上皮间质转化MACC1对结肠癌细胞HT-29增殖迁移和侵袭影响  

MACC1 regulate the proliferation,migration and invasion of colon cancer cell HT-29 through epithelial-mesenchymal transition

作　　者：赵为民[1] 金博 于震 王海江[1] ZHAO Wei-min;JIN Bo;YU Zhen;WANG Hai-jiang(Department of Gastrointestinal Surgery,Third Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第三附属医院胃肠外科,新疆乌鲁木齐830011

出　　处：《中华肿瘤防治杂志》2021年第14期1072-1079,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：新疆维吾尔自治区自然科学基金(2017D01C386)。

摘　　要：目的探讨基于上皮间质转化(EMT)研究结肠癌转移相关基因1(MACC1)对结肠癌细胞HT-29增殖迁移和侵袭的作用机制。方法人结肠癌HT-29细胞株购自中国科学院上海细胞研究所。转染shRNA敲降MACC1表达,RT-PCR和蛋白质印迹法筛选最佳敲降shRNA。根据随机对照法将细胞分为HT-29细胞对照组(HT-29组)、HT-29细胞转染shRNA-2组和HT-29细胞空白转染组。MTT法检测细胞增殖能力,Transwell法检测细胞迁移和侵袭能力,RT-PCR和蛋白质印迹法分别检测EMT标志物(E-cadherin和Vimentin)的mRNA和蛋白表达水平,免疫荧光法标记细胞骨架中的肌动蛋白和微管蛋白表达。结果当转染shRNA-2的干扰质粒后,MACC1基因的表达水平为0.31±0.08,与HT-29组(1.02±0.06)相比差异有统计学意义,F=46.43,P<0.01。与HT-29组的(0.02±0.04)%相比,转染shRNA-2后细胞在24、36、48、60和72h时增殖率分别被抑制(11.00±1.06)%、(28.33±3.79)%、(37.00±4.17)%、(41.33±4.04)%和(43.33±5.03)%,差异有统计学意义,F=27.52,P<0.01。Transwell细胞迁移和侵袭实验表明,与HT-29组(1.05±0.05,1.02±0.06)相比,转染shRNA-2后细胞迁移和侵袭数比率分别为0.41±0.03和0.24±0.04,差异有统计学意义,F值分别为56.01和110.82,均P<0.01。敲降MACC1后,E-cadherin mRNA表达量上调为1.59±0.06,而Vimentin mRNA表达量下调为0.36±0.05,与HT-29组(0.99±0.13,0.99±0.17)相比,差异有统计学意义,F值分别为42.40和34.03,均P<0.01。蛋白质印迹结果显示,E-cadherin蛋白表达量上调为2.09±0.02,Vimentin蛋白表达量下调为0.44±0.02,与HT-29组(1.02±0.02,1.04±0.03)相比,差异有统计学意义,F值分别为945.91和374.82,均P<0.01。敲降MACC1的表达后细胞的骨架蛋白Actin染色减弱,骨架排列紊乱;但细胞的微管蛋白增强。转染shRNA-NC组的细胞各项检测指标与HT-29组均差异无统计学意义,均P>0.05。结论敲降结肠癌细胞HT-29的MACC1可上调E-cadherin表达,下调Vimentin表达,改变细胞骨架�Objective To investigate the effects of metastasis-associated in colon cancer l(MACC1)on proliferation,migration and invasion in colon cancer HT-29 cells through epithelial-mesenchymal transition(EMT).Methods The expression of MACC1 in colon cancer HT-29 cells was knocked down by shRNAs transfected.The expression level of MACC1 was optimized by RT-PCR and western blot.Using ramdomized controlled method,the experiment was divided into HT-29 cell control group(HT-29 group),HT-29 cell transfection shRNA-2 group(HT-29+shRNA-2 group)and HT-29 cell blank transfection group(HT-29+shRNA-NC group).Cell proliferation was detected by MTT assay.Cell migration and invasion were tested by transwell assay.RT-PCR was employed to detect the E-cadherin and Vimentin mRNA expression.Western blot was used to detect the E-cadherin and Vimentin protein expression.Immunofluorescence was used to label the expression of Actin and Tubulin.Results The mRNA expression levels of MACC1 in HT-29+shRNA-2 groupwas significantly decreased to 0.31±0.08 compared to the HT-29 group(1.02±0.06,F=46.43,P<0.01).MTT assay showed that at 24,36,48,60 and 72 hours,the proliferation rate of HT-29+shRNA-2 group was significantly inhibited by(11.00±1.06)%,(28.33±3.79)%,(37.00±4.17)%,(41.33±4.04)%and(43.33±5.03)%respectively,compared to the HT-29 group(0.02±0.04)%(F=27.52,P<0.01).Transwell experiments showed that the ratio of cell migration and invasion was significantly decreased to 0.41±0.03 and 0.24±0.04 in HT-29+shRNA-2 group,respectively,compared to the HT-29 group(1.05±0.05,1.02±0.06,F=56.01,F=110.82,both P<0.01).RT-PCR results showed that the E-cadherin mRNA expression was significantly up-regulated to 1.59±0.06,while the Vimentin mRNA was down-regulated to 0.36±0.05 in HT-29+shRNA-2 group,compared to the HT-29 group(0.99±0.13,0.99±0.17,F=42.40,F=34.03,both P<0.01).Western blot results showed that the E-cadherin protein expression was significantly up-regulated to 2.09±0.02,while Vimentin protein expression was significantly down-regulate

关 键 词：MACC1 上皮间质转化 结肠癌细胞HT-29 细胞增殖 迁移 侵袭 

分 类 号：R735.3[医药卫生—肿瘤]