过表达PI3K p55γ N末端氨基酸可抑制MGC803胃癌细胞的黏附性

Overexpression of N-terminal amino acids of p55γ regulatory subunit of PI3K inhibits cell adhesion of human gastric carcinoma MGC803 cells

目的:研究磷脂酰肌醇3-激酶(PI3K)p55γ调节亚基N末端24个氨基酸(N24)过表达对胃癌细胞MGC803黏附性的影响,并初步探讨其作用的分子机制。方法:通过脂质体介导用p EGFP-N24和p EGFP-C1质粒分别进行基因转染,建立稳定表达融合蛋白GFP-N24和GFP的MGC803细胞系。倒置显微镜下观察转染细胞的形态学变化,免疫印迹实验鉴定转染基因的蛋白表达,细胞黏附实验检测转染细胞的黏附性,免疫印迹实验检测细胞黏附分子上皮细胞钙黏蛋白(E-cadherin)和钙紧张素(β-catenin)的表达,酶谱实验检测基质金属蛋白酶9(MMP9)和尿激酶型纤溶酶原活化因子(u PA)的表达。结果:建立了稳定表达融合蛋白GFP-N24的MGC803/GFP-N24细胞系和表达GFP的MGC803/p EGFP-C1细胞系,但GFP-N24的表达量明显低于GFP。基因转染使MGC803细胞的形态由铺路石样转变为成纤维细胞样,胞浆分泌颗粒明显增加。表达GFP-N24的MGC803细胞在纤黏连蛋白和胶原上的黏附性与对照组细胞相比明显下降(P<0.05)。GFP-N24的表达使MGC803细胞黏附分子E-cadherin的表达增高,而Wnt信号通路关键分子β-catenin的表达降低,但不影响肿瘤转移相关蛋白MMP9和u PA的表达与分泌。结论:PI3K p55γN末端氨基酸过表达通过影响E-cadherin和β-catenin的表达而抑制胃癌MGC803细胞的黏附性。

AIM：To investigate the effect of the N-terminal 24-amino acid （N24） overexpression in p55γre-gulatory subunit of phosphatidylinositiol 3-kinase （ PI3K） on the cell adhesion of human gastric carcinoma cell MGC 803. METHODS：MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone , were generated by transfec-tion with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively.The morphological change of the cells was ob-served under inverted microscope .The expression of GFP-N24 fusion protein was detected by Western blot .The adhesion of the cells was determined by cell adhesion assay .The effects of GFP-N24 fusion protein on the expression of E-cadherin andβ-catenin were analyzed by Western blot .The expression and secretion of matrix metalloproteinase 9 （MMP9） and u-rokinase-type plasminogen activator （ uPA ） in the MGC803 cells were detected by gelatin zymography .RESULTS：MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established , but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP.The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection , and the cytoplasm secretory granules were increased significantly .The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection .GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and de-creased the wnt signal pathway molecule β-catenin in the MGC803 cells.However, it did not affect the expression and se-cretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION：Overexpression of N24p55γinhibits cell ad-hesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.