Lu Fei, Liu Chuan-Fang, Ma Dao-Xin, Liu Yan-Ping, Kong Hai-Li, Zhang Jing-Jing
Department of Hematology, Qilu Hospital of Shandong University, Jinan 250012, China.
Zhonghua Xue Ye Xue Za Zhi. 2010 Apr;31(4):223-7.
To investigate the effect of DNA methylation in combination with histone deacetylase inhibitor on transcription regulation of Ras associated domain family gene 1(RASSF1A) tumor suppressor gene and the molecular biological behaviors in U266 cells.
The U266 cells were treated with different doses of 5-Aza-2'-deoxycytidine (5-Aza-CdR) and Valproate (VPA) each alone or in combination. Methylation-specific PCR (MSP) was used to detect CpG island methylation in RASSF1A promoter. Quantitative real-time reverse transcription polymerase chain reaction (RQ-PCR) was used to examine the expression of RASSF1A gene in U266 cells. MTT was used for cell proliferation. Cell apoptosis and cell cycle were analyzed by flow cytometry.
The methylation of RASSF1A gene promoter was detected in U266 cells, while there was little RASSF1A gene expressing in the control group. The demethylation effect could be detected in the 5-Aza-CdR treated and combined treatment groups but no in the VPA group. The expression level of RASSF1A was induced by 5-Aza-CdR in a concentration-dependent manner while VPA had no such effect. The expression level of RASSF1A mRNA was increased significantly in the combined treatment group. Higher growth inhibition and apoptosis effects were found in 5-Aza-CdR and VPA combination group than that in 5-Aza-CdR or VPA alone group (P < 0.05). After treatment with 5-Aza-CdR or VPA alone for 72 h, more cells were arrested in G(0)/G(1) phase as conpared with control group (P < 0.05), and even more cells were so arrested in combined treatment group (P < 0.05).
DNA methylation and histone deacetylase inhibitor can synergistically induce demethylation of the RASSF1A gene, re-express RASSF1A gene silenced in U266 cells, inhibit the proliferation of U266 cells and induce cell apoptosis.
探讨DNA甲基化联合组蛋白去乙酰化酶抑制剂对Ras相关结构域家族基因1（RASSF1A）抑癌基因转录调控及U266细胞分子生物学行为的影响。
用不同剂量的5-氮杂-2'-脱氧胞苷（5-Aza-CdR）和丙戊酸（VPA）单独或联合处理U266细胞。采用甲基化特异性PCR（MSP）检测RASSF1A启动子区CpG岛甲基化情况。运用定量实时逆转录聚合酶链反应（RQ-PCR）检测U266细胞中RASSF1A基因的表达。采用MTT法检测细胞增殖情况。通过流式细胞术分析细胞凋亡和细胞周期。
在U266细胞中检测到RASSF1A基因启动子甲基化，而对照组中RASSF1A基因表达极少。在5-Aza-CdR处理组和联合处理组可检测到去甲基化作用，而VPA组未检测到。5-Aza-CdR以浓度依赖方式诱导RASSF1A表达，而VPA无此作用。联合处理组RASSF1A mRNA表达水平显著升高。5-Aza-CdR与VPA联合组比单独使用5-Aza-CdR或VPA组具有更高的生长抑制和凋亡作用（P < 0.05）。单独用5-Aza-CdR或VPA处理72小时后，与对照组相比，更多细胞停滞于G(0)/G(1)期（P < 0.05），联合处理组停滞于该期的细胞更多（P < 0.05）。
DNA甲基化和组蛋白去乙酰化酶抑制剂可协同诱导RASSF1A基因去甲基化，使U266细胞中沉默的RASSF1A基因重新表达，抑制U266细胞增殖并诱导细胞凋亡。
