences of genes in renal tissue and 4 kinds of renal cells.Results:Ninepredicted genes associated with DN were obtained.The mRNA relative expression trend of 9 predicted genes in renal tissue was identical with the RNA-seq(p<0.05).The mRNA relative expressions of Rack1 and Polr2 e are consistent in 4 kinds of renal cells,and Rack1 was the most up-expression with high conservative were higher,which provided the basis for further analysis.Conclusions:By constructingDN related protein-protein interaction network can screen out the genes that may play an important role in the occurrence and development of DN.Rack1 is highly conservative,and the up-expression of Rack1 is found in renal tissuses of DN mice and four renal cells,which may play an important role in the occurrence and development of DN.Part ? Rack1 promotes the expression of inflammatory cytokines in diabetic nephropathyObjective:To explore the difference expression and basic characteristics of candidate gene Rack1 in kidney tissue of diabetic nephropathy mice and four kinds of renal cells under high glucose cultureand to explore the effects of Rack1 on the expression of inflammatory factors in Mesangial cell(MC)in mice with high glucose culture.Methods:Western blot assay was used to detect the protein expression level in glomerular mesangial cells(MC)under low-or high –glucose condition.Over-expression plasmid of pc DNA3.1(+)-Rack1 was constructed and Rack1 si RNA weredesigned and synthesized.Then the pc DNA3.1(+)-Rack1 and Rack1 si RNA were transfected respectively into MC under low-or high-glucose condition.q RT-PCR was employed to detect the m RNA levels of inflammatory cytokices.The protein levels of inflammatory cytokices were detected by ELISA.Results: Western blot results showed that the expression levels of Rack1 was significantly higher in the high-glucose cultured glomerulus mesangial cells than in low glucose condition(p<0.05).After the over-expression plasmid pc DNA3.1(+)-Rack1 was transfected into the MC under low glucose condition,the expressions of inflammation factors TNF-? and MCP-1 were enhanced with L-MC mock or L-MC pc DNA3.1 group(p<0.05).In addition,after the Rack1 si RNA were transfected into the high-glucose cells,the relative expressions of inflammation factors TNF-? and MCP-1 were decreased compared with H-MC or H-MC si RNA groups.The results of ELISA analysis were consistent with q RT-PCR.Conclusion:The expression of Rack1 was significantly increased in DN in vivo and in vitro(p<0.05).Rack1 is involved in the regulation of inflammatory of mesangial cells from diabetic nephropathy mouse(p<0.05).