Effect of miR-199a targeting regulation of HIF-1α on interstitial transdifferentiation of human renal tubular epithelial cells stimulated by high glucose
Objective To investigate the effect of miR-199a on interstitial transdifferentiation(EMT)of human renal tubular epithelial cells stimulated by high glucose.Methods Human renal tubular epithelial cells were cultured in vitro and divided into low glycemic group(LG),high osmotic group(HM)and high glycemic group(HG)according to different glucose concentrations.They were divided into miR-199a inhibition group(miR-199ai group),negative control group(miR-INC group)and liposome group(Mock group)according to whether they were transfected with miR-199a inhibitors(miR199ai).They were divided into si-hypoxia-inducible factor-1α(si-HIF-1α)group,miR-199ai group,miR-199ai+si-HIF-1α group and negative control group(NC group)according to whether they converted to si-HIF-1α and miR-199ai.The expressions of miR-199a,HIF-1α,fibronectin(FN)and α-smooth muscle actin(α-SMA)in different groups of cells were detected by real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)and Western blot.Dual luciferase target assay was used to verify the targeting relationship between miR-199a and HIF-1α gene.Results The mRNA expression level of miR-199a in the HG group was significantly lower than that in the LG group and the HM group(all P＜0.01),and the mRNA and protein expression levels of HIF-1α,FN and α-SMA in Hg group were significantly higher than those in LG group and HM group(all P＜0.01).The mRNA and protein expression levels of HIF-1α,FN and α-SMA in the miR-199ai group were significantly higher than those in the miR-iNC group and the Mock group(all P＜0.01).Target gene prediction by TargetScan and miRanda showed that miR-199a could target the 3'-untranslated region(3'-UTR)that binds HIF-1α.Dual luciferase reported results:Inhibition of miR-199a resulted in a significant increase in luciferase activity(P＜0.01),but there was no significant change in luciferase activity after 3'-UTR mutation of HIF-1α(P＞0.05).After transfection with si-HIF-1α,the mRNA and protein expression levels of HIF-1α,FN and α-SMA in si-HIF-1α group were significantly lower than those in NC group(all P＜0.05).After transfection with miR-199ai,the mRNA and protein expression levels of HIF-1α,FN and α-SMA in miR-199ai group were significantly higher than those in NC group(all P＜0.05).However,when co-transfected with si-HIF-1α and miR-199ai,the expression levels of mRNA and protein in miR-199AI group were significantly higher than those in NC group(all P＜0.05).The mRNA and protein expression changes of HIF-1α,FN and α-SMA induced by transfection of si-HIF-1α or miR-199ai alone can be recovered.Conclusions miR-199a can alleviate hyperglycemia-induced EMT in human renal tubular epithelial cells through targeted regulation of HIF-1α.
万方数据
维普
