凋亡蛋白酶活化因子-1基因甲基化在骨肉瘤细胞株中的作用

目的 检测骨肉瘤细胞株中凋亡蛋白酶活化因子(APAF)-1基因启动子区域甲基化状态及该基因mRNA表达,观察APAF-1基因对骨肉瘤形成的影响.方法 以骨肉瘤细胞株MG63和Hs888T为研究对象,提取DNA,经重亚硫酸钠处理后,采用限制性酶切图谱分析(COBRA)检测APAF-1基因CpG岛的甲基化情况,使用不同浓度(0、1×10~(-7)、3×10~(-7)mol/L)的DNA甲基转移酶抑制剂5-氮杂2'-脱氧胞苷(5-Aza-CdR)处理骨肉瘤细胞株4、10、20d,采用实时定量聚合酶链反应(QT-PCR)检测Apaf-1 mRNA表达水平的变化.结果 在Hs888T细胞株中APAF-1基因存在CpG岛甲基化并且随着5-Aza-CdR浓度的增加Apaf-1 mRNA表达水平也在增加.3×10~(-7)mol/L的5-Aza-CdR处理Hs888T细胞株20 d与对照组比较差异有统计学意义(P<0.05).结论 Hs888T细胞株中APAF-1基因异常表达与APAF-1基因启动子区域CpG岛甲基化有关,提示APAF-1基因甲基化可能参与某些骨肉瘤的发生.

The role of APAF-1 DNA methylation in osteosarcoma cell lines

Objective To explore the role of APAF-1 DNA methylation in osteosarcoma cell lines through the detection of the methylation status of the APAF-1 promoter and the expression of the Apaf-1 mRNA in osteosarcoma cell lines. Methods Two osteosarcoma cell lines MG63 and Hs888T were used in this study. The methylation pattern in the CpG island of APAF-1 promoter was analyzed by techniques of combined bisulfite restrict analysis (COBRA). The mRNA expression of APAF-1 gene in the two cell lines treated with different concentrations of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) (0,1×10~(-7) and 3×10~(-7) mol/L) was assayed by QT-PCR. Results CpG island methylation of APAF-1 gene was found in Hs888T cell line. The expression of Apaf-1 mRNA in Hs888T cell line was in-creased after the treatment of 5-Aza-CdR. There was significant difference between Hs888T cell line treated with 3×10~(-7) mol/L of 5-aza-CdR and controls (P<0.05). Conclusion CpG island methylation of APAF-1 gene in the osteosarcoma cell line Hs888T inhibits the expression of Apaf-1, which implies that APAF-1 DNA methylation might participate in the genesis of some osteosarcomas.