5-氮-2'脱氧胞苷对Reh细胞增殖及RUNX3基因启动子区甲基化状态的影响

Effects of 5-aza-2′-deoxycytidine on growth inhibition of Reh cell line and methylation of promoter in RUNX3 gene

目的:探讨5-氮-2'脱氧胞苷对Reh细胞增殖及其抑癌基因RUNX3启动子区甲基化状态的影响。方法:应用MTT法观察5-氮-2'脱氧胞苷对Reh细胞增殖的抑制能力;采用甲基化特异性PCR(MSP)检测5-氮-2'脱氧胞苷干预前后RUNX3基因启动子区的甲基化状况;应用RT-PCR检测干预前后RUNX3基因的mRNA表达水平的变化。结果:经5-氮-2'脱氧胞苷处理后,RUNX3基因启动子高甲基化的区域发生部分去甲基化,5-氮-2'脱氧胞苷处理前启动子高甲基化的RUNX3 mRNA未见表达,而在其处理后该基因可见重新表达,经5-氮-2'脱氧胞苷处理后,Reh细胞生长缓慢。结论:5-氮-2'脱氧胞苷能使Reh细胞RUNX3基因启动子区发生部分去甲基化,从而调控RUNX3基因表达并能抑制其细胞生长。启动子区异常甲基化是白血病Reh细胞RUNX3基因失活的主要原因之一。

Objective：To explore the proliferation and methylation status of RUNX3 promoter region in Reh cells in vitro exposed to the specific demethylation agent 5-aza-2′-deoxycytidine.Methods：Cell proliferation inhibition by 5-aza-2′-deoxycytidine was measured by MTT,and methylation-specific PCR（MSP） analysis was performed to evaluate the methylation status of the promoter region of RUNX3 gene in Reh cell line.RT-PCR was used to determine the mRNA expression of RUNX3 gene before and after methylation intervention.Results：RUNX3 gene promoter was hypermethylated in untreated Reh cell,methylated RUNX3 gene was partially demethylated after treatment of 5-aza-2′-deoxycytidine.RUNX3 mRNA expression failed to be found in RUNX3 gene prior to the promoter hypermethylation in Reh cells,and contrarily,expression of RUNX3 mRNA was seen after intervention with 5-aza-2′-deoxycytidine the growth of Reh cells became delayed.Conclusion：5-aza-2′-deoxycytidine may facilitate partial demethylation of RUNX3 on the promoter region in Reh cell lines to suppress the gene and cell proliferation,suggesting that high frequent hypermethylation would be one of role factors that play in inducing RUNX3 gene inactivation in Reh cell lines in leukemia.