肼屈嗪对人胃癌细胞株RUNX3基因甲基化及其表达的调节作用

Effect of Hydralazine on Methylation and Expression of RUNX3 Gene in Human Gastric Cancer Cell Lines

背景：甲基化所致的抑癌基因RUNX3表达沉默是胃癌发生的重要机制，以脱甲基化制剂恢复其表达可起到抗肿瘤作用。目的：研究脱甲基化制剂肼屈嗪对人胃癌细胞株RUNX3基因甲基化及其表达的调节作用，观察肼屈嗪对胃癌细胞生长和凋亡的影响。方法：分别以RT—PCR和甲基化特异性PCR（MSP）检测肼屈嗪和5-Aza-dC处理前后SGC7901、MKN28和MGC803细胞的RUNX3mRNA表达及其甲基化状态。以MTT法检测MKN28细胞增殖活性．以流式细胞术检测细胞周期和细胞凋亡。结果：MKN28细胞存在甲基化所致的RUNX3基因表达沉默。40μmol／L肼屈嗪作用72h后，MKN28细胞可扩增出RUNX3非甲基化条带．呈部分脱甲基化，RUNX3mRNA恢复表达，但相对表达量低于5-Aza—dC组（P〈0．05）。10μmol／L以上浓度的肼屈嗪对MKN28细胞生长具有抑制作用，可使细胞周期阻滞于G0／G1期，并诱导细胞凋亡（P〈0．05）。结论：肼屈嗪可通过脱甲基化恢复RUNX3基因表达并能抑制MKN28细胞生长，诱导细胞凋亡。有必要对肼屈嗪在胃癌治疗中的作用作进一步研究。

Background： Silencing of tumor suppressor gene RUNX3 by aberrant methylation is a crucial mechanism in the development of gastric cancer. Restoring the expression of RUNX3 by demethylation agents might achieve an anti-tumor effect. Aims： To investigate the effect of demethylation agent hydralazine on RUNX3 gene methylation and expression in human gastric cancer cell lines, and its influence on growth and apoptosis of gastric cancer ceils. Methods： RUNX3 mRNA expression and methylation status in SGC7901, MKN28 and MGC803 cells were examined by RT-PCR and methylation-speeific PCR （MSP） prior to and after hydralazine and 5-Aza-dC treatment. MTT and flow cytometry assays were used to observe the changes of proliferation activity, cell cycle, and apoptosis of MKN28 cells. Results： RUNX3 gene was silenced in MKN28 cells as a result of hypermethylation. After treated with 40 μmol/L hydralazine for 72 hours, unmethylated band representing partial demethylation could be detected in MKN28 cells, and the expression of RUNX3 mRNA was restored. The expression level of RUNX3 mRNA in hydralazine group, however, was inferior to 5-Aza-dC group （P〈0.05）. Proliferation inhibition, cell cycle arrest in G0/G1 phase and apoptosis could be observed in MKN28 cells when the dosage of hydralazine ≥ 10 μmol/L （P〈0.05）. Conclusions： Hydralazine restores RUNX3 gene expression by demethylation, and inhibits proliferation, induces apoptosis in MKN28 cells. The use of hydralazine in the treatment of gastric cancer is worthy of further investigation.