5-杂氮-2′-脱氧胞苷诱导EJ细胞RUNX3基因组蛋白甲基化改变

5-aza-2′-deoxycytidine induces changes of histone H3-lysine 9 methylation in bladder tumor cells

目的探讨DNA甲基化转移酶抑制剂5-杂氮-2′-脱氧胞苷（5-Aza-CdR）对人膀胱癌EJ细胞Runt相关转录因子3（RUNX3）基因组蛋白甲基化和基因表达的影响。方法培养人膀胱癌EJ细胞，用不同浓度5-Aza-CdR干预细胞。采用四甲基偶氮唑盐比色（MTT）法观察5-Aza-CdR对EJ细胞增殖活性的影响；通过染色质免疫沉淀技术分析干预前后RUNX3基因启动子区和第2个外显子区组蛋白H3第9位赖氨酸（H3-K9）的甲基化状态；以逆转录-聚合酶链反应（RT-PCR）技术检测干预前后RUNX3基因的表达。结果5-Aza—CdR对EJ细胞生长的抑制作用呈现剂量依赖性和时间依赖性，在浓度为0．1、0．5、1．0、2．0、5．0和10．0μmol／L时，细胞存活率分别为：98．1％，95．3％，75．9％，52．3％，16．2％和7．7％，在时间为12、24、36、48、72、96h的细胞存活率分别为：89．4％，85．2％，78．6％，37．1％，8．9％，7．1％，其中浓度为1．0、2．0、5．0、10．0μmol／L组与对照组（5-Aza-CdR0μm01）相比，差异有统计学意义（P〈0．05）。EJ细胞存在RUNX3基因组蛋白H3-K9的三甲基化和RUNX3基因表达的缺失；2．0μmol／L5-Aza-CdR干预细胞后RUNX3基因启动子区和第2个外显子区组蛋白H3-K9发生去甲基化，基因恢复表达。结论5-Aza-CdR对人膀胱癌EJ细胞生长增殖有明显的抑制作用；并可能通过诱导RUNX3基因组蛋白H3-K9的去甲基化使基因重新表达，组蛋白H3-K9三甲基化可能是该基因失活的重要原因之一。

Objective To investigate the effect of DNA methyhransferases inhibitor 5-aza-2′- deoxycytidine （5-Aza-CdR） on the histone H3-lysine 9 methylation status and gene expression of RUNX3 in human bladder tumor cells. Methods Human bladder tumor cells of the line EJ were cultured and treated with 5-Aza-CdR for 24 h. MTT test was used to observe the proliferation and growth of the EJ cells. Other EJ cells were cultured and treated with 5-Aza-CdR and then chromatin immunoprecipitation assay was used to analyze the histone H3-1ysine 9 methylation status of RUNX3 promoter and the second exon. The expression of RUNX3 was measured by RT-PCR. Results The survival rate of the EJ cells treated with 5-Aza-CdR of the concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 μmol/L were 98.1%, 95.3%, 75.9%, 52. 3% , 16. 2% , and 7.7% respectively. And the survival rates of the EJ cells treated with 5-Aza-CdR for 12, 24, 36, 48, 72, and 96 hours were 89.4%,85.2%,78.6%, 37.1%, 8.9%, and 7.1% respectively. The survival rates of the 1.0, 2. 0, 5.0, and 10. 0μmol/L group were significantly lower than that of the control group （all P 〈 0. 05）. Before the intervention, the amplified bands of the histone H3- lysine 9 methylation status of RUNX3 gene promoter and the second exon zone were obvious, but both disappeared after the treatment with 2.0 and 5.0 μmol/L 5-Aza-CdR. Before the intervention, RUNX3 gene was not expressed, and was expressed after treatment with 2. 0 μmol/L 5-Aza-CdR. Conclusion 5-Aza- CdR not only obviously inhibits the proliferation of human bladder cancer cells but also reactivates RUNX3 gene through demethylation of histone H3-lysine9 at RUNX3 promoter and the second exon. H3-lysine 9 trimethylation may be one of the most important reasons for gene inactivation of the RUNX3 gene.