TGF-β/Smads信号通路对胃肠间质瘤细胞增殖和侵袭能力的影响

Effects of TGF-β/Smads signaling pathway on proliferation and invasion of gastrointestinal stromal tumor cells

目的:探讨转化生长因子β(transforming growth factor-β,TGF-β)/Smads信号通路对胃肠间质瘤(gastrointestinal stromal tumor,GIST)细胞增殖、侵袭能力的影响及可能的作用机制。方法:分别采用外源性TGF-β1单独或联合不同浓度的SB-431542处理GIST882细胞,激活和阻断GIST882细胞的TGF-β/Smads信号转导通路;采用MTT法检测激活和阻断TGF-β/Smads信号通路对GIST882细胞增殖能力的影响,Transwell小室侵袭实验检测对GIST882细胞侵袭能力的影响;实时荧光定量PCR和蛋白质印迹法检测激活和阻断TGF-β/Smads信号通路后对GIST882细胞中Slug mRNA和蛋白表达的影响。采用siRNA干扰的方法沉默Smad3基因的表达,并采用实时荧光定量PCR和蛋白质印迹法检测Smad3基因的沉默效率,及对GIST882细胞中Slug mRNA及蛋白表达的影响。结果:TGF-β1激活TGF-β/Smads信号通路后,GIST882细胞的增殖活性和侵袭能力均明显增高(P值均<0.05),而SB-431542阻断TGF-β/Smads信号通路后GIST882细胞的增殖活性和侵袭能力均较TGF-β1单药组明显下降(P值均<0.05)。激活TGF-β/Smads信号通路后,GIST882细胞中转录因子Slug mRNA和蛋白的表达水平明显上调(P值均<0.01);而采用SB-431542阻断该信号通路后Slug mRNA和蛋白的表达水平均较TGF-β1单药组明显下调(P值均<0.05),且呈浓度依赖性。Smad3-siRNA2能有效沉默GIST882细胞中Smad3基因的表达,Slug mRNA和蛋白水平也明显下调(P<0.05和P<0.01)。结论:TGF-β/Smads信号通路能介导GIST882细胞的增殖和侵袭能力,调控Slug的表达,且该调控是Smad3依赖性的。Slug可能在TGF-β/Smads信号通路介导的GIST转移过程中发挥重要的作用。

Objective： To investigate the effects of transforming growth factor-β （TGF-β）/ Smads signaling pathway on proliferation and invasion of gastrointestinal stromal tumor （GIST） cells, and to explore the possible mechanisms. Methods： The exogenous TGF-J31 alone and in combination with different concentrations of SB-431542 were used to treat GIST882 cells for activating and blocking TGF-β/Smads signaling pathway, respectively. Then the proliferation of GIST882 cells was detected by MTT method, the invasive ability of GIST882 cells was detected by Transwell chamber invasion assay, and the expression levels of Slug mRNA and protein in GIST882 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The Smad3-siRNA was transfected into GIST882 cells for silencing the expression of Smad3 gene, then the expression levels of Slug mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results： After TGF-β/Smads signaling pathway was activated by TGF-β1, the proliferation and invasion of GIST882 cells were significantly increased （both P 〈 0.05）, and the expressions of Slug mRNA and protein were up- regulated in GIST882 cells （both P 〈 0.01）. After TGF-β/Smads signaling pathway was blocked by SB-431542, the proliferation and invasion of GIST882 cells were significantly decreased （both P 〈 0.05）, and the expressions of Slug mRNA and protein in GIST882 cells were significantly down-regulated in a concentration-dependent manner （both P 〈 0.05）. The expression of Smad3 gene in GIST882 cells was effectively silenced by Smad3-siRNA2, then the expression levels of Slug 3 mRNA and protein were significantly down-regulated （P 〈 0.05 and P 〈 0.01）. Condusion： TGF-β/Smads signaling pathway can mediate the proliferation and invasion of GIST882 cells, and regulate the expression of Slug which is dependent on Smad3 expression. Slug maybe play an important role in the process of TGF-β/Smads pathway-mediated metastasis of GIST882 cells.