Significance Of Urine Sediment MicroRNA Expression Profile In Non-invasive Diagnosis Of Diabetic Nephropathy

BackgroundDiabetic nephropathy（DN）is one of the important microvascular complications of diabetes and is considered to be a common cause of end-stage renal disease.However,not all patients with diabetes and kidney injury are diagnosed as diabetic nephropathy.This part of patients is non-diabetic renal disease（Non-Diabetic Renal Diseases,NDRD）the other part patients are diagnosed as Non-Diabetic Renal Diseases（NDRD）,both the treatment and clinical prognosis of them are different,NDRD includes diseases such as diabetes with membranous nephropathy（MN）,diabetes with IgA Nehropathy and so on.At present,the clinical diagnosis and monitoring of two types of diseases mainly rely on renal biopsy and urine microalbumin detection.However,renal biopsy is an invasive procedure,and urine microalbumin is not completely related to the severity of the disease.miRNA is a non-coding RNA with a length of about 22 nt.It is involved in the post-transcriptional regulation of multiple genes and plays a key role in a variety of physiological and pathological processes,including diabetic kidney disease and various kidney diseases.The miRNA in urine sediment is expected to become a promising noninvasive biomarker for the identification of DN and NDRD,and continuous monitoring of clinical treatment effects.ObjectiveThis study intends to use the miRNA chip found microRNAs,whose expression level have obvious differences between each group of selection queue,combined the technology of bioinformatics to select several specific microRNA biomarkers of DN,Then expand the validation samples for real-time Quantitative Polymerase Chain Reaction（RT-qPCR）validation,verification the result of the chip and screening several DN specific microRNA biomarkers,then,assess the role of these miRNAs in DN diagnosis and disease judgment.Further analysis f the correlation between the above-mentioned urinary sediment miRNA biomarkers and clinical indicators,laying a foundation for the discovery of non-invasive diagnostic biomarkers for DN.Method1.Urine samples were collected from DN,IgAN and MN patients admitted to the PLA general hospital from January 2016 to January 2018 with a history of diabetes,their pathology are confirmed by kidney biopsy.The experimental group was the urine sediment of DN,the healthy control group was the urine sediment of healthy people who underwent physical examination in the same hospital and the same time,the disease control group were patients with DM and NDRD（DM combined with IgA and DM combined with MN）.Urine samples were collected in the early morning of the day before kidney biopsy,a the urine sediment was obtained after centrifugation,then store samples in-80℃refrigerator.2.MicroRNA expression profile was determined by agilent miRNA V21.0 chip on urinary sediment of 5 HC,5 DM,6 DN and 9 NDRD（5 DM with IgA and 4 DM with MN）patients.Analyze microRNAs’expression levels comprehensively which have statistically significant differences in each group,GO（Gene Ontology,GO）database,KEGG（Kyoto Encyclopedia of Genes and Genomes）and biological information analysis techniques were used to the further screen.3.Using RT-qPCR technology to enlarge the validation group,validation group conclude24 patients with DN,49 patients with NDRD（23 cases of DM with IgA nephropathy,26cases of DM with membranous nephropathy）Test the expression levels of differential miRNAs selected by the chip between the experimental group and the control group,further draw receiver operating characteristic curve to to evaluate the sensitivity and specificity of the screened miRNAs markers for the diagnosis of DN.At last,analyse the correlation between clinical index of DN.Result1.We constructed microRNA expression profiles of urine sediment cells in patients with HC,DM,DN and NDRD.The microarray results showed that there were statistically significant differences in the expression levels of 109 miRNAs between the DN group and the HC group.There were statistically significant differences in the expression levels of a total of 126 miRNAs.Compared with the NDRD group,a total of 63 miRNAs in the DN group had statistically significant differences（P<0.05 and fold change>1.5 or<0.667）,2.Further verify the experimental results of the chip using RT-PCR technology.Results showed that the expression levels of miR-31-3p,miR-135b-5p and miR-1290 in urine sediment of patients with DN and NDRD had statistically significant differences（P=0.003,0.003,0.002）,and those three miRNAs were consistent with Screen microarray miRNA expression trends.The expression level of miRNAs were expressed by 2^-△△CT.The expression level of three miRNAs in DN group was higher than that in NDRD group（P<0.05）.Construct ROC with the three miRNAs.AUC represents the area under the curve.The areas under the miR-31-3p,miR-135b-5p,and miR-1290 curves were 0.691,0.698,and 0.653 respectively.The sensitivity were 0.542,0.792,0.583,respectively and the specificity were 0.792,0.551,0.864,respectively.There was a significant positive correlation between miR-135b-5p and ACR levels in DN.Conclusion1.Through miRNA chip,we found a large number of differentially expressed miRNAs among the four groups of HC,DM,DN,and NDRD.The verification cohort found that miR-31-3p,miR-135b-5p,and miR-1290 were highly expressed in the DN group than NDRD group,the differences between the groups were statistically significant.2.miR-31-3p,miR-135b-5p and miR-1290 have certain diagnostic efficacy,three miRNAs are expected to become noninvasive biodiagnostic markers of DN.There is a significant positive correlation between miR-135b-5p level and ACR level in DN,maybe it can be used as an important indicator of the severity of the disease.