c-Raf基因对大肠癌细胞生长的作用及其分子机制研究

Effect of C-Raf Gene on Growth of Colorectal Cancer Cells and Its Molecular Mechanism

[目的]通过转染siRNA沉默c-Raf基因以探讨其在大肠癌细胞生长中的作用及其相关分子机制。[方法]通过阳离子脂质体介导的方法将siRNA转染入大肠癌细胞HCT116和SW620,经Western blot验证siRNA的干扰效果。经MTT法、Transwell实验、细胞克隆形成实验和流式细胞术研究下调c-Raf基因对HCT116和SW620增殖、迁移和相关细胞活力的影响。采用Western blot法检测转染siRNA后相关细胞周期蛋白水平。[结果] HCT116和SW620转染后细胞活力明显下降,转染siRNA 48h后HCT116与SW620细胞的抑制率分别为34%、28%。流式细胞术发现HCT116和SW620在c-Raf基因下调后,较多细胞滞留在G1期(P<0.05)。Western blot实验发现转染siRNA后细胞p-Cdc2、E2F1、CyclinD1表达水平均有下降(P<0.05)。siRNA转染HCT116和SW620细胞后,细胞凋亡比例分别为9.68%±2.37%、7.29%±1.68%,均高于对照组(P<0.05)。siRNA转染HCT116和SW620细胞后Caspase-3相对水平分别为0.57±0.11、0.47±0.09,Bcl-2相对水平分别为0.16±0.05、0.23±0.04,与对照组相比差异均有统计学意义(P<0.05)。siRNA转染HCT116和SW620细胞后N-cadherin相对水平分别为0.24±0.07、0.22±0.04,E-cadherin相对水平分别为0.47±0.12、0.58±0.13,与对照组相比差异均有统计学意义(P<0.05)。[结论]通过下调c-Raf基因的表达可将大肠癌细胞阻滞在G1期,促使细胞发生凋亡,同时抑制大肠癌细胞发生EMT转化。

[Objective]To investigate the role of c-Raf gene in the growth of colorectal cancer cells and its molecular mechanism.[Methods]SiRNA was transfected into colorectal cancer HCT116 and SW620 cells with cationic liposome method,and the interference effect of siRNA was verified by Western blot.The effects of c-Raf gene on proliferation,migration,related cell viability of HCT116 and SW620 were studied by MTT assay,Transwell assay,cell clone formation assay and flow cytometry,respectively.The expression levels of p-Cdc2,E2F1 and CyclinD1 were detected by Western blot.[Results]The viability of HCT116 and SW620 decreased significantly after the siRNA transfection(P<0.05).And the inhibition rate of HCT116 and SW620 cells was34%and 28%respectively after transfection of siRNA for 48 h.The down-regulated c-Raf gene induced G1-phase arrest in HCT116 and SW620 cells.The expression of p-Cdc2,E2F1 and CyclinD1 decreased after siRNA transfection(P<0.05).After siRNA transfection,the percentage of apoptosis in HCT116 and SW620 cells was 9.68%±2.37%and 7.29%±1.68%respectively,which was significantly higher than that in the control group(P<0.05).After siRNA transfection the relative level of Caspase-3 in HCT116 and SW620 cells was 0.57±0.11 and 0.47±0.09,the relative level of Bcl-2 was 0.16±0.05 and 0.23±0.04,and there was significant difference with the control group(P<0.05),After siRNA transfection the relative level of N-cadherin was 0.24±0.07 and 0.22±0.04,the relative level of E-cadherin was 0.47±0.12 and 0.58±0.13 in HCT116 and SW620 cells,and the difference with the control group was significant(P<0.05).[Conclusion]The expression of c-Raf gene can arrest colorectal cancer cells in G1 stage,induce cell apoptosis and inhibit the EMT transformation of colorectal cancer cells.