Wang Xia, Lu Rong, Fang Jing-yuan
Renji Hospital, Medical College of Shanghai Jiaotong University, Shanghai Institute of Digestive Disease, Shanghai 200001, China.
Zhonghua Yi Xue Za Zhi. 2007 Apr 10;87(14):982-6.
To investigate the effects of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway activation on the proliferation and cell cycle associated genes in human colon cancer cells.
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.
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 cells 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 cells 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.
Reducing the G1phase 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.
探讨细胞外信号调节激酶/丝裂原活化蛋白激酶（ERK/MAPK）信号通路激活对人结肠癌细胞增殖及细胞周期相关基因的影响。
培养人结肠癌细胞系SW1116，分别用含上游分子靶点RAF的重组质粒pCMV-RAF、含上游分子靶点MEK的重组质粒pCMV-MEK或空白质粒pCMV转染，然后在含G418的培养液中筛选，获得稳定转染细胞克隆。采用MTT法检测细胞活力。通过软琼脂集落形成试验绘制细胞生长曲线。用流式细胞术分析细胞周期。采用实时荧光定量PCR检测肿瘤相关基因p21（WAF1）、p16（INK4A）和c-myc的mRNA表达。
MTT法和细胞生长曲线显示，转染pCMV-RAF和pCMV-MEK的SW1116细胞的活力和增殖速度均显著高于转染空白载体的细胞（均P<0.01）。转染pCMV-RAF和pCMV-MEK的SW1116细胞的集落数显著增多，集落大小也明显大于转染空白载体的细胞。光学显微镜显示，转染RAF和MEK的SW1116细胞为多倍体且有丝分裂。G0/G1期细胞减少，S期细胞增多（P<0.01），G2期细胞数量无明显变化。转染pCMV-RAF和pCMV-MEK的SW1116细胞中p21（WAF1）和p16（INK4A）的表达均下调，c-myc的表达上调。
ERK/MAPK信号通路激活通过减少G1期并加速G1/S转换、上调c-myc表达及下调p21（WAF1）和p16（INK4A）表达来促进癌细胞增殖。
