MAD2 RNA干扰载体的构建及其对胃癌细胞生长的影响

目的：构建MAD2（有丝分裂阻滞缺陷蛋白2）特异性RNA干扰真核表达载体,体外观察抑制MAD2基因表达对胃癌细胞生长和细胞周期的影响.方法：构建MAD2 siRNA真核表达载体,脂质体法将pSilencer3.1空载体和pSilencer3.1/MAD2-siRNA1和pSilencer3.1/MAD2-siRNA2真核表达载体分别导入人胃癌细胞系SGC7901.稳定转染后Western blot和RT-PCR检测胃癌细胞内MAD2蛋白及mRNA水平的表达情况,挑选出抑制效果最好的单个克隆.MTF法检测各实验组细胞生长情况,流式细胞术检测纺锤体抑制剂长春新碱作用后,胃癌细胞MAD2-siRNA转染组和空载体组细胞周期的分布.结果：成功构建MAD2 siRNA真核表达载体,转染胃癌细胞SGC7901可使其MAD2蛋白及mRNA表达显著下调.MAD2表达降低的胃癌细胞生长速度明显增快（P＜0.01）,经长春新碱作用后不能被阻滞于有丝分裂期.结论：小干扰RNA技术可以有效地特异性抑制胃癌细胞纺锤体检查点关键蛋白MAD2的表达.MAD2的抑制可使胃癌细胞SGC7901生长加速,增殖能力增强,同时减弱纺锤体抑制剂长春新碱阻滞细胞周期的作用.

Construction of eukaryotic expression vector of siRNA specific for MAD2 and its effect on the growth of gastric cell line SGC7901

AIM: To construct the RNA interference eukaryotic expression vector specific for human MAD2 gene and to observe its effect on the growth of gastric cancer cell line SGC7901. METHODS: The expression vectors of pSilencer3.1/MAD2-siRNA1 and pSilencer3.1/MAD2-siRNA2 were constructed by gene recombination and then were stably transfected into the gastric carcinoma cell line SGC7901 by liposome mediation. The expression of MAD2 on the levels of protein and mRNA was detected by Western blot and RT-PCR, and the monoclone with the highest inhibition efficiency was selected. The growth of the transfected cells was assessed by MTT. And the cells treated with 1.0 mg/L vincristine (VCR) for 24 h were analyzed by FCM for cell cycle. RESULTS: Sequence-specific siRNAs targeting MAD2 significantly down regulated the expression of MAD2 in SGC7901 cells. In MAD2-siRNA transfected cells, the rate of cell growth increased markedly and cell cycle couldn't be arrested in M phase induced by VCR, while the cells transfected with the mock vector could. CONCLUSION: Down regulation of MAD2 expression of SGC7901 bv sequence-specific siRNA could accelerate the cell growth and impair the mitosis arrest of SGC7901 induced by VCR.