地西他滨对白血病细胞DKK1基因去甲基化的实验研究

Effect of Decitabine on DKK1 Gene Demethylation in Leukemia Cells

目的:探讨地西他滨对急性髓系白血病细胞株HL-60中DKK1的表达及其下游Wnt信号通路的影响。方法:采用流式细胞术(FCM)和DNA Ladder分析检测不同浓度地西他滨对HL-60细胞凋亡的影响,甲基化特异性聚合酶链反应(MS-PCR)检测DKK1基因甲基化状态,qRT-PCR方法检测mRNA表达水平,Western-blot方法检测蛋白的表达变化。结果:FCM检测结果表明,不同浓度地西他滨作用于HL-60细胞48 h后,地西他滨组的早期凋亡细胞数明显增多(P<0.05);DNA梯度检测结果发现,地西他滨组出现明显的细胞凋亡特异性梯度条带;MSPCR检测发现,DKK1基因发生去甲基化,RT-PCR和Wertern blot检测显示mRNA表达水平增高,DKK1蛋白表达增加,β-catenin及C-MYC蛋白表达降低。结论:地西他滨可以通过DKK1基因去甲基化及抑制Wnt通路的作用,促进HL-60细胞凋亡。

Objective:To explore the effect of decitabine on Dickkopf-1(DKK1) gene expression level and its downstream Wnt signaling pathway in acute myeloid leukemia(AML) cell line HL-60.Methods:Flow cytometry and DNA ladder analysis were performed to detect apoptosis in HL-60 cell treated with different concentration of decitabine.Methylation- specific polymerase chain reaction(MS- PCR) was used to examine the methylation status of DKK1 gene.The expressions of mRNA and protein were determined by qRT-PCR and Western blot,respectively.Results:Flow cytometric detection showed that after treating HL-60 cell line with decitabine of different concentrations for 48 h,the early apoptosis of HL-60 cells increased significantly as compared with control group(P < 0.05).DNA ladder analysis showed that the DNA ladder and demethylation of DKK1 gene appeared.RT-PCR and Western blot showed that the expressions of mRNA and protein increased.The protein expressions of β-catenin and C-MYC decreased.Conclusion:The decitabine can promote the apoptosis of HL-60 cells throngh demethylation of DDK1 gene and inhibition of Wnt signalling pathway.