Yang Na, Li Zhi
Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, China.
Zhonghua Yi Xue Za Zhi. 2008 Aug 19;88(32):2295-8.
To investigate the effect of DNA methyltransferases 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.
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-lysine 9 methylation status of RUNX3 promoter and the second exon. The expression of RUNX3 was measured by RT-PCR.
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 micromol/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 10.0 micromol/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 micromol/L 5-Aza-CdR. Before the intervention, RUNX3 gene was not expressed, and was expressed after treatment with 2.0 micromol/L 5-Aza-CdR.
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.
探讨DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对人膀胱肿瘤细胞中组蛋白H3赖氨酸9甲基化状态及RUNX3基因表达的影响。
培养人膀胱肿瘤EJ细胞株，用5-Aza-CdR处理24小时。采用MTT法观察EJ细胞的增殖和生长情况。另取EJ细胞培养并用5-Aza-CdR处理，然后采用染色质免疫沉淀法分析RUNX3启动子及第二外显子的组蛋白H3赖氨酸9甲基化状态。通过RT-PCR检测RUNX3的表达。
用浓度为0.1、0.5、1.0、2.0、5.0和10.0 μmol/L的5-Aza-CdR处理EJ细胞后，其存活率分别为98.1%、95.3%、75.9%、52.3%、16.2%和7.7%。用5-Aza-CdR处理EJ细胞12、24、36、48、72和96小时后的存活率分别为89.4%、85.2%、78.6%、37.1%、8.9%和7.1%。1.0、2.0、5.0和10.0 μmol/L组的存活率均显著低于对照组（均P < 0.05）。干预前，RUNX3基因启动子及第二外显子区组蛋白H3赖氨酸9甲基化状态的扩增条带明显，但用2.0和5.0 μmol/L 5-Aza-CdR处理后均消失。干预前，RUNX3基因未表达，用2.0 μmol/L 5-Aza-CdR处理后表达。
5-Aza-CdR不仅能明显抑制人膀胱癌细胞的增殖，还能通过使RUNX3启动子及第二外显子处的组蛋白H3赖氨酸9去甲基化来重新激活RUNX3基因。组蛋白H赖氨酸9三甲基化可能是RUNX3基因失活的最重要原因之一。
