摘要
目的分析RNA干扰亚甲基四氢叶酸还原酶（methylenetetrahydrofolate reductase，MTHFR）基因表达后对人胃癌细胞生存活力及生长周期的影响。方法构建含人MTHFR靶点的小分子干扰RNA真核表达载体，将其转染入人胃癌MKN45细胞。以PCR检测转染结果，Wester印迹法证实转染影响MTHFR的蛋白表达，以四甲基偶氮唑盐微量酸反应比色法检测细胞生存活力，流式细胞术检测细胞周期改变。结果特异性干扰MTHFR表达使胃癌MKN45细胞生存活力明显降低，仅为对照组的65％（0．188±0．015比0．277±0．024，P〈0．01）。同时细胞形态发生改变，由原先饱满、边缘光整的多边形细胞变为细长梭形多触角细胞。流式细胞术检测发现细胞周期主要受阻在G2／M期（P〈0．01）。结论RNA干扰MTHFR表达后能影响胃癌细胞生存活力及阻滞细胞周期进程，可望成为肿瘤基因防治的新靶点。
Objective To investigate the effect of RNA interference mediated inhibition of methylenetetrahydrofolate reductase （MTHFR） on survival of human gastric cancer cells. Methods Human gastric cancer cell line, MKN45 was cultured. Recombinant plasmid containing MTHFR siRNA was constructed. Then the recombinant siRNA plasmid and scrambled siRNA plasmid were transfected into MKN45 cells respectively by using lipofectamine. PCR and Western blot were used to confirm the successful transfection. Cell viability was analyzed by 3-（ 4,5-dimethylthiazol-2-yl）- 2,5-diphenyl tetrazolium bromide. Cell cycle was determined by flow cytometry （FCM）. Results Specific antisense against MTHFR inhibited growth of gastric cancer cells by 65 % in comparison with control （0. 188 ± 0. 015 vs. 0. 277 ± 0. 024, P〈0.01）. And cell morphous changed from round polygon to slender and fusiform after RNA interference. FCM showed that MTHFR inhibition caused an arrest of the cell cycle at G2/M phase. Conclusions MTHFR influences the cell viability and the cell cycle arrest in human gastric cancer cell line MKN45, and might be a target for the prevention of gastric cancer.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2008年第1期30-33,共4页
Chinese Journal of Digestion
基金
国家自然科学基金资助项目（30470781）
关键词
胃肿瘤
亚甲基四氢叶酸还原酶
甲硫氨酸
RNA干扰
Stomach neoplasms
Methylenetetrahydrofolate reductase
Methionine
RNA interference
