沉默ZEB1基因对胶质瘤U87细胞上皮-间质转化的影响

目的：探讨沉默锌指E盒结合同源框1（ZEB1）基因对胶质瘤U87细胞间质标志物表达和细胞迁移的作用，阐明ZEB1对胶质瘤细胞上皮-间质转化（EMT）的影响。方法：将构建的ZEB1短发夹RNA（shRNA）干扰质粒（shZEB1#1和shZEB1#2）和对照质粒（shCtrl）转染至胶质瘤U87细胞，Western blotting法检测干扰效果。将胶质瘤U87细胞分为对照组（转染shCtrl的胶质瘤U87细胞）、EMT组[转染shCtrl的胶质瘤U87细胞用转化生长因子β1（TGF-β1）诱导EMT]和ZEB1基因沉默组（转染shZEB1的胶质瘤U87细胞用TGF-β1诱导EMT）。Western blotting法检测各组细胞中间质标志物（N-钙黏蛋白和波形蛋白）及基质金属蛋白酶9（MMP-9）的蛋白表达水平，划痕愈合实验检测各组细胞的迁移率。结果： Western blotting法检测，转染shZEB1#1和shZEB1#2的胶质瘤U87细胞中ZEB1蛋白表达水平较转染shCtrl的细胞明显降低（P<0.05或P<0.01），shZEB1#2对ZEB1表达的抑制效果更明显，表明ZEB1已稳定转染到U87细胞。与对照组比较，EMT组胶质瘤U87细胞中N-钙黏蛋白、波形蛋白和MMP-9蛋白表达水平均明显升高（P<0.05或P<0.01）；与EMT组比较，ZEB1基因沉默组N-钙黏蛋白、波形蛋白和MMP-9蛋白表达水平明显降低（P<0.05或P<0.01）。EMT组细胞迁移率明显高于对照组（P<0.01），而ZEB1基因沉默组细胞迁移率明显低于EMT组（P<0.01）。结论：沉默ZEB1基因表达可抑制胶质瘤U87细胞EMT，并降低细胞迁移能力，提示ZEB1可作为侵袭性胶质瘤治疗的重要靶标。

Effect of silencing ZEB1 gene on epithelial to mesenchymal transition in glioma U87 cells

Objective:To investigate the effects of silencing zinc finger E-box binding homeobox1(ZEB1) gene on the expressions of mesenchymal markers and cell migration in the glioma U87 cells, and to clarify the effect of ZEB1 on the epithelial to mesenchymal transition (EMT) in the glioma cells. Methods:The constructed ZEB1 shRNA interfering plasmid and control plasmid (shCtrl) were transfected into the glioma U87 cells and the interfering effects were detected by Western blotting method. The glioma U87 cells were divided into control group (the glioma U87 cells were transfected with shCtrl), EMT group (EMT was induced by TGF-β1 in the glioma U87 cells transfected with shCtrl) and ZEB1 silence group (EMT was induced by TGF-β1 in the glioma U87 cells transfected with ZEB1 shRNAs plasmid). The protein expression levels of mesenchymal markers (N-cadherin, Vimentin), and matrix metalloproteinase-9 (MMP-9) in the glioma U87 cells were measured by Western blotting method. The scratch-healing assay was performed to examine the migration ability of glioma cells. Results:The Western blotting results showed that the expression levels of ZEB1 in the glioma U87 cells transfected with shZEB1#1 and shZEB1#2 were significantly lower than that in the cells transfected with shCtrl (P<0.05 or P<0.01), and the inhibitory effect of shZEB1#2 on the ZEB1 expression was more obvious, indicating that ZEB1 was stably transfected into the U87 cells. Compared with control group, the expression levels of mesenchymal markers N-cadherin, Vimentin, and MMP-9 in EMT group were significantly increased (P<0.05 or P<0.01).Compared with EMT group, the expression levels of the above proteins in ZEB1 silencing group were markedly reduced (P<0.05 or P<0.01).The cell migration rate in EMT group was obviously elevated compared with control group (P<0.01), and the cell migration rate of the glioma U87 cells in ZEB1 silence group was significantly lower than that in EMT group(P<0.01). Conclusion:Silencing ZEB1 gene expression can inhibit the EMT in the glioma U87 cells and reduce the cell migration abilities, suggesting ZEB1 as an important therapeutic target of invasive glioma.