Study On The Value Of MiR-223-3p In The Diagnosis Of Type 2 Diabetic Nephropathy

[Objective]Explore changes in plasma miRNA in patients with DKD to find miRNAs that have a diagnostic role for DKD.[Methods](1)Three DKD patientsverifiedby the clinical diagnosiswas recruitedas the chip case groupfrom Dec.2015 to Jan.2016in the Department of Nephrology of the First People's Hospital of Yunnan Province.Anotherthreepeople who had undergone physical examination in our hospital were selected at the same time,and theywere no DMand hypertensionhistory,family history,and normal glucose tolerance and their other test results were normal controls.The general clinical data and biochemical indicators of the study subjects were collected;(2)Agilent miRNA microarray and Affymetrix HTA2.0 LncRNAs microarray analysis were performed simultaneously on plasma samples from 3 DKD patients and 3 normal controls;(3)The co-expression analysis was used to co-express and analyze the differentially expressed miRNAs and LncRNAs on the two chips,and the miRNAs with distinct expression in plasma were screened out;(4)RT-qPCR assay was used to detect the expression of miRNAs in the plasma after co-expression analysis.[Results](1)Agilent miRNA results showed that there were 88 miRNAs with significant changes in plasma,of which 47 were up-regulated and 41 were down-regulated;127 lncRNAs were identified by Affymetrix HTA 2.0 chip results,of which 101 were up-regulated and 26 were Down-regulation;(2)Co-expression analysis of microarray differences evaluated that the expression of miR-223-3p and miR-106b-5p was most significantly different;(3)RT-qPCR detectedmiR-223-3p and miR-106b-5p expression in3 cases and 3 controls..The miR-223-3p amplification curve was smooth,with the Ct value<35,and the dissolution curve was a single sharp fluorescence peak.The amplification curve of miR-106b-5p was rough with a Ct value>35.Compared with the control group,the miR-223-3p level in the blood of the case group decreased significantly(P=0.05).[Conclusion]The chip co-expression analysis showed that compared with the normal control group,the miRNA expression in the plasma of the case group changed significantly,and the co-expression coefficient of the differential expression of miR-223-3p was the most obviuosly,whichsuggestedthat miR-223-3p may play an important rolein developmentand progressof DKD.[Objective]To understand the expression of miR-223-3p in plasma and urine of different stage DKD patients,and to investigate the diagnostic value of miR-223-3p in DKD.[Methods](1)We collected 180 patients with type 2 diabetes mellitus who were admitted to the Department of Endocrinology and Senile endocrinology of the First Affiliated Hospital of Kunming Medical University from May 2016 to December 2017.The subjects were divided into two types by randomized ACR:type 2 60 patients with diabetes non-nephropathy group(DM group);60 patients with diabetes microalbuminuria group(Micro-DKD group);60 patients with diabetes macroproteinuria group(Macro-DKD);60 patients with normal control group(Control group);(2)The general clinical data and biochemical parameters of all subjects were collected.The expression levels of miR-223-3p in plasma and urine of different groups were detected by SYBR Green fluorescent dye RT-qPCR in plasma and urine;(3)SPSS 19.0 software was used to analyze the expression of miR-223-3p in plasma and urine of each group and other indicators.Logistic regression analysis was performed and the receiver operating curve(ROC curve)was plotted.(4)miR-223-3p target genes were predicted online using Target Scan,miRanda,PITA,and miRDB byBioinformatics method.The results were converged byusing Venny intersections and screened through literature search for target genes that are associated with the development of DKD.[Results](1)Compared with the control group,miR-223-3p plasma expression levels in the DM group,Micro-DKD group and Macro-DKD group were significantly reducedgradually;and with the severity of the disease exacerbated,miR-223-3p declinationwas statistically significant(P<0.01);(2)Compared with the Control group,the expression level of miR-223-3p in the urine of the case group showed a downward trend,but there was no statistical difference among the groups(P>0.05);(3)By Logisitic regression analysis,it was found that ACR is a risk factor for DKD,and miR-223-3p is a protective factor for DKD(P<0.01,OR=1.1,0.48);(4)The ROC curve was plotted for the expression of miR-223-3p in plasma and urine.The AUC>0.7 in plasma had DM/Control,Micro-DKD/Control,Macro-DKD/Control,DM/Macro-DKD.There was a statistically significant difference between Micro-DKD/Macro-DKD(P<0.01)and AUC in each group of urine<0.7,and there was no significant difference among the groups(P>0.05);(5)Target gene prediction resultsfromfour software online predicted miR-223-3p target genes,Target Scan predicts 412 target genes;miRanda predicts 2,561 target genes;PITA predicts 174 target genes,and miRDB predicts 242 target genes.Finally,19 co-expressed target genes were screened using Venny intersections.It was found that miR-223-3p may participate in the occurrence and development of DKD by regulating the target genes IL6ST and PRKCE.[Conclusion:1(1)The expression level of miR,223-3p in plasma of DKD patients decreased significantly,and the severity of the disease progressed.The expression level of miR-223-3p in plasma was expected to become a new diagnosis markers for DKD.;the significance of miR-223-3p expression In urine need to be further verified byenlarge samples size;(2)ACR is a risk factor for DKD;miR-223-3p is a protective factor for DKD;(3)miR-223-3p may participate in the development of DKD by regulating the target genes IL6ST and PRKCE.