5Aza-dc对癌细胞TIMP-3启动子去甲基化的作用

目的：观察5-氮杂-2'-脱氧胞苷（5-Aza-2'-deoxycytidine，5Aza-dc）对癌细胞TIMP-3启动子甲基化的影响。 方法： 用5Aza-dc处理TIMP-3启动子甲基化的H2M肝癌细胞和A431表皮癌细胞，用Transwell检测癌细胞的侵袭及运动能力，用Western blot 检测TIMP-3的蛋白表达，用RT-PCR检测TIMP-3 mRNA的表达，用甲基化特异性PCR检测TIMP-3基因启动子的甲基化。 结果： （1）5Aza-dc作用后的H2M和A431细胞的侵袭及运动能力降低；（2）5Aza-dc作用后的H2M和A431细胞的TIMP-3蛋白及mRNA表达量增加；（3）5Aza-dc作用后的H2M和A431细胞的TIMP-3启动子区未检测到甲基化。 结论： 5Aza-dc可以使肝癌和表皮癌细胞TIMP-3启动子区去甲基化，使TIMP-3得以重新表达，恢复其抑制肿瘤侵袭和移动的能力。

Effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells

AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells.