miR-23b过表达对高糖诱导近端肾小管上皮细胞EMT及PI3K-AKT通路的影响

Effects of overexpression of MiR-23b on high glucose induced EMT and PI3K-AKT pathway in renal tubular epithelial cells

目的研究过表达miR-23b对高糖诱导近端肾小管上皮细胞间充质转化(EMT)及PI3K-AKT通路的影响。方法将人近端肾小管上皮细胞HK-2分为4组:正常葡萄糖(NG)组、高糖(HG)组、miR-23b阴性对照(NC)组和miR-23b过表达(mimic)组,转染48 h后,采用实时荧光定量PCR(qRT-PCR)检测miR-23bmRNA的表达,光学显微镜观察细胞形态,qRT-PCR检测波形蛋白(VIM)、α-平滑肌肌动蛋白(α-SMA)、E-钙黏素(E-CD) mRNA的表达,蛋白免疫印迹(WB)检测VIM、α-SMA、E-CD、PI3K、Akt、p-Akt蛋白的表达。结果与NG组比较,HG组中miR-23b表达显著下调,差异有统计学意义(P <0. 05);转染miR-23b mimic后,HK-2细胞中miR-23b表达显著上调(P <0. 05)。与NG组比较,HG组、NC组和mimic组HK-2细胞出现大量纺锤形细胞,细胞中VIM、α-SMA mRNA和蛋白表达显著上调(P <0. 05),E-CDmRNA和蛋白表达显著下调(P <0. 05),PTEN蛋白表达上调(P <0. 05),PI3K、p-Akt蛋白表达下调(P <0. 05);与HG组、NC组比较,mimic组HK-2细胞纺锤形细胞数量明显减少,细胞中VIM、α-SMA mRNA和蛋白表达下调(P <0. 05),E-CD、PI3K mRNA和蛋白表达上调(P <0. 05),PTEN蛋白表达下调(P <0. 05),PI3K、p-Akt蛋白表达上调(P <0. 05)。结论过表达miR-23b抑制高糖诱导近端肾小管上皮细胞间充质转化作用,其机制可能与抑制PI3K-AKT通路激活有关。

Objective To investigate the effects of overexpression of miR-23b on high glucose induced endothelial-mesenchymal transformation(EMT)and PI3K-AKT pathway in renal tubular epithelial cells.Methods The renal tubular epithelial cells HK-2 were divided into four groups:normal glucose group(NG group),high glucose group(HG group),miR-23b negative control group(NC group)and miR-23b overexpression group(mimic group).On 48h after transfection,real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of miR-23bmRNA,and the morphous of cells was observed by optical microscopy,moreover,qRT-PCR was used to detect the expression levels of vimentin(VIM),α-smooth muscle actin(α-SMA)and E-cadherin(E-CD)mRNA,and protein immunoblotting(WB)was used to detect the expression levels of VIM,α-SMA,E-CD,PI3K,Akt and p-Akt protein.Results As compared with those in NG group,the expression levels of miR-23b in HG group were significantly down-regulated(P<0.05).After transfection of miR-23b mimic,the expression levels of miR-23b in HK-2 cells were significantly up-regulated(P<0.05).As compared with NG group,a large number of spindle cells appeared in HK-2 cells in HG group,NC group and mimic group,moreover,the expression levels of VIM,α-SMA mRNA and protein in the cells were significantly up-regulated(P<0.05),however,the expression levels of E-CDmRNA and protein were significantly down-regulated(P<0.05),and the expression levels of PTEN protein were significantly up-regulated(P<0.05),and the expressions of PI3K and p-Akt protein were obviously down-regulated(P<0.05).As compared with those in HG group and NC group,the numbers of spindle cells in mimic group were decreased significantly,the expressions of VIM,α-SMA mRNA and protein were obviously down-regulated(P<0.05),and the expressions of E-CD,PI3K mRNA and protein were up-regulated(P<0.05),and the expression levels of PTEN protein were down-regulated(P<0.05),moreover,the expression levels of PI3K and p-Akt protein were significantly up-regulated(P<0.05).Conclusion The overexpression of miR-23b can inhibit high glucose induced endothelial-mesenchymal transformation of renal tubular epithelial cells,its action mechanism may be correlated with the inhibition of the activation of PI3K-AKT pathway.