The Molecular Mechanism Of Mir-124-3p In Regulating Renal Fibrosis By Targeting ITGB1

BackgroundRenal fibrosis is the main pathological basis for the development of chronic kidney disease(CKD)to end-stage,which is characterized by abnormal deposition of extracellular matrix(ECM),and its pathological mechanism includes epithelialmesenchymal transition(EMT).Due to the lack of timely and effective diagnosis and treatment methods,the prevalence of CKD is increasing year by year,and it has been paid more and more attention by the medical community.Therefore,exploring the molecular mechanism of renal fibrosis makes sense to the clinical treatment.In this study,bioinformatics analysis was conducted on blood samples from patients with CKD,and unilateral ureteral ligation obstruction(UUO)model,and TGF-β 1-induced TCMK1 model were used to explore the molecular mechanism of miR-124-3p in regulating renal fibrosis by targeting ITGB1.Methods1.The CHIP GSE62792 with blood samples from CKD patients and the chip GSE162794 with kidney tissue samples from UUO mice were downloaded by GEO database.Key genes in dataset GSE62792 were screened by RStudio software,STEM and CTD,and their upstream miRNAs were screened by Targetscan and StarBase online database.Differentially expressed miRNAs in dataset GSE162794 were screened out by RStudio software and take the intersection between differentially expressed miRNAs with upstream miRNAs of key genes.2.In TGF-β1-stimulated renal tubular epithelial cells,overexpression of miR124-3p and inhibition of ITGB1 were performed,the expression level of miR-124-3p,ITGB1 and fibrosis-related indicators were detected by qRT-PCR and Western blot.3.The dual luciferase activity assay verifies that ITGB1 is the target gene of miR-124-3p and determines their binding sites.4.Chemically synthesized ITGB1 siRNA and miR-124-3p agomir were injected intraperitoneally after UUO operation,and the expression levels of ITGB1 were detected by Western blot.The deposition of collagen fibers in mouse kidney tissue was detected by Sirius Red staining,and the expression of fibrosis-related proteins were detected by Western blot and immunofluorescence methods.Result1.Bioinformatics analysis was performed on the GSE62792 dataset of the GEO to screen out differential genes.ITGB1 was significantly up-regulated in the blood of CKD patients,which may be regulated by miR-124-3p.The expression of ITGB1 was significantly up-regulated and the expression of miR-124-3p was significantly downregulated in the UUO renal fibrosis model.2.In the cell model,TGF-β1 induced low expression of miR-124-3p and high expression of ITGB11 overexpression of miR-124-3p inhibited the expression of ITGB1.Overexpression of miR-124-3p and inhibition of ITGB1 expression attenuated TGF-β1 induced renal fibrosis,and the expression of fibrosis-related indicators decreased.3.The results of TargetScan bioinformatics analysis showed the binding site between the 3’UTR of ITGB 1 and miR-124-3p,and we verified that miR-124-3p directly targets ITGB1 through dual luciferase reporter genes.4.Chemically synthesized ITGB1 siRNA and miR-124-3p agomir were injected intraperitoneally after UUO operation,the expression level of ITGB1 protein was significantly decreased,Sirius Red staining results showed the deposition of collagen fibers were reduced.At the same time,the expression levels of fibrosis related indicators were decreased.ConclusionThe expression of ITGB 1 was significantly increased in the blood of CKD patients and in the renal fibrotic tissue of mice,and was negatively correlated with miR-124-3p.miR-124-3p inhibited EMT by targeting ITGB1 and delayed the progression of renal fibrosis,thus exerting a protective effect on renal function.