Numb对结肠癌细胞迁移和侵袭的影响

Effect of Numb on migration and invasion of colorectal cancer cells and its mechanism

目的 :探讨Numb基因对结肠癌细胞迁移和侵袭的影响及其可能的作用机制。方法:应用蛋白质印迹法检测结肠上皮细胞HCEpi及结肠癌SW480、SW620和HCT-8细胞中Numb蛋白的表达。将含Numb过表达重组载体的慢病毒感染Numb表达水平较低的HCT-8细胞(HCT-8-Numb组),应用蛋白质印迹法检测空白对照组(HCT-8细胞未进行感染)、阴性对照组(HCT-8细胞感染含空载体的慢病毒)和HCT-8-Numb组HCT-8细胞中Numb蛋白的表达水平,并应用蛋白质印迹法、细胞划痕实验和Transwell法检测空白对照组、阴性对照组、HCT-8-Numb组和Ras相关C3肉毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)抑制剂NSC23766处理组HCT-8细胞中活化的Rac1蛋白的表达水平以及细胞迁移和侵袭能力。结果:结肠上皮细胞HCEpi中Numb蛋白的表达水平明显高于结肠癌SW480、SW620和HCT-8细胞(P值均<0.05),且HCT-8细胞中Numb蛋白的表达水平最低。HCT-8-Numb组细胞中Numb蛋白的表达水平明显高于空白对照组和阴性对照组(P值均<0.05)。HCT-8-Numb组细胞中活化的Rac1蛋白的表达水平明显低于空白对照组和阴性对照组(P值均<0.05),而与NSC23766处理组细胞中活化的Rac1蛋白的表达水平相比差异无统计学意义(P>0.05)。HCT-8-Numb组细胞的迁移和侵袭能力明显低于空白对照组和阴性对照组(P值均<0.05),而与NSC23766处理组细胞的迁移和侵袭能力相比差异无统计学意义(P>0.05)。结论 :过表达Numb蛋白可抑制结肠癌HCT-8细胞的迁移和侵袭,这一作用可能与抑制Rac1蛋白的活化有关。

Objective： To investigate the effects of Numb on invasion and migration of colorectal cancer cells, and to explore its possible mechanism.Methods： The expression levels of Numb in colonic epithelial cell line HCEpi and colorectal cancer cell lines SW480, SW620 and HCT-8 were detected by Western blotting. A recombinant lentiviral vector for Numb overexpression （LV-GV218-Numb-EGFP） was infected into HCT-8 cells with low expression of Numb （named as HCT-8-Numb group）. The expression levels of Numb protein in the blank control （HCT-8 cells without any infection）, negative control （HCT-8 cells infected with lentivirus containing an empty vector, LV-GV218-EGFP） and HCT-8-Numb groups were detected by Western blotting. The expression levels of the activated Ras-related C3 botulinum toxin substrate 1 （Rat1） and the abilities of migration and invasion of colonic epithelial cells and colorectal cancer cells in the blank control, negative control, HCT-8- Numb and NSC23766 （Racl inhibitor） treatment groups were detected by Western blotting, wound scratch assay and Transwell invasion assay, respectively.Results： The expression level of Numb protein in colonic epithelial cell line HCEpi was higher than those in colonic cancer cell lines SW480, SW620 and HCT-8 （P 〈 0.05）. The Numb expression level in HCT-8 cells was lowest. The expression level of Numb protein in HCT- 8-Numb group was higher than those in the blank control and the negative control groups （both P 〈 0.05）. The expression level of activated Racl protein in HCT-8-Numb group was significantly lower than those in the blank control and the negative control groups （both P 〈 0.05）, but there was no significant difference between NSC23766 treatment group and HCT- 8-Numb group （P 〉 0.05）. The abilities of migration and invasion in HCT-8-Numb group were decreased as compared with those in the blank control and the negative control groups （all P 〈 0.05）, but there was no significant difference between NSC23766 treatment group and HCT-8-Numb group （P 〉 0.05）.Conclusion： Numb overexpression can inhibit the invasion and migration of colonic cancer HCT-8 cells. This effect may be related to inhibiting the activation of Racl.