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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MAD2siRNA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Westernblot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。MTT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MAD2siRNA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。
Objective To construct eukaryotic expression vector of MAD2 (mitotic arrest defect protein 2) specific RNA interference and observe the effect of inhibiting MAD2 gene expression on the growth and cell cycle of gastric cancer cells in vitro. Methods The eukaryotic expression vector of MAD2 siRNA was constructed. The empty vector pSilencer3.1 and the eukaryotic expression vector pSilencer3.1 / MAD2-siRNA1 and pSilencer3.1 / MAD2-siRNA2 were respectively introduced into human gastric cancer cell line SGC7901 by lipofectamine. After stable transfection, the expression of MAD2 protein and mRNA in gastric cancer cells was detected by Western blot and RT-PCR, and the single clone with the best inhibitory effect was selected. The cell growth of each experimental group was detected by MTT assay. The distribution of cell cycle in gastric cancer cell MAD2-siRNA transfected group and empty vector group was detected by flow cytometry after vincristine was used as spindle inhibitor. Results The eukaryotic expression vector of MAD2siRNA was successfully constructed and the expression of MAD2 protein and mRNA was significantly down-regulated in SGC7901 cells. The growth of gastric cancer cells with decreased expression of MAD2 was significantly increased (P <0.01), and could not be arrested in mitosis after vincristine treatment. Conclusion Small interfering RNA technology can effectively inhibit the expression of MAD2, a key protein in the spindle checkpoint of gastric cancer cells. Inhibition of MAD2 can accelerate the growth of SGC7901 gastric cancer cells, enhance the proliferative capacity, and weaken the inhibitor of vincristine block cell cycle.