丙戊酸钠协同5-氮-2＇-脱氧胞苷对U266细胞RASSF1A基因表达调控的影响

Synergistic effect of DNA methylation inhibitor and histone deacetylase inhibitor on RASSF1A gene expression in U266 cells

目的 探讨DNA甲基转移酶抑制剂5-氮-2＇-脱氧胞苷（5-Azs-CdR）联合组蛋白去乙酰化酶抑制剂丙戊酸钠（VPA）对人多发性骨髓瘤细胞株U266细胞中Ras相关区域家族1基因（RASSF1）的表达调控以及对细胞生物学活性的影响.方法 5-Aza-CdR、VPA单独或联合干预U266细胞,甲基化特异性PCR（MS-PCR）法和实时荧光定量-PCR（RQ-PCR）法检测药物干预前后RASSF1A基因甲基化状态和RASSF1A mRNA的表达;MTT法检测细胞增殖活性;流式细胞术分析细胞凋亡及细胞周期的改变.结果 未经药物处理的U266细胞检测到RASSF1A基因启动子区域的高甲基化,RASSF1A基因微弱表达,5-Aza-CdR可逆转RASSF1A基因CpG岛高甲基化.诱导U266细胞RASSF1A基因呈剂量依赖性表达（P〈0.05）,VPA不能诱导U266细胞RASSFlA基因表达,联合用药组U266细胞RASSF1A mRNA的表达明显增强（P〈0.05）;与5-Aza-CdR、VPA单药组相比,联合用药组对细胞增殖的抑制作用更强,细胞凋亡率明显升高（P〈0.05）;与对照组相比,5-Aza-CdR或VPA作用于U266细胞72 h后,细胞阻滞于G0/G1期,联合用药组比单独用药组G0/G1期细胞阻滞作用更明显（P〈0.05）.结论 VPA联合5-Aza-CdR能有效逆转U266细胞RASSF1A基因的异常甲基化,可显著诱导因高甲基化而沉默的RASSF1A基因再表达,并明显增强5-Aza-CdR对U266细胞的增殖抑制及诱导凋亡作用.

Objective 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.Methods The U266 cells were treated with different doses of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） and Valproate （VPA） each alone or in combina-tion.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 ex-pression of RASSF1A gene in U266 cells.MTT was used for cell proliferation.Cell apoptosis and cell cycle were analyzed by flow cytometry.Results 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 ）.Conclusion 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.