Regulation of CTGF Expression by miR-133b for the Treatment of Renal Interstitial Fibrosis in Old UUO Rats

Introduction: Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated the high expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) from old rats, which mediated the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.Methods: miR-133b expression induced during the EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and time points (0, 24, 48, and 72 h) was detected using real-time polymerase chain reaction. The target genes of miR-133b were validated using a dual-luciferase reporter assay. In vitro experiments were performed to observe mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-week-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. miR-133b、 target gene and other indexes mentioned above mRNA and protein levels and renal interstitial fibrosis were detected at 7 and 14 days.Results: miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. Dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. miR-133b mimic transfection inhibited the TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mouse kidneys after injection of the miRNA-133b overexpression complex, significantly alleviating renal interstitial fibrosis in UUO mice.Conclusion: miR-133b exerted targeted inhibitory effects on CTGF expression, consequently reducing the TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in old UUO mice.