N-乙酰氨基葡萄糖转移酶-V过表达对人肝癌细胞迁移及粘附分子表达的影响

GnT-V OVEREXPRESSION IN HUMAN HEPATOCARCINOMA CELLS AFFECTS ITS MIGRATION AND EXPRESSION OF CELL ADHESION MOLECULES

为了探讨转染N-乙酰氨基葡萄糖转移酶Ⅴ(GnT-V)正义cDNA后对7721细胞迁移影响及其机制,我们研究了GnT-V/7721和pcDNA3/7721(对照组)两株细胞的迁移力及其与侵袭转移能力密切相关的细胞表面重要粘附分子整合蛋白和E-钙粘蛋白的表达情况。通过琼脂滴法检测两株细胞的迁移力;间接免疫荧光法测定细胞表面整合蛋白α5和β1的含量及用RT-PCR方法检测细胞整合蛋白α5和β1的mRNA水平;免疫细胞化学ABC法检测了细胞E-钙粘蛋白表达水平;Western杂交方法检测β-连环蛋白含量。结果发现,7721细胞经转染GnT-V cDNA后,迁移力明显增高;整合蛋白α5亚基的含量比对照组增加2.9倍;β1亚基未见明显变化。α5亚基mRNA水平为对照细胞的2.1倍,β1亚基的mRNA含量无明显改变。GnT-V/7721细胞E-钙粘蛋白及β-连环蛋白表达也有不同程度的升高。本文结果提示与N-糖链加工有关的GnT-V过表达,可促进7721细胞表面整合蛋白的表达以及E-钙粘蛋白β-连环蛋白的表达,从而增加肿瘤细胞的迁移能力。

The purpose of this paper is to study the effect of N-acetylglucosaminyltransferase V (GnT-V) overexpression on the migration of 7721 cells and its mechanism. The abilities of migration of both 7721 cells transfected with GnT-V cDNA and 7721 cells transfected with pcDNA3 was detected, the expressions of integrin and E-cadherin which are important adhesion molecules on surface membrane and closely related to the abilities of invasion and metastasis. Cell migration abilities were measured by the agarose drop explant method. Flow cytometric analysis (FACS) was applied to determine the relative amounts of integrin α5 and β1 subunits on the cell surface while RT-PCR was carried out to determine the expressions of their mRNA. The expression of E-cadherin was examined by the immunocytochemical ABC method. Western blot analysis was carried out to examine the expression of β-catenin. GnT-V overexpression enhanced evidently the migration ability of 7721 cells and increased the amount of integrin β5 subunit to 2. 9 times of that of control while the amount of β1 subunits was not significantly changed. Besides, the expressions of E-cadherin and β-catenin were enhanced at different levels in GnT-V/7721 cells compared with mocked. The results suggested that the over-expression of GnT-V related to the production of N-linked sugar chains could promote the expressions of integrin, E-cadherin and β-catenin on 7721 cells so that the migration ability of tumor cells was enhanced.