摘要
目的探讨Smad4基因对人结肠癌细胞中Wnt/β-catenin信号通路的影响。方法将Smad4真核表达质粒以脂质体介导方法转染结肠癌SW480细胞,G418筛选稳定表达株,TOPFLASH/FOPFLASH双荧光素酶报告基因方法检测转染前后SW480细胞中Wnt信号通路活性,并检测Wnt通路关键调节因子β-catenin及通路下游Claudin-1蛋白的表达变化,免疫荧光细胞化学法观察β-catenin在SW480细胞中的分布变化。结果成功构建Smad4稳定表达的结肠癌细胞模型,转染组Wnt通路信号活性较转染前明显降低,Smad4重建能降低β-catenin mRNA及蛋白表达水平,并导致β-catenin发生自胞浆、胞核向胞膜的转位,同时能降低Claudin-1的蛋白表达水平。结论 Smad4基因能抑制结肠癌细胞中Wnt/β-catenin信号通路的活性,提示Smad4除了参与经典的TGF-β信号通路外,还可能通过Wnt信号途径发挥肿瘤抑制作用。
【Objective】To analyze the effect of Smad4 restoration on Wnt/β-catenin signaling pathway in human colon cancer cell line SW480. 【Methods】Colon cancer cell line SW480 were transfected with the eukaryotic ex- pression plasmid using lipofectamine reagent, the stable expression cells were screened by G418. Subsequently, Wnt pathway activities were measured by TOPFLASH/FOPFLASH reporter plasmids, western blotting were carried out with antibodies toβ-catenin, Claudin-1, at last, immuno-fluorescence staining analysis of β-catenin were done. 【Results】Stable colon cancer cells that have increased Smad4 expression were created, relative luciferase activities were markedly inhibited in Smad4-SW480 cells as compared with SW480. Smad4 restoration decreased β-catenin expression and alters its localization from cytoplasm to the plasma membrane in SW480 cells, at the same time, up- regulation of Smad4 also decreased the expression of Claudin-1. 【Conclusions】Smad4 restoration significantly in- hibits the activity of Wnt signaling pathway in colon cancer cells. Results from the study demonstrate that a novel mechanism underlying the Smad4 tumor-suppressive function through the regulation of the Wnt signaling pathway.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2013年第33期11-16,共6页
China Journal of Modern Medicine
