Research On The Regulation Of The Establishment Of Early Diabetic Nephropathy Cycle MICRORNA Fingerprinting And Related MIRNA Gene Promoter Methylation

Objective To establish the Circulating microRNAs (miRNAs)fingerprinting in early diabetic nephropathy (DN), and to provide the basisfor the early molecular diagnosis and treatment of diabetic nephropathy.Methods Three cases of each early diabetic nephropathy anddiabetes’fresh blood were collected by PAXgene blood RNA tubes,totalRNA was extracted from the fresh blood using Trizol reagent.YM-100micro-centrifugal filter column enriched small RNA fragments which areless than300nt, hybridized with RNA microarray which containing thelatest version of the probe sequence(version10.0the microRNA databasehttp://microrna.sanger.ac.uk/sequences/) and dyed by specific fluorescentdyes after modificated..After a software analysis and dataprocessing,obtained the differential expression profile of early diabeticnephropathy and diabetes. Using TaqMan MicroRNA Assaysquantitative detect the expression level of let-7a in six samples to verifythe results reliability of RNA chip. Results The detection results of RNA chip displayed that22miRNAwere differential expressed between early diabetic nephropathy anddiabetic.Among them there were ten miRNAs which were significantdifferential expressed,within5miRNAs(hsa-miR-4429, hsa-miR-4454,hsa-miR-320e,hsa-miR-320d,hsa-miR-320b) were upregulated and5miRNAs(hsa-let-7f, hsa-miR-363, hsa-let-7a, hsa-let-7d, hsa-miR-26a)were downregulated.The expression levels of let-7a detected byquantitative RT-PCR confirmed the differences in the microarrayresults,indicating that the chip results are reliable.Conclusion Part of the circulating miRNAs expression in earlydiabetic nephropathy and diabetic patients were significant different,andthe establishment of circulating miRNA fingerprint in early diabeticnephropathy may provide new ideas for the early molecular diagnosis andtreatment of diabetic nephropathy. Objective To detect let-7a-3gene promoter methylation level inwhole blood DNA of early diabetic nephropathy (DN group), diabetesmellitus (DM group) and normal (CON group) and explore the effect ofgene promoter methylation on let-7a expression.To provide new clues forthe study of molecular mechanisms that regulate the development of earlydiabetic nephropathy.Methods Let-7a-3gene promoter methylation rate of PCR productsfrom20whole blood DNA samples of DN group, DM group and CONgroup was detected by fluorescence quantitative methylation specific PCR(qMSP),and the accuracy of the PCR reaction was verified by agarose gelelectrophoresis and sequencing.Results QMSP showed that DN group of let-7a-3gene promotermethylation average rate was96.2%,DM group was76.6%, and normalgroup was63.2%.After statistical analysis, DN group average methylationrate was significantly higher than DM group and normal group (P<0.05),but DM group increased not significantly (P>0.05) compared with normal group.Conclusion Let-7a-3gene promoter methylation rate of early diabeticnephropathy Patients was significantly increased, which affected theexpression of let-7a-3gene and decreased let-7a expression (just as RNAchips and quantitative RT-PCR analysis confirmed).It may be one of themolecular mechanisms that modulates the occurrence and development ofearly diabetic nephropathy.