Analysis Of Urinary Exosomal MiRNA Expression In Patients With Idiopathic Membranous Nephropathy

Objective:To investigate the expression profile of miRNA in urine exosomes of patients with idiopathic membranous nephropathy(IMN)and to screen urine biomarkers of IMN patients,and to explore their possible involvement in IMN signaling pathway.Methods:(1)Urine samples were collected from 9 patients with idiopathic membranous nephropathy(IMN)and 9 healthy volunteers(NC)in the Affiliated Hospital of Youjiang Medical University For Nationalities.They were divided into screening sequence(IMN-A group,n = 3;NC-A group,n = 3)and confirmation sequence(IMN-B group,n = 6;NC-B group,n = 6).(2)Urine exosomes of patients in the screening sequence were extracted by hypervelocity centrifugation method,and exosomes were observed by transmission electron microscopy and identified by particle size analysis.Urineexosome small RNA library was constructed after passing exosome RNA extraction and detection.(3)The miRNA expression profiles of urinary exosomes in the screening sequence patients were obtained by miRNA-seq technique,and significantly differentially expressed miRNAs were obtained according to the screening criteria with expression changes of more than 2 times and P value less than0.05.(4)Bioinformatics was used to analyze the GO annotation and KEGG signaling pathway of the differentially expressed target miRNA genes to explore their possible involvement in IMN signaling pathway.(5)The target miRNA was verified by q RT-PCR in the confirmation sequence,and the correlation analysis between the target miRNA and 24 h urinary protein and serum albinin was conducted to obtain candidate biomarkers in the urine of IMN patients.Results:(1)A total of 2048 urinary exosome miRNAs were detected in the screening sequence,and 48 miRNAs were found to have significant expression differences(expression changes were more than 2 times and P value was less than 0.05),among which 36 miRNAs were up-regulated and 12 were down-regulated.(2)KEGG significant enrichment analysis showed that differential miRNA target genes were mainly involved in signal transduction pathways such as MAPK signaling pathway,Wnt signaling pathway and insulin signaling pathway.(3)Urine exosome miR-184 was selected for verification in the patients of confirmed sequence according to the change multiple,mean expression level and literature retrieval results.(4)Verification results of q RT-PCR showed that the expression of miR-184 in IMN-B group’s urinary exosomes was higher than that of NC-B group,and the difference was statistically significant(P<0.05),which was consistent with the sequencing results.(5)Correlation analysis results showed that miR-184 was negatively correlated with serum albumin in IMN-B group(r=-0.814,P=0.049).Conclusion:(1)The expression of miRNA in urine exosomes of patients with idiopathic membranous nephropathy is specific.Among them,miR-184 may be a candidate biomarker for IMN.(2)The differentially expressed urinary exosome miRNA target genes may participate in MAPK signaling pathway,Wnt signaling pathway,insulin signaling pathway and other signal transduction pathways to regulate the podocyte injury process of IMN.