Hydralazine逆转胃癌细胞株p16基因甲基化及增强其表达的研究

目的: 通过使用甲基转移酶抑制剂肼屈嗪(Hydralazine)作用于胃癌细胞株BGC-823,观察Hydralazine对MTS1/p16基因启动子区CpG岛甲基化、p16 mRNA、p16蛋白表达及细胞增殖的影响;分析p16基因启动子区CpG岛甲基化与p16基因表达两者间的关系,以此考察胃癌细胞株BGC-823 p16基因的失活机制是否与启动子区CpG岛甲基化有关。方法: 对体外培养的胃癌细胞株BGC-823进行分组实验,实验组加入水溶性的Hydralazine,浓度0、10、30、100 μmol/L,对照组不加Hydralazine,96 h 后对两组分别行甲基化特异性PCR法(MSP)、RT-PCR及Western blot检测,以观察p16基因甲基化状态、mRNA、蛋白表达的变化;应用6种不同浓度的水溶性Hydralazine(0、3、10、30、100、300 μmol/L)处理胃癌细胞BGC-823,72 h 后行MTT检测,以观察对细胞增殖的影响。结果: P16基因在胃癌细胞株BGC-823中,启动子区CpG岛呈高甲基化状态,mRNA及蛋白呈低水平表达。Hydralazine阻断甲基转移酶作用后,p16基因启动子区CpG岛呈去甲基化状态,Hydralazine浓度越高,CpG岛去甲基化越明显。Hydralazine阻断甲基转移酶作用后,用药后p16 mRNA表达较前明显增强(P<0.05)。Hydralazine阻断甲基转移酶作用后,用药后p16蛋白表达较前明显增强(P<0.01)。MTT检测到不同浓度的Hydralazine(0、3、10、30、100、300 μmol/L)对细胞增殖的抑制率差异有统计学意义(P<0.05),其量效关系反映出抑癌基因p16去甲基化程度越高,对胃癌细胞株BGC-823的增殖抑制越显著。结论: P16基因动子区CpG岛甲基化是p16基因失活的重要机制。

Hydralazine reverses human gastric cancer cell lines p16 methylation and reactivates its expression

AIM: To investigate the mechanism of p16 gene silencing and evaluate the effects of Hydralazine on gastric cancer cell lines in vitro.METHODS: Experimental group BGC-823 cells were treated with 10, 30, 100 μmol/L Hydralazine and control group cells were not treated. After 96 h, methylation specific PCR was used to detect the CpG island methylation state of p16 gene. The expression of p16 gene mRNA and protein were analyzed by RT-PCR and Western blot. BGC-823 cells were treated with six different concentrations of Hydralazine(0, 3, 10, 30, 100, 300 μmol/L)for 72 h, and MTT assay was used to observe the change of proliferation activity.RESULTS: P16 gene promoter region was hypermethylation in control group. P16 mRNA and protein were expressed at a low level in control group. In Hydralazine-treated group, p16 promoter region exhibited a demethylation state, and its mRNA and protein were expressed up-regulation.CONCLUSION: Promoter region hypermethylation is an important mechanism of p16 silencing. Hydralazine, an effective inhibitor of p16 methylation, can inhibit cell growth in human gastric cancer in vitro and be potentially used for the clinical treatment of human gastric cancer.