MASPIN真核表达载体的构建及对胃癌细胞SGC7901的诱导凋亡作用

Construction of a Eukaryotic Vector Expressing MASPIN Gene and Its Effect on Cell Apoptosis in Gastric Cancer Cell Line SGC7901

背景与目的:MASPIN基因与多种恶性肿瘤的发生密切相关,在细胞的生长、浸润、转移和凋亡中起着十分重要的作用。本研究通过构建MASPIN基因真核表达载体,分析MASPIN过表达对胃腺癌细胞株SGC7901凋亡的影响。方法:构建MASPIN/PCR2.1表达载体,转染胃癌细胞株SGC7901。RT-PCR和Western bolt检测MASPIN基因、Bax/Bcl-2基因表达变化;琼脂糖凝胶电泳检测凋亡特征性DNA梯形带;采用AnnexinV-FIFC/PI检测凋亡率。结果:酶切证实成功构建真核表达质粒MASPIN/PCR2.1;转染重组质粒组MASPIN mRNA(33.6±1.2)和蛋白水平(23.4±1.6)的表达明显高于未处理组(13.7±2.0、12.0±1.3)和空质粒组(15.0±1.5、12.3±1.5,P<0.05);凝胶电泳观察到特征性DNA梯带;各组细胞均有凋亡率的增加且呈时间依赖性:转染重组质粒∕TRAIL作用组最高,12、24、48h分别达到8.0%、16.3%、25.8%,转染重组质粒组为3.0%、8.2%、14.4%,TRAIL作用组为4.1%、9.8%、15.9%(P<0.05);48h转染重组质粒∕TRAIL组BaxmRNA和蛋白表达(55.3±2.1、75.4±1.3)高于转染重组质粒组(34.3±1.2、40.7±1.8,P<0.05)和TRAIL作用组(43.2±1.8、36.2±1.3,P<0.05);48h转染重组质粒∕TRAIL组、转染重组质粒组、TRAIL作用组Bcl-2mRNA表达为28.3±2.5、34.3±1.2、32.8±2.1(P<0.05),蛋白表达为17.4±1.5、45.1±2.1、42.8±1.5(P<0.05)。结论:上调MASIPIN基因的表达能显著增加胃癌细胞对凋亡刺激的敏感性。其机制可能通过上调Bax,下调Bcl-2基因的表达诱导胃癌细胞凋亡。

BACKGROUND ＆ OBJECTIVE： MASPIN gene is closely associated with carcinogengsis and plays a key role in cell proliferation, adhesion, migration and apoptosis. This study was to construct a recombinant eukaryotic vector expressing MASPIN, and explore the effect of MASPIN overexpression on the apoptosis in human gastric carcinoma cell line SGC7901. METHODS. A eukaryotic expression vector MASIPIN/PCR2.1 was constructed and transfected into SGC7901 cells. RT-PCR and Western blot were used to detect the expression changes of MASPIN and Bax/Bcl-2. TNF- related apoptosis inducing ligand （TRAIL） （50ng/ml） was used to induce apoptosis at different time courses. DNA apoptotic ladders were determined using agarose gel electrophoresis （AGE）. Cell apoptosis was measured by flow cytometry （FCM）. RESULTS： Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein levels of MASPIN were significantly higher in the MASPIN/PCR2.1 group （33.6±1.2, 23.4±1.6） than in the PCR2.1（15.0±1.5, 12.3±1.5）and the untreated group （13.7±2.0, 12.0±1.3） （P〈0.05）. After transfection of MASIPIN/PCR2.1, DNA apoptotic ladders appeared in SGC7901 cells and the induction of apoptosis was in a time-dependent manner. The apoptosis rates were 8.0%, 16.3% and 25.8%in the MASPIN/PCR2.1 plus TRAIL group, 3.0%, 8.2%, 14.4% in the MASPIN/PCR2.1 group, and 4.1%, 9.8%,15.9% in the TRAIL group at 12, 24, and 48 h （P〈0.05）. The expression levels of Bax mRNA and protein at 48 h after MASIPIN/PCR2.1 transfection were significantly higher in MASPIN/PCR2.1 plus TRAIL group（55.3±2.1, 75.4±1.3） than in the PCR2.1 group （34.3±1.2, 40.7±1.8） and the TRAIL group （43.2 ±1.8,36.2±1.3） （P〈0.05）. The expression of Bcl-2 mRNA in the MASPIN/PCR2.1 plus TRAIL group, PCR2.1 group and TRAIL group were 28.3±2.5, 34.3±1.2, 32.8±2.1, respectively （P〈0.05）, and those of Bcl-2 protein were 17.4±1.5, 45.1± 2.1, 42.8±1.5 in the three groups, respectively （P〈0.05）. CONCLUSIONS. upregulation of MASPIN/PCR2.1 can significantly enhance the sensibility of gastric cancer cell line SGC7901 to the apoptosis inducer. This maybe related to the upregulation of Bax and downregulation of Bcl-2.