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.
引文
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