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目的:探讨Grb2协同结合蛋白2(Grb2binding protein-2,Gab2)对人肺癌A549细胞体外迁移和侵袭的影响及其机制。方法:将质粒pGCsilencerU6/GFP/Neo-RNAi-Gab2#1、pGCsilencerU6/GFP/Neo-RNAi-Gab2#2和对照空载质粒转染到A549细胞中,建立Gab2低表达的siGab2/A549#1、siGab2/A549#2细胞和对照SCR/A549细胞。应用RT-PCR和蛋白质印迹法检测小分子干扰RNA(siRNA)干扰Gab2表达后的基因和蛋白表达水平。趋化运动实验检测细胞的迁移能力;体外侵袭实验检测细胞的侵袭能力;蛋白质印迹法检测siRNA干扰Gab2表达后,A549细胞在胰岛素样生长因子-1(insulinlike growth factor-1,IGF-1)刺激下基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metal-loproteinase-9,MMP-9)的表达及磷酸化Akt(pAkt)、磷酸化人雷帕霉素靶蛋白(phosphorylated mammalian target of rapamy-cin,pmTOR)的活化情况。结果:siRNA干扰Gab2表达后,RT-PCR测定A549细胞Gab2mRNA相对表达量为1.340±0.009,Scr/A549细胞为1.201±0.074,siGab2#1/A549细胞为0.315±0.008,siGab2#2/A549细胞为0.289±0.007。蛋白印迹法检测A549细胞Gab2蛋白表达量为0.205±0.003,Scr/A549细胞为0.241±0.004,siGab2#1/A549细胞为0.128±0.002,siGab2#2/A549细胞为0.066±0.001。siGab2#2/A549细胞与A549细胞比较Gab2基因表达显著降低,t=168.032,P<0.001;Gab2蛋白表达也显著降低,t=64.352,P<0.001。siGab2#2/A549细胞的体外迁移和侵袭能力明显低于SCR/A549细胞,t值分别为5.101和10.812,P值分别为0.029和0.002。在IGF-1刺激下,siGab2/A549细胞的MMP-2、MMP-9蛋白的表达量不再有明显变化,t值分别为-2.051和-2.652,P值分别为0.054和0.064。siGab2/A549细胞中pAkt、pmTOR的活化也显著抑制。结论:Gab2可能通过调节Akt/mTOR信号通路,促进A549细胞的侵袭能力。
AIM: To investigate the effect of Grb2binding protein-2 (Gab2) on the migration and invasion of human lung cancer A549 cells in vitro and its mechanism. METHODS: The plasmid pGCsilencerU6 / GFP / Neo-RNAi-Gab2 # 1, pGCsilencerU6 / GFP / Neo-RNAi-Gab2 # 2 and control vector were transfected into A549 cells to construct a low expression of siGab2 / siGab2 / A549 # 2 cells and control SCR / A549 cells. The expression of Gab2 gene and protein was detected by RT-PCR and Western blotting. Chemotaxis assay was used to detect the migration ability of cells. In vitro invasion assay was used to detect the invasion ability of cells. Western blotting was used to detect the effect of siRNA on Gab2 expression induced by insulin-like growth factor-1 (IGF-1) The expression of matrix metalloproteinase-2 (MMP-2), matrix metal-loproteinase-9 (MMP-9) and phosphorylated Akt (pAkt) Activation of phosphorylated mammalian target of rapamy-cin (pmTOR). Results: The relative expression level of Gab2 mRNA in A549 cells was 1.340 ± 0.009 by RT-PCR, 1.201 ± 0.074 in Scr / A549 cells, 0.315 ± 0.008 in siGab2 # 1 / A549 cells and 0.289 ± 0.007. The expression of Gab2 protein in A549 cells was 0.205 ± 0.003 by Western blotting, 0.241 ± 0.004 in Scr / A549 cells, 0.128 ± 0.002 in siGab2 # 1 / A549 cells and 0.066 ± 0.001 in siGab2 # 2 / A549 cells. Compared with A549 cells, the expression of Gab2 gene in siGab2 # 2 / A549 cells was significantly decreased (t = 168.032, P <0.001). The expression of Gab2 protein was also significantly decreased, t = 64.352, P <0.001. The ability of siGab2 # 2 / A549 cells to migrate and invade in vitro was significantly lower than that of SCR / A549 cells with t values of 5.101 and 10.812, respectively, with P values of 0.029 and 0.002, respectively. Under the stimulation of IGF-1, the expression of MMP-2 and MMP-9 in siGab2 / A549 cells showed no significant change, with t values of -2.051 and -2.652, respectively, with P values of 0.054 and 0.064, respectively. Activation of pAkt and pmTOR was also significantly inhibited in siGab2 / A549 cells. Conclusion: Gab2 may promote the invasiveness of A549 cells by regulating the Akt / mTOR signaling pathway.