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  

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

作　　者：梁德勇[1] 康延海[1] 富伟能[2] 郭艳[2] 

机构地区：[1]中国医科大学附属第一医院骨科,沈阳110001 [2]中国医科大学遗传学教研室

出　　处：《中华实验外科杂志》2009年第11期1530-1532,共3页Chinese Journal of Experimental Surgery

基　　金：辽宁省教育厅资助项目（051520）

摘　　要：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^-7moL／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^-7moL／L的5-Aza—CdR处理Hs888T细胞株20d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。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 methyhransferase 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 increased after the treatment of 5-Aza-CdR. There was significant difference between Hs888T cell line treated with 3×10^-7mol/L of 5-aza-CdR and controls （ P 〈 0.05 ）. Conclusion CpG island mcthylation 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.

关 键 词：骨肉瘤 凋亡蛋白酶活化因子-1 甲基化 

分 类 号：R738.1[医药卫生—肿瘤]