膀胱癌T24细胞中RUNX3基因对Smad4表达的影响

Effect of RUNX3 on the Expression of Smad4 in T24 Bladder Cancer Cell Line

目的观察RUNX3基因重组质粒转染人膀胱癌T24细胞后细胞增殖及凋亡的变化以及Smad4mRNA表达的变化,探讨RUNX3基因与转化生长因子-β(TGF-β)/Smad信号通路在膀胱癌发生机制中的作用。方法构建pIRES-EGFP-RUNX3质粒,将T24细胞分空白对照组(不转染)和空载体质粒组以及重组质粒组。空载体质粒组和重组质粒组细胞分别转染pIRES-EGFP和pIRES-EGFP-RUNX3,转染24h后采用荧光显微镜观察细胞形态的变化;流式细胞仪检测细胞凋亡情况;RT-PCR检测各组细胞RUNX3、Smad4基因mRNA表达水平。结果成功构建pIRES-EGFP-RUNX3重组质粒,显微镜观察发现转染组均出现细胞死亡,重组质粒组出现凋亡细胞;转染24h后空白对照组凋亡率为(3.23±0.45)%,空载体质粒组为(8.98±1.62)%,重组质粒组为(43.61±2.69)%;RUNX3mRNA仅重组质粒组有表达(2.79±0.36),重组质粒组Smad4mRNA较另两组表达上调(P<0.05)。结论转染RUNX3基因可上调膀胱癌T24细胞中Smad4mRNA的表达,且能抑制细胞增殖并促进其凋亡,抑癌基因RUNX3可能通过TGF-β/Smad信号通路参与对膀胱肿瘤细胞增殖与凋亡的调控。

Objective To examine the effects of RUNX3 on cell proliferation and apoptosis and the expression level of Smad4 mRNA in the bladder cancer cell line of T24 by transfection with recombinant plasmid of pIRES- EGFP-RUNX3. Methods The recombinant plasmid of pIRES-EGFP-RUNX3 was constructed successfully. Cultured T24 cells were divided into three groups, including control group, empty vector group, and recombinant piasmid group. The cells in empty vector group and recombinant plasmid group were respectively transfected by plRES-EGFP and pIRES-EGFP-RUNX3. The cells were harvested at 24 h after the transfection, the variation of cell morphology was examined by fluorescence microscopy. The cell apoptosis was detected by flow cytometry. The expression level of RUNX3 and,Smad4 mRNA was measured by RT-PCR. Results Cell death was observed in two transfection groups. At 24 h after transfection,the apoptosis rate was （3.23±0.45）% in control group, （8.98± 1.62）% in empty vector group and （43. 61 ±2. 69）% in recombinant plasmid group. The expression level of RUNX3 mRNA was 2.79± 0. 36 , detected only in recombinant plasmid group, which was significantly up-regulated compared with the other two groups （P〈 0. 05）. Conclusion The expression level of Smad4 mRNA was up- regulated by transfection with pIRES-EGFP-RUNX3, which also inhibited cell proliferation and promoted cell apoptosis. The tumor suppressor gene of RUNX3 could regulate the bladder cancer cell proliferation and apoptosis by TGF-β/Smad signaling pathway.