The Effect Of Rno-miR-433-3p On Renal Fibrosis Induced By High Glucose In Vitro

Research background andObjective:Previous studies have shown that miR-433 is an important regulator of renal fibrosis induced by TGF-β/smad3 in obstructive nephropathy(UUO).MiR-433 amplifies TGF-β/smad3 signaling pathway-induced renal fibrosis by its promoter binding to smad3,and it was found that down-regulating miR-433 expression can significantly reduce UUO renal fibrosis.Renal fibrosis is one of the pathological characteristic of diabetic kidney disease.The objective of this study is to investigate whether rno-miR-433-3p has an effect on renal fibrosis under conditions of high glucose stimulation in vitro.object and methods:First,the qRT-PCR method was used to detect the expression changesof rno-miR-433-3p on rat tubular epithelial cells(TECs)and glomerular mesangial cells(MMCs)stimulated by high glucose of different concentrations and different glucose exposure time,and the optional highglucoseexposure time and concentrations were determined.The qRT-PCR method was used to detect the expression of renal fibrosis indices(Col-1,Col-4,fibronectin,a-SMA)in high glucose-stimulated MMCs and TECs.Second,the rno-miR-433-3p overexpression plasmid and the rno-miR-433-3p small hairpin RNA(rno-miR-433-3p shRNA)plasmid were transfected into rat MMCs and TECs in vitro.The qRT-PCR method was used to detectthe expression changes of rno-miR-433-3p and renal fibrosis markers(Col-1,Col-4,fibronectin,a-SMA)in high glucose-exposed MMCs and TECs of rats.Western blotting method was used to detect the expression of renal fibrosis index(Col-1 Col-4,fibronectin,a-SMA)in MMCs and TECs induced by high glucose.Research result:1.Theexpression of mo-miR-433-3p was significantly increased in high glucose-treated rats renal TECs(p<0.01).Glucose-stimulated expression of rno-miR-433-3p reached to maximum level under 25 mM glucoseand 48 hours.Renal fibrosis indices(Col-1,Col-4,FN,a-SMA)were significantly increased in high glucose-stimulated MMCs and TECs(p<0.01).2.The expression of rno-miR-433-3p was significantly increased in rat MMCs and TECs transfected with rno-miR-433-3p overexpression plasmid(p<0.01).The expression of rno-miR-433-3p was significantly decreased in MMCs and TECs transfected with rno-miR-433-3p shRNA plasmid in vitro(p<0.01).Renal fibrosis markers(Col-1 Col-4,fibronectin,a-SMA)expression were significantly increased in rat MMCs and TECs transfected with rno-miR-433-3p overexpression plasmid(p<0.01)and the renal fibrosis expression were significantly decreased in rat MMCs and TECs transfected with rno-miR-433-3p shRNA plasmid(p<0.01).3.Western Blotting results showed that Col-1,Col-4,fibronectin and a-SMA were significantly increased in rat MMCs and TECs transfected with rno-miR-433-3p overexpression plasmid and that the renal fibrosis index expression were significantly decreased in rat MMCs and TECs transfected with rno-miR-433-3p shRNA plasmid.Conclusion:1.High glucose may up-regulate theexpression of rno-miR-433-3p and fibrosis markers inrenal MMCs and TECs in a time-dependent and dose-dependent manner,and the expression of rno-miR-433-3p and fibrosis markers were significantly up-regulated in the high glucose expose,suggesting that high glucose may induce renal fibrosis by up-regulating expressionofrno-miR-433-3p.2.The rmo-miR-433-3p overexpression plasmid/rno-miR-433-3p shRNA plasmid had high transfection efficiency for MMCs and TECs in rat,and could promote/inhibit the expression of rno-miR-433-3p in MMCs and TECs,respectively.rno-miR-433-3p is an important regulator of renal fibrosis induced by high glucose.Up-regulation of rno-miR-433-3p expression in vitro can enhance high glucose-induced renal fibrosis.Down-regulation of rno-miR-433-3p expression in vitro can inhibit high glucose-induced renal fibrosis.