Bioinformatics Analysis Of MiRNA-mRNA Regulatory Networks In Neuropathy Of Type 2 Diabetes

ObjectiveThe expression differences of blood micro RNA(miRNA)in diabetic peripheral neuropathy patients were analyzed by bioinformatics,and further verified in diabetic peripheral neuropathy patients,so as to explore the role of miRNA in the pathogenesis of diabetic peripheral neuropathy.Methods(1)Retrieve the keyword of “neurologic complications of type 2diabetes ”from the GEO database of NCBI,and select the human data gene chip for analysis.According to the source of samples,the microarray was divided into three groups:(1)healthy control group(CN,n=6),(2)Diabetic group without peripheral neuropathy(NDPN,n=6),(3)Diabetic peripheral neuropathy group(DPN,n=6).The classified data were divided into three groups.The analysis tool GEO2 R was used for comparative analysis of DPN vs CN and DPN vs NDPN,and the differentially expressed genes(DEGs)were screened.(2)Functional and pathway enrichment analysis was performed for the differentially expressed genes.(3)Protein Protein Interaction network analysis was performed on the differentially expressed genes.(4)Correlation of the screened differentially expressed key genes with diabetic peripheral neuropathy was predicted in the database.(5)The locked DEGs related to diabetic peripheral neuropathy obtained in step(4)were used to predict the targeted miRNA relationships in the micro RNA database,and further analysis and discussion were made on the possible roles of miRNAs that have targeted binding relationships with the locked genes in the pathogenesis of DPN,and the miRNA-mRNA regulatory network was constructed.(6)In this study,7 healthy control patients,8 diabetic patients without neurological complications,and 8diabetic peripheral nerve patients were collected.RT-PCR was used to analyze the expression of the identified key genes to further verify the results of bioinformatics analysis.Results(1).Chip analysis shows that in the adj.P.Value<0.05 &|log2FC|>2,Compared with healthy controls,there were 1367 plasma differentially expressed mRNAs in diabetic peripheral neuropathy patients,of which 1127 were up-regulated and 240 were down-regulated.Compared with diabetic patients without neurological complications,161 mRNAs were differentially expressed in plasma of diabetic peripheral neuropathy patients,among which 79 mRNAs were up-regulated and 82 mRNAs were down-regulated.(2).Through the analysis of the function of enrichment,is closely related to the development of DPN occurred three terms,respectively is "ganglioside catabolic process","cellular response to oxidative stress" and "oligosaccharide catabolic process ",and then from the three terms in the five key genes(NEU4 HEXB PRDX3,ZC3H12 A and SLC25A24).(3).Through protein protein interaction(PPI)network analysis,15 proteins with the closest interaction relationship were obtained,and 3 key genes(CCR2,CXCL5 and RB1)were identified through literature research.(4).Through the CTD database,the correlation between genes and the disease of diabetic peripheral neuropathy was predicted,and the two genes,PRDX3 and CXCL5,were finally targeted with the Score of diabetic neuropathy > 15 as the boundary.(5).According to bioinformatics analysis,2 up-regulated mRNAs(PRDX3 and CXCL5)and 10 corresponding targeted miRNAs were targeted to establish a miRNA-mRNA regulation network,namely CXCL5 and hsa-miR-616-3p,hsa-miR-8063,hsa-miR-6728-3p,hsa-miR-4748,hsa-miR-3529-3p and hsa-miR-4284.PRDX3 and hsa-miR-501-5p,hsa-miR-181A-5p,hsa-miR-26a-5p and hsa-miR-383-5p.Current studies have shown that PRDX3 and the corresponding targeted hsa-miR-181A-5p and hsa-miR-26a-5p are most likely to be related to the incidence of diabetic peripheral neuropathy,so the above genes were verified in this study.(6).The RT-PCR results of PRDX3,hsa-miR-181A-5p and hsa-miR-26a-5p showed that the expression of PRDX3 in the diabetic peripheral neuropathy group was higher than that in the healthy control group and the diabetic group,and there was a significant difference between the diabetic peripheral neuropathy group and the diabetic group(P<0.01).Hsa-miR-26a-5p was decreased in healthy control group,diabetic group and diabetic peripheral neuropathy group.The expression level of hsa-miR-181a-5p in diabetic peripheral neuropathy group was lower than that in healthy control group and diabetic group.The results of small sample clinical validation experiment are consistent with the results of bioinformatics analysis.ConclusionsIn this study,it was found that mRNA-miRNAs(PRDX3 and hsa-miR-181-a-5p,hsa-miR-26a-5p)differentially expressed in DPN patients compared with CN and NDPN.Through bioinformatics studies,it was found that the expressions of PRDX3 and CXCL5 were increased in patients with DPN,and the corresponding miRNA was decreased.The genes(PRDX3,hsa-miR-181A-5p,hsa-miR-26a-5p)found to be most likely related to the incidence of DPN were verified.It was proved that the results of clinical case verification were consistent with the results of bioinformatics,suggesting that the above genes play a certain role in the pathogenesis of diabetic peripheral neuropathy and may be involved in the occurrence and development of DPN.