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目的恶性黑色素瘤主要由皮肤黑色素细胞的恶性转化引起。Grb2协同结合蛋白2(Grb2-associated binding protein-2,GAB2)是一个脚手架蛋白,通过招募受体酪氨酸激酶等膜受体进行信号传递,已经证实GAB2蛋白与多种肿瘤的发生和转移有关,但GAB2蛋白抑制黑色素瘤的机制还不清楚。本研究通过沉默GAB2基因对SK-Mel28细胞转移能力的影响,探讨GAB2基因促进SK-Mel28黑色素瘤细胞转移的细胞信号转导通路变化。方法采用RNA干扰技术沉默GAB2基因表达。采用Matrigel法检测SK-Mel28细胞在沉默GAB2基因表达后48h对细胞转移能力的影响;采用细胞划痕实验检测SK-Mel28细胞在沉默GAB2基因表达后细胞迁移能力的改变;蛋白质印迹法检测PI3K和MAPK信号通路的关键蛋白、pAKT、pERK、pPDK1和β-catenin的蛋白表达水平。结果沉默GAB2基因可使SK-Mel28细胞的转移能力和迁移能力减弱,统计学分析显示,SK-Mel28细胞的转移能力降低了54.5%,与scramble RNA对照组比较差异有统计学意义,t=5.053,P<0.001;SK-Mel28细胞在转染GAB2siRNA 18h后细胞的迁移能力减弱,与scramble RNA对照组比较迁移距离减少了43.0%,差异有统计学意义,t=3.877,P=0.001。SK-Mel28黑色素瘤细胞的GAB2蛋白表达水平降低后,PI3K信号通路的关键分子pAKT和pPDK1表达下调,而T-AKT和T-PDK1无明显变化,细胞膜表面粘附因子β-catenin蛋白表达下降。结论 GAB2蛋白的表达水平与SK-Mel28黑色素瘤的转移相关,其机制与GAB2蛋白调控下游的PI3K信号通路并抑制转移相关因子β-catenin有关。
Purpose Malignant melanoma is mainly caused by the malignant transformation of cutaneous melanocytes. Grb2-associated binding protein-2 (GAB2) is a scaffolding protein that has been shown to be involved in the development and metastasis of multiple tumors by signaling through membrane receptors such as receptor tyrosine kinases , But the mechanism by which GAB2 protein inhibits melanoma is unclear. In this study, we investigated the effect of GAB2 gene on SK-Mel28 cell metastasis by silencing the expression of GAB2 gene, and discussed the changes of cell signal transduction pathways that GAB2 gene promoted the SK-Mel28 melanoma cell metastasis. Methods RNA interference technique was used to silence GAB2 gene expression. Matrigel assay was used to detect the effect of GAB2 gene silencing on SK-Mel28 cells 48h after 48h silencing of cell migration. Cell viability was detected by cell scratch assay in SK-Mel28 cells after silenced GAB2 gene expression. Western blotting was used to detect PI3K and MAPK signaling pathway of key proteins, pAKT, pERK, pPDK1 and β-catenin protein expression levels. Results The silencing of GAB2 gene reduced the metastatic potential and migration ability of SK-Mel28 cells. Statistical analysis showed that the metastatic potential of SK-Mel28 cells decreased by 54.5%, which was significantly different from that of scramble RNA control group (t = 5.053 , P <0.001. The migration ability of SK-Mel28 cells after transfection of GAB2 siRNA 18h was weaker than that of scramble RNA control group (43.0%), the difference was statistically significant (t = 3.877, P = 0.001). The decrease of GAB2 protein expression in SK-Mel28 melanoma cells resulted in the down-regulation of pAKT and pPDK1, but no significant change of T-AKT and T-PDK1 expression, while decreased the expression of β-catenin on cell membrane. Conclusions The expression level of GAB2 protein is related to the metastasis of SK-Mel28 melanoma. The mechanism of GAB2 protein is related to the regulation of PI3K signaling pathway and the inhibition of metastasis-related factor β-catenin.