Comprehensive Analysis Of Differentially Expressed Genes Associated With Diabetic Peripheral Neuropathy Based On Microarray Technology

Background: Diabetic peripheral neuropathy(DPN)is a common complication associated with diabetes mellitus with a pathogenesis that is incompletely understood.LncRNAs and miRNAs are reported to be linked to various biological processes,and play vital roles in disease occurrence and development.However,the relationship between miRNAs or lncRNAs and the progress of DPN still lacks a thorough exploration.Our study was divided into two parts,to study their importance to the pathophysiological processes of DPN.Part one: Differentially expressed miRNAs and target mRNAs in dorsal root ganglia from streptozotocin-induced diabetic ratsPurpose: To identify the diabetic peripheral neuropathy(DPN)-related miRNAs and target mRNAs.Methods: Twenty healthy eight-week-old male Sprague-Dawley(SD)rats,weighing 180-220 g,were purchased and randomly divided into diabetic and control groups(n = 10 per group).After a 12-hour fast,the diabetic group rats were induced by a single intraperitoneal injection of streptozotocin at a dose of 65 mg/kg body weight,the controls received an injection of an equal volume of vehicle(citrate buffer)alone.Seven days afterSTZ injection,nonfasting blood glucose levels were measured on tail vein blood to confirm diabetic status,and the diabetic group rats with glucose levels less than 16.7 mM were excluded from this study.Rats were kept for 8 weeks following STZ injection,then we checked whether the diabetic rats had developed DPN using two methods(Electronic Von Frey test and electron microscopy examination).Subsequently,the rats were killed and bilateral L3-L6 DRGs were collected to extract total RNA.The Affymetrix GeneChip miRNA 4.0 Array and Affymetrix GeneChip Rat Transcriptome Array 1.0 were employed to identify the differentially expressed miRNAs and mRNAs,respectively.The miRanda database was utilized to predict the potential target genes of the differentially expressed miRNAs.The intersection genes between differentially expressed mRNAs and predicted target genes were chosen,and then we collected those which were inversely correlated with their predicted miRNA matches.We analyzed the collected mRNAs through GO analysis and pathway analysis,to identify the significant functions and signaling pathways.The miRNA-gene-network analysis was performed to identify the key genes in the regulatory network.Results: One rat from diabetic group was excluded from this study because the glucose level did not meet our criterion.The withdrawal threshold of diabetic rats had decreased significantly relative to that of control rats.Electron microscopy examination indicated that the sciatic nerve from diabetic group exhibited lamellar separation and demyelination.Through microarray analysis,37 miRNAs and 1357 mRNAs were found to be significantly differentially expressed in the diabetic group.GO analysis showed a total of 399 GO terms were enriched;specifically,142 GO terms were upregulated and 257 GO terms were downregulated.According to KEGG pathway analysis,there were 23 downregulated and 6 upregulated enriched pathways.The miRNA-mRNA-network showed that rno-miR-330-5p,rno-miR-17-1-3p and rno-miR-346 had a high degree of connectivity;meanwhile,Podxl and Hoxa1 were the most common target mRNAs.Conclusion: We successfully induced DPN rats,and identified the differentially expressedmiRNAs and mRNAs in DRGs.Through GO analysis,pathway analysis and miRNA-mRNA-network analysis,we found that these genes may be important in the development of DPN.Our results may be the underlying framework of future studies regarding the effect of the aberrantly expressed genes on the pathophysiology of DPN.Part two: Microarray analyses of lncRNAs and mR NAs expression profiling associated with diabetic peripheral neuropathy in rats and ceRNA network constructionPurpose: To identify the biological functions of the aberrantly expressed lncRNAs and mRNAs,and their roles in the development of DPN.Meanwhile,the ceRNA network was built.Methods: The diabetic rat induction and tissue collection were the same as the part one.The Affymetrix GeneChip Rat Transcriptome Array 1.0 was employed to identify the expression changes of lncRNAs and mRNAs.GO analysis and pathway analysis were applied to identify the biological processes and functions of the dysregulated mRNAs associated with DPN.Furthermore,we used the intersecting genes between GO analysis and pathway analysis to perform the signal-net analysis.The lncRNA-mRNA network analysis was performed based on the correlations of the aberrantly expressed lncRNAs and their co-expressed mRNAs.According to the competing endogenous RNA theory(ceRNA),we further built the ceRNA network based on the differentially expressed lncRNAs,miRNAs and mRNAs,to explore the importance of ceR NA network on the pathophysiological processes of DPN.Results: We identified 983 lncRNAs and 1357 mRNAs which were differentially expressed.GO analysis was conducted to uncover the associated functions of these aberrantly expressed mRNAs.A total of 558 GO terms were significantly enriched,including 190 upregulated GO terms and 368 downregulated GO terms.By using the pathway analysis,we identified 94 significantly enriched pathways,including 37upregulated pathways and 57 downregulated pathways.Signal-net analysis revealed that Plcd4 and Itgb3 had the highest betweenness centrality.Besides,our results indicated that integrin receptors,including the members of Itgb3,Itgb1,Itgb8 and Itga6,had the highest degrees of connectivity.The lncRNA-mRNA network included 474 lncRNAs and 169 mRNAs.There were 1426 connections in this network,the number of negative interactions and positive interactions were 569 and 857,respectively.Among this network,the lncRNA speegaw.aSep08-unspliced and the m RNAs Scd1,Map4k4 and Tyrp1 had the highest degree of centrality.The ceR NA network revealed that the sphingolipid metabolic and biosynthetic process and the pathways such as PPAR signaling pathway,IL-17 signaling pathway and Wnt signaling pathway were involved.Conclusion: Our study revealed that the aberrantly expressed lncRNAs play important roles in the pathogenesis of DPN by controlling their co-expressed mRNAs.In the meantime,the ceR NA network showed various pathophysiological process and pathways may be involved,indicating its importance to DPN.