| Background:Diabetic nephropathy(DN)is a serious threat to the health of diabetic patients,and also the leading cause of end stage renal disease(ESRD)worldwide.The pathogenesis of DN is complex and hasn’t been fully understood.The renal tubular epithelial-mesenchymal transition(EMT)is regarded as a key point of renal interstitial fibrosis.A large amount of evidences indicated that miRNAs participated in the pathogenesis of DN by regulating the renal tubular EMT.Objectives:To investigate mechanism,by which miR-30b/SNAIL regulate high glucose-induced EMT in human renal proximal tubule cell(HK-2).The data might provide a new clue in prevention and treatment of DN.Methods:Using bioinformatics to predict the candidates of miRNAs which potentially target SNAIL,then we confirmed the expressions of all of miRNA candidates in renal cortex of db/db mice.Relevant analysis was performed between miRNAs and Snail respectively.We selected the miRNA in this study which showed the most significantly negative relevance with Snail.In vitro the HK-2 cells were divided into four groups:control group(D-glucose 5.5mmol/L),high glucose group(D-glucose 30mmol/L),and mimic group(D-glucose 30mmol/L+candidate miRNA mimic),scramble group(D-glucose 30mmol/L+scramble siRNA).72h after transfection,qRT-PCR,Western Blot and Immunofluorescence were performed to detect the expression levels of miRNA,SNAIL and EMT-related genes of E-cadherin,α-SMA.Results:We have found a total of 6 miRNA candidates potentially targeting SNAIL,which were miR-30b-5p,miR-25-3p,miR-199a-5p,miR-128-3p,miR-22-3p,and miR-27-3p.Among these candidates,miR-30b-5p showed a significantly negative relevance with Snail,R~2=0.77549.Down-regulation of miR-30b-5p was detected in HK-2 cells exposure to high glucose whereas the level of SNAIL up-regulated and the epithelial marker protein of E-cadherin was decreased.Meanwhile the mesenchymal marker proteins ofα-SMA and Vimentin were enhanced in the presense of high glucose.Overexpression of miR-30b-5p decreased the levels of SNAIL,whereas E-cadherin increased in cultured HK-2 cells instead.Meanwhile decline in the levels of Vimentin andα-SMA were detected by the exogenous transfection of mimics in high glucose cultured HK-2 cells.Conclusion:miR-30b/SNAIL is involved in the regulation of high glucose-induced renal tubule epithelial cell EMT.Overexpression of miR-30b-5p could protect HK-2cells from EMT induced by high glucose. |
