TRPM8对人膀胱移行细胞癌细胞T24凋亡和细胞增殖的影响

Effect of TRPM8 on proliferation and apoptosis of human bladder transitional cell carcinoma cell T24

目的 探讨瞬时受体电位M8（TRPM8）对人膀胱癌细胞T24细胞增殖和凋亡的影响.方法 利用荧光定量PCR检测人膀胱移行细胞癌细胞MGH-U1、T24、BIU-87以及EJ中TRPM8的表达水平;设计并合成靶向TRPM8的特异性shRNA,通过脂质体转染法转染TRPM8表达最高的人膀胱癌细胞T24以构建稳定低表达TRPM8细胞株,通过免疫印迹法和荧光定量PCR来验证shRNA的干扰效率;通过四甲基偶氮唑蓝（MTT）比色法检测干扰后细胞的增殖能力;通过流式细胞仪检测细胞凋亡情况;免疫印迹法检测PI3K、细胞周期素DI（Cyclin D1）以及Bcl-2的表达水平.结果 T24的TRPM8表达量在4株膀胱移行细胞癌细胞中最高;特异性靶向TRPM8的shRNA能够有效下调T24细胞中TRPM8的表达;特异性shRNA干扰组细胞较其他两组细胞增殖显著减慢（F=64.73,P＜0.01）;此外,干扰组的凋亡率显著高于空白组和对照组（F =84.73,P＜0.01）;最后,TRPM8干扰组细胞的PI3K、Cyclin D1以及Bcl-2蛋白的表达较空白组和对照组显著下调.结论 采用RNA干扰技术能够有效沉默T24细胞的TRPM8基因,并致使其细胞增殖能力下降以及细胞凋亡率升高,其中可能的机制为通过调节细胞因子PI3K来调控人膀胱移行细胞癌细胞的增殖和凋亡.TRPM8在膀胱癌的发生发展中起着较为重要作用,TRPM8可能成为治疗膀胱癌的新靶点.

Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 （TRPM8） on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24.Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation.The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium （MTT） assay and flow cytometry.Expression of extracellular regulated kinase （ERK）, cyclin D1, and Bcl-2 were detected with Western blot.Results TRPM8-targeted shRNA downregulated TRPM8 expression of T24 cells.MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups （P 〈0.01）.Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group （P 〈 0.01）.In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group.Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K.It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer.