稳定干扰ITGB1抑制人乳腺癌BT549细胞迁移和侵袭

Inhibition of the Migration and Invasion of Human Breast Cancer Cell Line BT549 by Stable Interference of ITGB1

在人乳腺癌细胞系BT549中稳定干扰整合素β1(integrinβ1,ITGB1),研究其对乳腺癌细胞迁移和侵袭的影响。构建靶向干扰ITGB1基因的sh RNA慢病毒,感染BT549细胞,通过筛选成功获得稳定干扰ITGB1蛋白的BT-549细胞系,实验设空白对照组(Blank)、病毒感染阴性对照组(shNC)、ITGB1-shRNA慢病毒感染组(shITGB1);Real-time PCR和Western blotting法检测稳定干扰后ITGB1 mRNA和蛋白的表达情况;Western blotting法检测稳定干扰ITGB1后上皮间质转化(epithelial-mesenchymal transition,EMT)相关标志蛋白(E-cadherin,Vimentin,N-cadherin,Fibronectin)的表达情况;利用划痕试实验和Transwell侵袭试实验分别检测稳定干扰ITGB1后细胞的迁移及侵袭能力。Real-time PCR和Western blotting结果显示,shITGB1组细胞ITGB1 mRNA和蛋白的表达水平显著低于Blank组和shNC组细胞(p<0.01,p<0.01)。Western blotting结果显示,稳定干扰ITGB1可部分逆转乳腺癌细胞BT549的EMT化;稳定干扰ITGB1后,上皮标志蛋白E-cadherin表达显著上调(p<0.01),间质标志蛋白Vimentin(p<0.05)、N-cadherin(p<0.01)和Fibronectin(p<0.01)的表达显著降低。划痕和Transwell侵袭试验实验结果显示,稳定干扰ITGB1后可显著降低乳腺癌细胞的迁移及侵袭能力(p<0.05,p<0.05)。在乳腺癌BT549细胞中稳定干扰ITGB1后,通过逆转EMT化抑制细胞迁移和侵袭。

This study aimed to stably interfere the ITGB 1 gene in BT549 human breast cancer cells to investigate its effects on migration and invasion of breast cancer cell. shRNA lentiviral vector targeting ITGB 1 gene was constructed to infect the BT549 cell, and BT-549 cell line that could stably interfere the ITGB1 protein was successfully obtained by screening. The experiment set up blank control group （Blank）, viral infection negative control group （shNC） and ITGB1 lentivirus infection group （shITGB1）. Expressions of ITGB1 mRNA and protein after stable interference were determined by Real-time PCR and Western blotting. Besides, expressions of EMT marker proteins （E-cadherin, Vimentin, N-cadherin, Fibronectin） in different treated cells were detected by Western blotting. The cell migration and invasion abilities were further analyzed by assays of scratching and Transwell invading after stable interference of ITGB1. Real-time PCR and Western blotting results indicated that the expression levels of ITGB1 mRNA and protein in shITGB1 group were significantly lower than those in Blank group and in shNC group （p〈0.01, p〈0.01）. Western blotting revealed that stable interference of ITGB1 could partially set-back the EMT of breast cancer cells BT549. In addition, the expression of epithelial marker E-cadherin protein was up-regulated （p 〈0.01）, but the mesenchymal markers Vimentin （P〈0.05）, Fibronectin （p 〈0.01） and N-cadherin （p 〈0.01） were down-regulated significantly after ITGB 1 was stably interfered. The outcomes of scratching and Transwell invading tests showed that the migration and invasion abilities of shITGB 1 group cells could be obviously decreased after stable interference of ITGB1 （p 〈0.05, p 〈0.05）. In conclusion, the stable interference of ITGB 1 in breast cancer BT549 cells could reverse EMT to restrain cell migration and invasion.