用锌指转录因子Snail诱导肺癌细胞A549发生上皮细胞-间质转化（epithelial-mesenchymal transition, EMT），检测肺癌发生EMT后细胞侵袭能力的变化，为临床筛选分子靶向药物提供依据．构建pcDNA3.1-snail载体，用pcDNA3.1-snail及空pcDNA3.1载体转染肺癌A549细胞后,进行G418筛选；光镜观察培养后细胞形态学的改变、免疫细胞化学与免疫荧光检测细胞表达E-钙黏着蛋白（E-cadherin）、波形蛋白（vimentin）的改变，Western印迹检测细胞中E-钙黏着蛋白和波形蛋白的改变，Transwell侵袭小室法进行细胞体外侵袭能力检测．采用pcDNA3.1-snail转染细胞后，A549细胞变得细长，细胞融合度降低．免疫细胞化学、免疫荧光、Western印迹结果显示,E-钙黏着蛋白表达降低，波形蛋白表达升高；transwell侵袭小室法结果显示,过表达Snail的A549细胞穿透matrigel胶的细胞数明显增多．结果提示，Snail能有效诱导肺癌发生EMT，并且能增强肺癌的体外侵袭能力．
Abstract
The invasion change of human lung cancer cell A549 was detected with overexpression of snail transcription factor inducing epithelial mesenchymal transition (EMT) to supply evidence for selection of new targeting molecular drugs. pcDNA3.1-snail vector was constructed and transfected to number of A549 cell, then cells being selected with G418 to construct the EMT cell model of lung cancer cell line. The morphological change and the expression change of E-cadherin, vimentin were observed by immunocytochemistry, immunofluorescence and Western blotting. Invasive capacity of A549 cell was detected by transwell invasion chamber in vitro. After being transfected with pcDNA3.1-snail, the A549 cells become long and thin, sparse, the degree of cell fusion decreased. The results indicated that the expression of E-cadherin was down regulated and the expression of vimentin was up regulated. Results of transwell assay showed that number of A549 cells penetrated to the membrane were obviously increased. In short, snail can induce non-small lung cancer to EMT and enhance non-small lung cancer in vitro invasion capability.
关键词
转录因子Snail /
上皮细胞-间质转化 /
肺癌 /
侵袭能力
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Key words
snail transcription factor /
epithelial mesenchymal transition /
lung cancer /
invasive capacity
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中图分类号：
R734.2
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参考文献
[1]Boyer B, Vallés AM, Edme N. Induction and regulation of epithelial-mesenchymal transitions[J]. Biochem Pharmacol, 2000, 60(8): 1091-1099
[2］Ahlstrom J D, Erickson C A. New views on the neural crest epithelial-mesenchymal transition and neuroepithelial interkinetic nuclear migration[J]. Commun Integr Biol, 2009, 2(6): 489-493
[3］Fernando R I, Litzinger M, Trono P, et al. The T-box transcription factor Brachyury promotes epithelial- mesenchymal transition in human tumor cells[J]. J Clin Invest, 2010, 120(2): 533-544
[4］C me C, Arnoux V, Bibeau F, et al . Roles of the transcription factors snail and slug during mammary morphogenesis and breast carcinoma progression[J]. J Mammary Gland Biol Neoplasia, 2004, 9(2): 183-193
[5］Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer[J].Am J Pathol, 2002, 161(5): 1881-1891
[6］Miyoshi A, Kitajima Y, Kido S, et al. Snail accelerates cancer invasion by upregulating MMP expression and is associated with poor prognosis of hepatocellular carcinoma[J]. Br J Cancer, 2005, 92(2):252-258
[7］Pálmer HG, Larriba MJ, García JM, et al. The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer[J]. Nat Med, 2004, 10(9):917-919
[8］王二云, 赵丽丽, 高倩, 等. 脂肪间充质干细胞体外分化成心肌样细胞[J]. 中国生物化学与分子生物学报(Wang Er-Yun, Zhao Li-Li, Gao Qian, et al. In vitro differentiation of rat adipose-derived stem cells into cardiomyocytes[J]. Chin J Biochem Mol Biol), 2009, 25(9): 855-860
[9］Ries C, Egea V, Karow M, et al. MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines[J]. Blood, 2007, 109(9):4055-4063
[10］Fritzenwanker JH, Saina M, Technau U. Analysis of forkhead and snail expression reveals epithelial- mesenchymal transitions during embryonic and larval development of Nematostella vectensis[J]. Dev Biol, 2004,275(2):389-402
[11］Natalwala A, Spychal R, Tselepis C. Epithelial-mesenchymal transition mediated tumourigenesis in the gastrointestinal tract[J]. World J Gastroenterol, 2008, 14(24): 3792-3797
[12］谢林伸, 张蕾, 樊均明. 转录因子Snail调控肾脏上皮-间质转化的机制研究[J]. 现代预防医学(Xie Lin-Shen, Zhang Lei, Fan Jun-Ming. Mechanism of transcription factor snail regulating kidkey epithelial-mesenchymal transition[J]. Mod Prev Med), 2009, 36（9）: 1787-1788, 1790
[13］Yang D, Lu H, Hong Y, et al. Interpretation of X chromosome dose at sex-lethal requires non-E-box sites for the basic helix-loop-helix protein SISB and daughterless[J]. Mol Cell Biol, 2001,21(5):1581- 1592
[14］Jorda M, Olmeda D, Vinyals A, et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor[J]. J Cell Sci, 2005,118(Pt 15):3371-3385
[15］卓文磊，张云嵩，王彦，等．RNA干扰抑制Snail表达对A549细胞上皮-间充质转分化及体外侵袭的影响[J]．现代肿瘤医学(Zhuo Wen-Lei, Zhang Yun-Song, Wang Yan, et al. Inhibition of snail expression by RNA interference repress epithelial-mesenchymal transition and invasion ability of human lung carcinoma cell A549 in vitro[J], J Mod Oncol), 2008, 16（6）: 889-893
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脚注
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基金
山东省自然科学基金(No.Zr2009cm047)资助项目
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