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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 investigate the changes of cell proliferation and apoptosis and the expression of Smad4 mRNA in human bladder cancer T24 cells transfected with RUNX3 gene recombinant plasmids and to explore the mechanism of RUNX3 gene and TGF-β / Smad signaling pathway in bladder carcinogenesis In the role. Methods pIRES-EGFP-RUNX3 plasmid was constructed and T24 cells were divided into blank control group (without transfection), empty vector plasmid group and recombinant plasmid group. The transfected pIRES-EGFP and pIRES-EGFP-RUNX3 were transfected into the empty vector plasmid group and the recombinant plasmid group respectively, and the morphological changes of the cells were observed by fluorescence microscopy after 24h transfection. The apoptosis of cells was detected by flow cytometry and RT- Group RUNX3, Smad4 mRNA expression levels. Results The recombinant plasmid pIRES-EGFP-RUNX3 was successfully constructed. The apoptosis of transfected cells was observed by microscope. The apoptotic cells appeared in the recombinant plasmid group after transfection for 24 h. The apoptosis rate of blank control group was (3.23 ± 0.45)%, (8.98 ± 1.62)% in the plasmid group and (43.61 ± 2.69)% in the recombinant plasmid group. The expression of RUNX3 mRNA in the recombinant plasmid group was (2.79 ± 0.36). The expression of Smad4 mRNA in the recombinant plasmid group was higher than that in the other two groups (P <0.05). CONCLUSIONS: Transfection of RUNX3 gene can up-regulate the expression of Smad4 mRNA in bladder cancer T24 cells and inhibit the proliferation and promote the apoptosis of tumor cells. RUNX3 may be involved in the proliferation and apoptosis of bladder tumor cells through TGF-β / Smad signaling pathway Regulation.