Li Jing-nan, Li Xiao, Qian Jia-ming, Lu Xin-qing, Yang Hong
Department of Gastroenterology, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2010 Feb;32(1):46-50. doi: 10.3881/j.issn.1000-503X.2010.01.012.
To explore the effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity.
K-ras wild-type colon cancer cell line Caco-2 was transiently transfected by phr-GFP vector (control group), transfected by mutant K-ras gene phr-K-ras (Val12) vector (transfection group), transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific MAPK pathway inhibitor PD98059 (MAPK inhibition group), or transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific PI-3K pathway inhibitor LY294002 (PI-3K inhibition group), respectively. Cell migration was tested by Transwell experiment. E-cadherin and beta-catenin protein expression and intracellular location were detected by cell immunofluorescence method. Intracellular p120 protein expression was detected by Western blot. beta-catenin protein level which combined with E-cadherin was detected by immunoprecipitation. RhoA activity was analyzed by Pull-down assay.
The Caco-2 cell migration rate was (19.8 +/- 5.6) % in transfection group, which was significantly higher than that in control group [(14.0 +/- 4.2) %] (P = 0.001) and in MAPK inhibition group [(15.8 +/- 1.2) %] (P = 0.044), but was not significantly different from that in PI-3K inhibition group [(17.5 +/- 2.8) %] (P = 0.095). Immunofluorescence method showed that the E-cadherin and beta-catenin stain located in the cell membrane decreased in transfection group. Western blot showed that the total intracellular p120 protein decreased in transfection group and PI-3K inhibition group. Immunoprecipitation data showed that beta-catenin protein level combined with E-cadherin decreased in transfection group and PI-3K group. Pull-down test showed that RhoA protein activity was up-regulated in transfection group.
K-ras gene mutation stimulates the migration of colon cancer cell Caco-2, which may be achieved by decreasing the E-cadherin/beta-catenin/p120 protein complex formation via MAPK pathway and increasing the RhoA protein activity.
通过调节E-钙黏蛋白/β-连环蛋白/p120蛋白复合物的形成以及RhoA蛋白活性，探讨K-ras基因突变对结肠癌细胞系Caco-2转移的影响。
将K-ras野生型结肠癌细胞系Caco-2分别用phr-GFP载体瞬时转染（对照组）、用突变型K-ras基因phr-K-ras（Val12）载体转染（转染组）、用突变型K-ras基因phr-K-ras（Val12）载体转染并经特异性MAPK通路抑制剂PD98059处理（MAPK抑制组）、用突变型K-ras基因phr-K-ras（Val12）载体转染并经特异性PI-3K通路抑制剂LY294002处理（PI-3K抑制组）。采用Transwell实验检测细胞迁移。用细胞免疫荧光法检测E-钙黏蛋白和β-连环蛋白的蛋白表达及细胞内定位。用蛋白质免疫印迹法检测细胞内p120蛋白表达。用免疫沉淀法检测与E-钙黏蛋白结合的β-连环蛋白蛋白水平。用下拉分析法分析RhoA活性。
转染组Caco-2细胞迁移率为（19.8±5.6）%，显著高于对照组[（14.0±4.2）%]（P = 0.001）和MAPK抑制组[（15.8±1.2）%]（P = 0.044），但与PI-3K抑制组[（17.5±2.8）%]差异无统计学意义（P = 0.095）。免疫荧光法显示，转染组细胞膜上的E-钙黏蛋白和β-连环蛋白染色减少。蛋白质免疫印迹法显示转染组和PI-3K抑制组细胞内总p120蛋白减少。免疫沉淀数据显示，转染组和PI-3K组中与E-钙黏蛋白结合的β-连环蛋白蛋白水平降低。下拉试验显示转染组RhoA蛋白活性上调。
K-ras基因突变促进结肠癌细胞Caco-2的迁移，这可能是通过MAPK通路减少E-钙黏蛋白/β-连环蛋白/p120蛋白复合物的形成并增加RhoA蛋白活性来实现的。
