Rap1 GTP酶激活蛋白对结肠癌细胞增殖、侵袭和迁移作用的影响

Effects of Rap1 GTP-activating protein on proliferation,invasion and migration of colon cancer cells

目的探讨Rap1 GAP在结肠癌组织中的表达及与临床病理特征和预后的相关性。方法采用免疫组化EnVision两步法检测125例结肠癌组织中Rap1 GAP蛋白表达水平,应用Western blot法检测结肠癌细胞系(LOVO、HCT116、SW480)和正常结肠上皮细胞HCoEPiC中Rap1 GAP蛋白表达。通过慢病毒转染LOVO、HCT116和SW480细胞,分别下调、上调Rap1 GAP的表达,根据不同处理分为空载组(sh-NON、LV-NON)、sh-Rap1 GAP组(低表达Rap1 GAP)和LV-Rap1 GAP组(过表达Rap1 GAP),Western blot法验证细胞转染效率;采用MTT实验和Transwell实验分别检测各组细胞的增殖、侵袭和迁移能力。结果125例结肠癌标本中,Rap1 GAP缺失表达者83例(66.4%),高于癌旁对照组织(7.2%)(P<0.001)。Rap1 GAP缺失表达率与肿瘤分化程度(χ^(2)=6.152,P=0.011)、是否伴黏液腺癌(χ^(2)=4.908,P=0.028)有关,与患者性别、年龄、肿瘤发生部位、临床分期、淋巴结转移均无关(P>0.05)。Western blot结果显示,与HCoEPiC(0.189±0.081)细胞相比,LOVO(0.238±0.008)细胞中Rap1 GAP蛋白表达增高,HCT116(0.064±0.002)和SW480(0.152±0.026)细胞中Rap1 GAP蛋白表达降低(F=159.6,P<0.05)。LOVO细胞转染Rap1 GAP低表达慢病毒后,sh-Rap1 GAP-1组(0.733±0.071)、sh-Rap1 GAP-2组(0.559±0.136)和sh-Rap1 GAP-3组(0.606±0.037)Rap1 GAP蛋白表达水平明显低于LOVO组(1.880±0.129)(F=49.57,P<0.05)。与sh-NON组(1.260±0.109)相比,sh-Rap1 GAP-2组(1.569±0.059)和sh-Rap1 GAP-3组(1.548±0.087)细胞72 h增殖能力明显提高(F=28.36,P<0.05),其侵袭和迁移能力明显增高(P<0.05)。HCT116细胞转染Rap1 GAP过表达慢病毒后,与LV-NON组(0.485±0.097)相比,LV-Rap1 GAP组(1.395±0.137)Rap1 GAP蛋白表达明显较高(P<0.05)。MTT实验结果显示,与LV-NON组(0.652±0.047)相比,LV-Rap1 GAP组(1.212±0.038)细胞增殖能力降低,其侵袭和迁移能力明显降低(P<0.05)。SW480细胞的转染结果及增殖、侵袭和迁移能力与HCT116细胞一致。结论Rap1 GAP缺失表达率与结肠癌分化程度、是否伴黏液腺癌有关,上调Rap1 GAP表达能抑制结肠癌细胞增殖、侵袭和迁移能力,可为探究结肠癌的发生、发展机制提供理论依据。

Purpose To investigate the correlation between Rap1 GAP expression in colon cancer tissues and clinicopathological features and prognosis.Methods Immunohistochemistry was used to detect Rap1 GAP protein expression in 125 cases of colon cancer,and its correlation with clinicopathological features and prognosis was analyzed.Rap1 GAP protein expression in colon cancer LOVO,HCT116,SW480 cells and normal colon epithelial HCoEPiC cells was detected by Western blot.The expression of Rap1 GAP was down-regulated and up-regulated in LOVO,HCT116 and SW480 cells by lentivirus transfection,and divided into no-load group(sh-NON,LV-NON),sh-Rap1 GAP group(low expression Rap1 GAP)and LV-Rap1 GAP group(overexpression Rap1 GAP)according to different treatments.The transfection efficiency was verified by Western blotting.MTT assay and Transwell assay were used to detect cell proliferation,invasion and migration in each group.Results In 125 colon cancer samples,83 cases(66.4%)had the loss of Rap1 GAP expression,which was higher than that in paracancer control(7.2%,P<0.001).The rate of loss of Rap1 GAP expression was correlated with the degree of tumor differentiation(χ^(2)=6.152,P=0.011)and the presence of mucinous adenocarcinoma(χ^(2)=4.908,P=0.028),but not with gender,age,tumor location,tumor stage,or lymph node metastasis(P>0.05).Western blotting results showed that compared with HCoEPiC(0.189±0.081)cells,Rap1 GAP protein expression was increased in colon cancer LOVO(0.238±0.008)cells.Rap1 GAP protein expression was decreased in HCT116(0.064±0.002)and SW480(0.152±0.026)cells(F=159.6,P<0.05).After LOVO cells were transfected with Rap1 GAP low expression lentivirus,the expression level of Rap1 GAP in sh-Rap1 GAP-1 group(0.733±0.071)and sh-Rap1 GAP-2 group(0.559±0.136)and sh-Rap1 GAP-3 group(0.606±0.037)was significantly lower than that in LOVO group(1.880±0.129)(F=49.57,P<0.05).Compared with sh-NON(1.260±0.109)group,the proliferation ability of sh-Rap1 GAP-2(1.569±0.059)and sh-Rap1 GAP-3(1.548±0.087)cells was significantly increased at 72 h(F=28.36,P<0.05).Its invasion and migration ability were significantly increased(P<0.05).After HCT116 cells transfected with overexpression lentivirus,the expression of Rap1 GAP protein in LV-Rap1 GAP group(1.395±0.137)was relatively higher than that in LV-NON group(0.485±0.097)(P<0.05).The results of MTT assay showed that compared with LV-NON(0.652±0.047)group,the proliferation ability of cells in LV-Rap1 GAP(1.212±0.038)group was decreased,and the invasion and migration ability were significantly decreased(P<0.05).The transfection results,proliferation,invasion and migration of SW480 cells were consistent with those of HCT116 cells.Conclusion The loss rate of Rap1 GAP expression is related to the differentiation degree of colon cancer and whether it is accompanied by mucinous adenocarcinoma.The up-regulation of Rap1 GAP expression can inhibit the proliferation,invasion and migration of colon cancer cells,providing a theoretical basis for exploring the occurrence and development of colon cancer.