ILK siRNA 对高糖刺激的人肾小管上皮细胞GSK-3β及β-catenin 表达的影响

摘要

AIM:To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β(GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kid-ney proximal tubular epithelial cell line HKC induced by high glucose .METHODS: HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control ) group and HG+ILK siRNA group.The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP).The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting.The expression of p-GSK-3βandβ-catenin was observed by immunocytochemical staining .The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, totalβ-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting.RESULTS:GFP was observed in HKC cells, indicating that the transfection was successful.Both the protein and mRNA of ILK were down-regulated in HG +ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group .Silencing of ILK down-regulated the expression of p-GSK-3βand nuclear β-catenin.No difference of total GSK-3βor total β-catenin was observed among the 4 groups.CON-CLUSION:These data support a functional role of ILK , GSK-3βandβ-catenin in tubular EMT induced by high glucose . ILK may promote tubular EMT by regulating the activity of GSK-3βandβ-catenin, the downstream effectors of the Wnt/β-catenin pathway.%目的：探讨高糖诱导肾小管上皮细胞转分化中整合素连接激酶小干扰RNA（ILK siRNA）对糖原合成酶激酶3β（GSK-3β）磷酸化和β-连环蛋白（β-catenin）核内表达的影响及意义。方法：体外培养人近端肾小管上皮细胞系HKC，分为正常对照组（NG）、高糖组（HG）、高糖＋阴性转染对照组（HG＋HK）和高糖＋ILK siRNA组（HG＋ILK siRNA）。倒置荧光显微镜下观察绿色荧光蛋白表达。 RT-PCR及Western blotting 检测ILK mRNA及蛋白表达水平；免疫细胞化学检测磷酸化GSK-3β（p-GSK-3β）和β-catenin表达。 Western blotting 检测总GSK-3β、p-GSK-3β、核β-catenin、总β-catenin、E-钙黏蛋白（E-cadherin）和α-平滑肌肌动蛋白（α-SMA）的表达水平。结果：（1）倒置荧光显微镜下可见绿色荧光蛋白表达，证实构建的siRNA重组质粒成功转染HKC细胞；（2）与HG和 HG＋HK组相比，HG＋ILK siRNA组ILK mRNA及蛋白水平下降，但较NG组表达仍高；（3）HG＋ILK siRNA组ILK基因沉默后，p-GSK-3β与核β-catenin蛋白表达较HG及HG＋HK组均下降，但较NG组表达仍高。而总GSK-3β与总β-catenin 在各组表达无明显差异。结论：ILK、GSK-3β和β-catenin可能参与了高糖介导的肾小管上皮细胞转分化过程。 ILK可能通过调节Wnt/β-catenin途径下游效应蛋白GSK-3β和β-catenin的表达而促使肾小管上皮细胞转分化。