5-氮杂胞苷对Eca109细胞增殖及RUNX3基因表达的影响

Effect of 5-azaC on Growth Inhibition of Eca109 Cell Line and Expression of the RUNX3 Gene

目的探讨去甲基化制剂5-氮杂胞苷(5-azaC)干预对Eca109细胞增殖及其抑癌基因RUNX3表达的影响。方法应用MTT法来观察5-azaC对Eca109细胞增殖的抑制能力;采用10μmol/L、20μmol/L和50μmol/L3种不同浓度的5-azaC处理Eca109细胞株,采用MSP检测干预前后RUNX3基因启动子区的甲基化状况;应用RT-PCR、Western blotting检测干预前后RUNX3基因的mRNA和蛋白表达水平的变化。结果 Eca109细胞RUNX3基因启动子区处于异常的高甲基化状态,经5-azaC处理后,高甲基化的RUNX3基因启动子部分去甲基化;处理前启动子高甲基化的RUNX3基因其mRNA或蛋白均不表达,去甲基化处理后能重新激活该基因表达,经3种不同浓度5-azaC处理后,Eca109细胞生长减慢。结论启动子区高甲基化是食管癌Eca-109细胞RUNX3基因失活的主要原因之一,5-azaC能逆转RUNX3基因甲基化状态,从而调控RUNX3基因表达并抑制细胞生长。

Objective To explore the transcription and protein expression effects of promoter demethylation on RUNX3 gene in Eca109 cell line in vitro exposed to the specific demethylation agent-5-azaC. Methods Eca109 cell line was treated by 10 μmol/L,20 μmol/L,and 50 μmol/L 5-azaC.MSP analysis was evaluated for the promoter region methylation status of the RUNX3 gene in Eca109 cell line.RT-PCR and Western blotting were used to determine the mRNA expression of RUNX3 gene and protein expression. Results RUNX3 gene promoter was hypermethylated in untreated Eca109 cell,methylated RUNX3 gene was demethylated partly after treated with 5-azaC.No expression of RUNX3 mRNA or protein was found in RUNX3 gene which promoter hypermethylated in Eca109 cell not treated by 5-azaC.On the contrary,expression of RUNX3 mRNA or protein was found after Eca109 cell treated by 5-azaC.After 5-azaC treatment,RUNX3 restoration was accompanied by growth inhibition,which was partly mediated through upregulation of RUNX3 expression and this may be one of the mechanisms for the tumor cell growth inhibition by 5-azaC. Conclusion 5-azaC can reactivate the RUNX3 gene transcription and protein expression by demethylation,slow the growth of Eca109 cell.