CAJ | 학술논문

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<O.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G_0/G_1期,联合用药组比单独用药组G_0/G_1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.
목적 탐토DNA갑기전이매억제제5-담-2'-탈양포감(5-Azs-CdR)연합조단백거을선화매억제제병무산납(VPA)대인다발성골수류세포주U266세포중Ras상관구역가족1기인(RASSF1)적표체조공이급대세포생물학활성적영향.방법 5-Aza-CdR、VPA단독혹연합간예U266세포,갑기화특이성PCR(MS-PCR)법화실시형광정량-PCR(RQ-PCR)법검측약물간예전후RASSF1A기인갑기화상태화RASSF1A mRNA적표체;MTT법검측세포증식활성;류식세포술분석세포조망급세포주기적개변.결과 미경약물처리적U266세포검측도RASSF1A기인계동자구역적고갑기화,RASSF1A기인미약표체,5-Aza-CdR가역전RASSF1A기인CpG도고갑기화.유도U266세포RASSF1A기인정제량의뢰성표체(P<0.05),VPA불능유도U266세포RASSFlA기인표체,연합용약조U266세포RASSF1A mRNA적표체명현증강(P<O.05);여5-Aza-CdR、VPA단약조상비,연합용약조대세포증식적억제작용경강,세포조망솔명현승고(P<0.05);여대조조상비,5-Aza-CdR혹VPA작용우U266세포72 h후,세포조체우G_0/G_1기,연합용약조비단독용약조G_0/G_1기세포조체작용경명현(P<0.05).결론 VPA연합5-Aza-CdR능유효역전U266세포RASSF1A기인적이상갑기화,가현저유도인고갑기화이침묵적RASSF1A기인재표체,병명현증강5-Aza-CdR대U266세포적증식억제급유도조망작용.
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.