MiR-23a-3p Regulates Albumin-induced Tubulointerstitial Inflammation And Fibrosis By Targeting Egr1

Objective:Diabetic kidney disease(DKD)has become a common cause of chronic kidney disease.Proteinuria is generally considered as one of the clinical indicators of DKD,and it’s also closely related to the progression of DKD.Accumulating evidence indicates that proteinuria induces renal tubular inflammation and fibrosis,but the mechanism remains unclear.Many studies showed that early growth response 1(Egr1)played a key role in tubulointerstitial injury in DKD.However,the upstream regulatory mechanism of Egr1 in the development of DKD is poorly understood.microRNAs(miRNAs)are a group of small,highly conserved noncoding RNAs with a length of 19-24 nucleotides that are involved in a variety of biological processes.A variety of miRNAs are involved and play an important role in the progression of DKD.We speculate that miRNAs may act as upstream regulatory molecules of Egr1,and bind to the 3’untranslated region(3’UTR)of Egrl and then regulate the progression of DKD.However,which miRNA can target Egr1 is unclear.miR-23a-3p was screened for possible target regulation of Egr1.In this study,we aimed to discuss the underline mechanisms that regulate renal tubular inflammatory response and fibrosis in DKD.Methods:1.RNA and protein samples were extracted from renal cortex of DKD mice induced by high-fat diet combined with low-dose streptozotocin(HFD/STZ).The expression levels of Egr1,inflammatory cytokines and fibrosis markers were measured by qRT-PCR and Western blot.Human proximal tubular epithelial cell line(HK-2)was treated with bovine serum albumin(BSA).The expression levels of Egr1,inflammatory cytokines and fibrosis markers were detected by qRT-PCR and Western blot.HK-2 cells were transiently transfected with si-Egrl and pENTER-Egr1 plasmid,respectively.The expression levels of inflammatory cytokines and fibrosis markers were detected by qRT-PCR and Western blot.2.Analysis of public available algorithms was performed to identify which miRNAs could target Egr1 3’UTR.The expression levels of miR-23a-3p were detected in DKD mice and BSA-induced HK-2 cells.Transfection of miR-23a-3p mimic or inhibitor was performed to detect the expression level of inflammatory cytokines and fibrosis markers.3.In BSA-induced HK-2 cells,the expression of mRNA and protein of Egr1 was detected by qRT-PCR and Western blot methods after transfection of miR-23a-3p mimic or inhibitor,respectively.HK-2 cells were co-transfected with miR-23a-3p inhibitor and si-Egr1,and the changes of mRNA and protein expression of inflammatory cytokines and fibrosis markers were detected by qRT-PCR and Western blot.Further,the targeting regulation of Egr1 by miR-23a-3p was verified by dual-luciferase reporter gene assay.Results:1.The expression levels of Egr1,inflammatory cytokines and fibrosis markers were significantly upregulated in the renal tissues of DKD mice and BSA-induced HK 2 cells.The mRNA and protein expression levels of inflammatory cytokines and fibrosis markers were significantly decreased after transfection with si-Egr1.In contrast,the mRNA and protein expression levels of inflammatory cytokines and fibrosis markers were significantly increased after transfection with pENTER-Egr1 plasmid.2.Through bioinformatics analysis,we proposed that there may be a binding site between miR-23a-3p and 3’UTR of Egr1.The expression of miR-23a-3p was significantly reduced in the renal tissues of DKD mice and BSA-induced HK-2 cells.A miR-23a-3p mimic suppressed the expression levels of inflammatory cytokines and fibrosis markers in albumin-stimulated HK-2 cells,and a miR-23a3p inhibitor accelerated these events.3.In HK-2 cells,both mRNA and protein levels of Egr1 were significantly decreased after transfection with miR-23a-3p mimic,while the expression of Egr1 was significantly increased after transfection with miR-23a-3p inhibitor.The induction of inflammatory response and fibrosis by the miR-23a-3p inhibitor was abolished by Egr1 silencing.Dual-luciferase activity assay showed that miR-23a-3p can directly target the 3 ’UTR of Egr1.Conclusion:In summary,miR-23a-3p attenuates inflammatory response and fibrosis by inhibiting the expression of Egr1 in BSA-induced HK-2 cells.