激活细胞外信号调节激酶-丝裂原蛋白激酶信号通路对人结肠癌细胞增殖及相关基因的影响

Effects of extracellular signal-regulated kinase/mitogren-activated protein kinase signaling pathway activation on proliferation and cell cycle associated genes in human colon cancer cells

目的探讨野生型 RAF 和 MEK 持续激活细胞外信号调节激酶-丝裂原蛋白激酶信号通路(ERK-MAPK)对人结肠癌细胞增殖及细胞周期相关基因的影响。方法培养人结肠癌细胞系SW1116,脂质体介导 RAF、MEK 基因和空质粒转染,G418筛选阳性克隆。流式细胞仪分析细胞周期,光学显微镜下观察细胞形态学变化,四甲基偶氮唑蓝(MTT)法检测细胞活力,生长曲线和软琼脂集落形成检测细胞增殖,实时定量 PCR 检测 p21^(WAF1)和 p16^(INK4A)及癌基因 c-myc 转录水平。结果建市稳定表达 RAF 或 MEK 的细胞系,RAF 或 MEK 高表达均能明显降低 G_0/G_1期细胞百分比,促进细胞周期由 G_1向 S 期进行,增加 DNA 合成的 S 期细胞百分比,细胞活力和增殖力升高3～4倍,细胞分裂增殖旺盛。转染 RAF/MEK 质粒后,细胞均呈现 p21^(WAF1)和 p16^(INK4A)转录水平降低,c-myc 转录水平升高。结论 ERK-MAPK 信号通路激活可下调细胞周期相关基因 p21^(WAF1)和 p16^(INK4A)表达,并上调 c-myc表达,减少 G_0期时相,加速 G_1/S 期转化,促进细胞增殖。

Objective To investigate the effects of extracellular signal-regulated kinase/mitogrenactivated protein kinase （ERK/MAPK） signaling pathway activation on the proliferation and cell cycle associated genes in human colon cancer cells. Methods Human colon cells of the line SW1116 were cultured, transfected with the recombinant plasmids pCMV-RAF containing the upstream molecular target RAF, pCMV-MEK containing the upstream molecular target MEK, or blank plasmid pCMV respectively, and then screened in the culture fluid with G418, thus obtaining a stable transfected cell clone. MTT method was used to detect the cell viability. The cell growth curve was drawn by using soft agar colony formation test. Flow cytometry was used to analyze the cell cycle. Real-time PCR was performed to detect the mRNA expression of the tumor-associated genes p21^WAF1,p16^INK4A, and c-myc. Results MTT method and cell growth curve showed that the viability and proliferation speed of the SW1116 cells transfected with pCMV- RAF and pCMV-MEK were all significantly greater compared with those of the cells transfected with blank vector （all P 〈 0.01 ）. The numbers of colony of the SW1116 ceils transfected with pCMV-RAF and pCMV- MEK were significantly greater and the sizes of the colonies were ignorantly bigger than those of the cells transfected with blank vector. Light microscopy showed that the SW1116 cells transfected with RAF and MEK were polyploidy with mitochysis. The ceils at the G0/G1 phase decreased and the cells at the S phase increased （P 〈 0.01 ）, and the cells at the G2 phase did not change significantly in number. The expression of p21^WAF1 and that of p16^INK4A were both down-regulated and the expression of c-myc was up-regulated in the SW1116 cells transfected with pCMV-RAF and pCMV-MEK. Conclusion Reducing the G1 phase and accelerating G1/S transition, up-regulating the expression of c-myc and down-regulating the expression of p21^WAF1 and p16^INK4A, ERK/MAPK signaling pathway activation promotes proliferation of cancer cells.