高表达ITGB1促进人乳腺上皮MCF10A细胞EMT及迁移  

ITGB1 induces epithelial-mesenchymal transition and cell migration in breast epithelial cell line MCF10A

作　　者：郎磊[1] 周明莉[1,2] 杨佳佳[1,2] 杜燕娥[1] 文思阳 柳满然[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [2]成都市第三人民医院检验科,四川成都610031

出　　处：《基础医学与临床》2016年第11期1511-1516,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(31171336)

摘　　要：目的探讨ITGB1基因过表达对人乳腺上皮MCF10A细胞迁移和侵袭的影响。方法构建重组质粒p BABEpuro-myc-ITGB1,在人胚肾上皮细胞系293T细胞中包装反转录病毒,转染人乳腺上皮MCF10A细胞系,构建ITGB1基因过表达的稳定细胞系;实验设空白对照组(MCF10A)、阴性对照组(MCF10A-vector)和ITGB1基因过表达组(MCF10A-ITGB1);经嘌呤霉素筛选后,实时荧光定量PCR和蛋白质印迹法检测ITGB1 mRNA及蛋白的表达;Transwell迁移和侵袭实验分别检测细胞株迁移和侵袭能力;蛋白质印迹法进一步检测上皮间质转换(EMT)相关蛋白E-cadherin、vimentin、fibronectin、N-cadherin以及ITGB1下游ILK、p-FAK和FAK蛋白的表达。结果成功构建ITGB1基因稳定过表达人乳腺上皮MCF10A细胞系;ITGB1基因过表达组(MCF10A-ITGB1)与空白对照组(MCF10A)和阴性对照组(MCF10A-vector)细胞相比,迁移能力(P<0.05)和侵袭能力(P<0.01)显著增强;EMT相关蛋白E-cadherin显著下调(P<0.05),vimentin、fibronectin、N-cadherin以及ITGB1下游ILK,p-FAK蛋白显著上调(P<0.05)。结论在人乳腺上皮MCF10A细胞中,过表达ITGB1基因诱导细胞EMT并促进其迁移和侵袭。Objective To investigate the effect of overexpressed-integrin beta 1（ ITGB1） gene on epithelial-mesenchymal transition（ EMT） and cell migration in breast epithelial cell line MCF10 A. Methods Recombinant plasmid p BABE-puro-myc-ITGB1 was constructed and retrovirus was packaged in HEK293 T cells. MCF10 A cells were transfected with the retrovirus carrying ITGB1 gene or the controls. Blank group（ MCF10A）,negative control group（ MCF10A-vector）,ITGB1 overexpression group（ MCF10A-ITGB1） were set up. After treatment with puromycin for 3 days,the expression of ITGB1 in MCF10 A cells was determined by quantitative real-time PCR and Western blot assays. The ability of migration and invasion were analyzed by Transwell assays. The expression of EMT marker proteins（ E-cadherin,vimentin,fibronectin and N-cadherin） and ITGB1 downstream proteins（ ILK,p-FAK and FAK） in MCF10 A cells were detected by Western blot. Results ITGB1 gene was successfully deliveredinto MCF10 A cells,resulting in a stable expression of ITGB1 mRNA and protein. Compared with MCF10 A and MCF10A-vector cells,the ability of migration and invasion were notably enhanced in MCF10A-ITGB1 cells（ P 0. 05）. The expression of the epithelial marker E-cadherin protein was downregulated（ P 0. 01）,the mesenchymal marker proteins（ vimentin,fibronectin and N-cadherin） and ITGB1 downstream proteins（ ILK and p-FAK）were upregulated in MCF10A-ITGB1 cells（ P 0. 01）. Conclusions ITGB1 induces EMT and promotes cell migration and invasion in breast epithelial cell line MCF10 A.

关 键 词：ITGB1 上皮间质转换 细胞迁移 细胞侵袭 

分 类 号：R737.9[医药卫生—肿瘤] R34[医药卫生—临床医学]