[摘要] 目的：探讨Krüppel 样因子4（KLF4）调控膀胱癌细胞EMT 及迁移的作用及其机制。方法：在膀胱癌5637 和T24 细胞中构建稳定过表达KLF4 的实验组（LV-KLF4）和阴性对照组（LV-NC），用qPCR 和WB 实验验证KLF4 mRNA 和蛋白的表达水平。用Transwell 小室法检测LV-KLF4 组、LV-NC 组细胞的迁移能力变化，用WB 检测EMT 相关标志物上皮钙黏蛋白、神经钙黏蛋白、波形蛋白及Wnt 信号通路相关蛋白的表达水平，用免疫荧光技术检测过表达KLF4 后细胞内β -catenin 的分布变化情况。结果：成功构建KLF4 过表达的5637 和T24 细胞。与LV-NC 组比较，LV-KLF4 组细胞中KLF4 mRNA 及蛋白表达水平升高（均P<0.01），上皮钙黏蛋白的表达水平升高（P<0.01），神经钙黏蛋白和波形蛋白的表达水平降低（均P<0.01），总β -catenin、核β-catenin、MMP9 及c-Myc 表达水平显著降低（均P<0.01），细胞的迁移能力显著下降（P<0.01），细胞内β -catenin 蛋白荧光表达减弱。结论：过表达KLF4 可能通过调控Wnt/β -catenin 信号通路抑制EMT过程，从而抑制膀胱癌5637和T24 细胞的迁移。

[Abstract] Objective: To investigate the role and mechanism of Krüppel-like factor 4 (KLF4) in regulating epithelial-mesenchymal transition (EMT) and migration of bladder cancer cells. Methods: Bladder cancer 5637 and T24 cell lines that stably over-expressing KLF4 (LV-KLF4, experiment group) were constructed, and the negative control group (LV-NC) was also established; the mRNA and protein expressions of KLF4 were verified by qPCR and WB, respectively. Transwell chamber assay was used to detect the migration ability of cells in LV-KLF4 and LV-NC groups. WB was performed to detect the expression levels of EMT-related markers (E-cadherin, N-cadherin, Vimentin) and Wnt signaling pathway-related proteins. Immunofluorescence technique was used to detect the distribution of β-catenin in cells after over-expression of KLF4. Results: The 5637 and T24 cell lines over-expressing KLF4 gene were successfully constructed. Compared with the LV-NC group, the mRNA and protein expressions of KLF4 increased in LV-KLF4 groups (all P<0.01); the expression of E-cadherin increased (P<0.01), while the expressions of N-cadherin, vimentin, and the expression levels of total β - catenin, nuclear β - catenin, MMP 9 and c-Myc decreased (all P<0.01); moreover, the migration ability of cells decreased significantly (P<0.01); the fluorescence expression of β -catenin in cells also decreased significantly in LV-KLF4 group as compared to LV-NC group. Conclusion: Over-expression of KLF4 gene in bladder cancer cells may inhibit EMT process by regulating Wnt/ β -catenin signaling pathway, and further inhibit the migration of bladder cancer 5637 and T24 cells.

贵州省科技厅基金资助项目（No. 黔科合LH字[2015]7493）；贵州省遵义市红花岗区科技基金（No. 遵红科合社字[2015]12）