miR-30b-5p modulate renal epithelial-mesenchymal transition in diabetic nephropathy by directly targeting SNAI1

Object: Renal tubulointerstitial fibrosis plays a significant role in the development of diabetic nephropathy (DN). SNAl1 is a main activator of epithelial-to-mesenchymal transition (EMT) in the process of fibrosis. This study aimed to investigate the effect of miR-30b-5p targeting SNAIL on the EMT in DN. Methods: Bioinformatics and miRNAs microarray analyses were used to predict the candidate miRNA targeting SNAIL, that is miR-30b-5p. The db/db mice was as DN animal model and renal tissues of mice were stained with PAS. The miR-30b-5p expression in mouse and human renal tissue were examined by quantitative RT-PCR (qRT-PCR) and fluorescence in situ hybridization (FISH), while SNAIL expression was determined by qRT-PCR and immunohistochemistry. Luciferase reporter gene assay was used to confirm miR-30b-5p directly target 3'-UTR of the SNAIL mRNA. In vitro, HK-2 cells were treated with high glucose to establish hyperglycemia cell model and transfected with miR-30b-5p mimics to overexpress miR-30b-5p. Expression of miR-30b-5p, SNAI1 and EMT related indicators (E-cadherin, a-SMA and Vimentin) in HK-2 cells under different treatments were determined by qRT-PCR and/or western-blot. In addition, immunofluorescence was performed to evaluate a-SMA expression in HK-2 cells under different treatments. Results: Bioinformatics analyses revealed miR-30b-5p had complementary sequences with SNAIL mRNA and the seed region of miR-30b-5p was conserved in human and a variety of animals, including mice. Microarray analysis showed miR-30b expression decreased in DN mice, which was further verified in db/ db mice by qRT-PCR and in human DN by FISH. Contrary to miR-30b-5p, SNAI1 expression level was upregulated in db/db mice. Correlation analysis suggested SNAI1 mRNA level was negatively with miR30b-5p level in renal tissue of db/db mice. Luciferase reporter gene assay confirmed miR-30b-5p directly targeted SNAI1 mRNA. In high glucose induced HK-2 cells, expression levels of miR-30b-5p and E-cadherin were decreased, while SNAI1, a-SMA and Vimentin were increased. Overexpression miR-30b-5p in high glucose induced HK-2 cells could reverse that phenomenon to some extent. Conclusion: These findings suggest that miR-30b-5p play a protective role by targeting SNAI1 in renal EMT in DN. (C) 2020 Elsevier Inc. All rights reserved.