EZH2抑制剂对膀胱癌T24细胞上皮间质转化的影响及其机制研究

Effect of EZH2 inhibitor on epithelial-mesenchymal transition of T24 bladder cancer cells and its mechanism

摘要:

目的 探讨Zeste基因增强子同源物2（EZH2）抑制剂对膀胱癌T24细胞上皮间质转化（EMT）的影响及其作用机制。方法 取对数生长期T24细胞，随机分为空白组、GSK126低剂量组、GSK126中剂量组、GSK126高剂量组，每组设置5个复孔，分别用终浓度为0 μmol/L、10 μmol/L、20 μmol/L、40 μmol/L的GSK126细胞培养基培养。采用MTT法检测培养24 h、48 h、72 h细胞增殖能力，Transwell法和划痕实验检测培养48 h细胞侵袭和迁移能力，qRT-PCR和Western blotting检测培养48 h细胞中上皮间质标志物E-cadherin、Vimentin、β-catenin及EZH2 mRNA和蛋白相对表达量。结果 空白组及GSK126低、中、高剂量组细胞培养24 h、48 h、72 h的增殖能力比较，采用重复测量设计的方差分析，结果 ①不同时间细胞增殖能力有差异（F =15.498，P =0.000）；②4组细胞增殖能力有差异（F =5.162，P =0.013）；③4组细胞增殖能力变化趋势有差异（F =12.314，P =0.000）。与空白组比较，GSK126低、中、高剂量组穿膜细胞数、迁移率，以及EZH2、Vimentin、β-catenin mRNA和蛋白相对表达量均降低（P <0.05），且随GSK126剂量升高而降低（P <0.05）。与空白组比较，GSK126低、中、高剂量组E-cadherin mRNA和蛋白相对表达量均升高（P <0.05），且随GSK126剂量升高而升高（P <0.05）。与空白组比较，GSK126低、中、高剂量组p-JAK2/JAK2、p-STAT3/STAT3蛋白相对表达量均降低（P <0.05），且随GSK126剂量升高而降低（P <0.05）。结论 EZH2抑制剂可有效抑制膀胱癌T24细胞EMT，其调控机制可能与抑制JAK2/STAT3信号通路中JAK2和STAT3蛋白磷酸化有关。

Abstract:

Objective To investigate the effect and mechanism of enhancer of zeste homolog 2 (EZH2) inhibitor on the epithelial-mesenchymal transition (EMT) of T24 bladder cancer cells.Methods T24 cells in the log phase were randomly divided into blank group, low-dose GSK126 group, medium-dose GSK126 group, and high-dose GSK126 group in quintuplicate, with the final concentrations of GSK126 being 0 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L, respectively in the cell culture medium. MTT assay was used to detect the proliferation ability of cells cultured for 24 h, 48 h, and 72 h. Transwell assay and scratch assay were performed to evaluate the invasion and migration ability of cells cultured for 48 hours. The mRNA and protein expressions of biomarkers for EMT including E-cadherin, vimentin and β-catenin as well as those of EZH2 were determined by qRT-PCR and Western blotting, respectively.Results The repeated measures analysis of variance was performed to compared the proliferation ability of cells in blank group, low-dose GSK126 group, medium-dose GSK126 group, and high-dose GSK126 group after 24 h, 48 h and 72 h of culture. The results showed that the proliferation ability of cells was different among distinct time points (F =15.498, P =0.000) and among distinct groups (F = 5.162, P = 0.013), and that the change trend of proliferation ability of cells was different among the groups (F = 12.314, P = 0.000). Compared with the blank group, the number of cells passing the porous membranes, the migration rate, and the mRNA and protein expressions of EZH2, vimentin and β-catenin were lower in low-dose GSK126 group, medium-dose GSK126 group, and high-dose GSK126 group and decreased as the dose of GSK126 increased (P < 0.05). In contrast, the mRNA and protein expressions of E-cadherin were higher in low-dose GSK126 group, medium-dose GSK126 group, and high-dose GSK126 group than those in the blank group, and increased with the increase of the dose of GSK126 (P < 0.05). Besides, the relative protein expressions of p-JAK2/JAK2 and p-STAT3/STAT3 were lower in low-dose GSK126 group, medium-dose GSK126 group, and high-dose GSK126 group compared with the blank group, and decreased as the dose of GSK126 increased (P < 0.05).Conclusions EZH2 inhibitor can effectively suppress the EMT of T24 bladder cancer cells, and the underlying mechanism may be related to the blockade of JAK2 and STAT3 phosphorylation in the JAK2/STAT3 signaling pathway.