N-乙酰氨基葡萄糖转移酶Ⅴ过表达对人肝癌细胞粘着斑复合物介导的信号转导作用

Modulation of Focal Adhesion Complex Mediated Signaling in Human Hepatocarcinoma Cells through Over-expression of N-Acetylglucosaminyltransferase Ⅴ

为了探讨过表达N 乙酰氨基葡萄糖转移酶Ⅴ (GnT Ⅴ )后 772 1细胞侵袭、迁移等行为改变的机制 ,检测了GnT Ⅴ 772 1及pcDNA3 772 1两组细胞中与恶性表型密切相关的粘着斑激酶 (focalad hesionkinase ,FAK)、PTEN蛋白、蛋白激酶B(PKB)等重要信号分子的表达水平 ,同时测定了 2组细胞非贴壁依赖生长的能力 .利用Western印迹方法检测FAK、PTEN、PKB的表达或磷酸化水平 .利用poly hema使细胞非贴壁生长 ,2组细胞悬浮无血清培养 2 0h ,采用流式细胞仪方法检测细胞的失巢凋亡 (anoikis) .研究发现 ,转染GnT Ⅴ后的肝癌细胞的FAK表达无明显变化 ,FAK的酪氨酸磷酸化水平增高 70 %;而PTEN的表达下降了 4 9%;PKB的磷酸化增加 2 0 0 %;pcDNA3 772 1细胞已有明显凋亡 ,而转染GnT Ⅴ的 772 1细胞未发生凋亡 .结果提示 ,转染GnT Ⅴ后的肝癌细胞迁移力增强 ,可能与其FAK的磷酸化程度升高 ,激酶活力增强有关 ;而能逃逸失巢凋亡是因为PTEN的表达下降 ,PTEN蛋白的磷酸酶活性降低 ,细胞Akt PKB磷酸化水平保持在较高水平 .

To explore the machanisms that cause phenotype changes including increase of cell migration and invation after transfection of N-acetylglucosaminyltransferase Ⅴ(GnT-Ⅴ) cDNA into 7721 cells, the expressions and activities of focal adhesion kinase(FAK), PTEN and PKB were examined, which are commonly associated with phenotype such as migration and invation,and the growing ability independent of anchorage in the GnT-Ⅴ/7721 cells and pcDNA3/7721 cells was detected. Western blot assay was adopted to examine the expression or phosphorylation level of FAK, PTEN and PKB. Poly-hema was used to let the GnT-Ⅴ/7721 cells and pcDNA3/7721 cells grow independent of anchorage, and the cells were cultured in poly-hema dish without serum for 20 hours, Flow cytometry assay was used to detect anoikis. It was shown that FAK expression in the GnT-Ⅴ/7721 cells had no obvious changes while the phosphorylation level of FAK increased 70%, GnT-Ⅴ/7721 produced 49% PTEN in the control; the phosphorylation level of PKB enhanced 200% compared with the control; the apoptotic percentage in pcDNA3/7721 cells was 16.8% while no sub-G1 peak was observed in GnT-Ⅴ/7721 cells. The results proved that GnT-Ⅴ could increase the phosphorylation level of FAK,which, in turn could increase the kinase activity of FAK and promote migration and invation of GnT-Ⅴ/7721 cells; Reduction of PTEN level in GnT-Ⅴ/7721 cells caused a significant increase of the phosphorylation level of PKB,Which could prevent anoikis of tumor cells.