亚甲基四氢叶酸还原酶对胃癌细胞基因表达的影响

Effect of eukaryotic expression plasmid containing methylenetetrahydrofolate reductase gene on transcriptional level of tumor-related genes in human gastric cancer cell line

目的分析亚甲基四氢叶酸还原酶(MTHFR)基因的真核表达质粒对人胃癌细胞生存力、DNA甲基化酶基因及肿瘤相关基因转录水平的影响。方法分别构建含有人野生型(正义)MTHFR(W)和反义MTHFR(A)的真核表达质粒,将其转染入人胃癌MKN45细胞,噻唑蓝(MTT)法检测细胞生存活力,定量PCR检测DNA甲基化酶及胃癌相关基因的转录水平。结果转染野生型MTHFR质粒的胃癌细胞生存活力增高(P＜0．01),而转染反义质粒者生存活力降低(P＜0．01)。野生型MTHFR质粒下调c-myc、p21^(WAFI)和hMLH1基因表达,反义MTHFR质粒的转染则无明显的基因调控作用。结论重组MTHFR表达质粒可影响细胞生存活力及人胃癌细胞相关基因的表达,可望成为肿瘤基因防治的新靶点。

Objective To analyze the effect of eukaryotic plasmids containing wild （sense） or antisense methylenetetrahydrofolate reductase （MTHFR） gene on cell viability and transcription level of tumor related genes in human gastric cancer cell line, Methods Human gastric cancer cell line MKN-45 was cultured. Recombinant plasmids containing wild MTHFR （W） or antisense MTHFR （A） gene, pCMV-W and pCMV-A, were constructed. Then pCMV-W, pCMV-A and pCMV blank plasmid were transfected into MKN45 cells respectively by using lipofect. Cell viability was analyzed by 3-（4, 5-bime- thylthiazolyl-2）-2, 5-diphenyltetrazolium dromide（MTT）. The transcription levels of Dnmt 1, c-myc, p21wan and hMLH1 genes were detected by real-time polymerase chain reaction（PCR）. Results Cell viability remarkably increased in those transfected with wild MTHFR （P 〈 0, 01）, which was contrary to those transfected with antisense MTHFR（P 〈 0.01）. The expression of those tumor related genes mRNAs were all remarkably decreased in the MKN45-W cells in comparison with those in the MKN45-pCMV cells, No significant difference in the expressions of those tumor related genes mRNAs were found between the MKN45 cells transfeeted with pCMV-A and blank pCMV. Conclusion MTHFR influences cell viability and the expression level of tumor related genes in human gastric cancer cell line MKN45.