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目的:探讨去甲基化药物1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮(Zebularine,Zeb,折布拉林)处理HL-60及K562细胞后WWOX甲基化水平的变化,分析Zeb在白血病治疗中的意义。方法:Zeb处理HL-60及K562细胞,以5-氮杂-2脱氧胞苷(5-Aza-CdR,地西他滨)作为阳性对照,MSP法检测WWOX基因启动子及第一外显子甲基化状态;QRT-PCR检测WWOXmRNA的表达,并分析不同细胞株药物处理组与处理前组甲基化情况与mRNA表达的关系。结果:1.药物处理前,K562细胞WWOX基因启动子甲基化PCR扩增出阳性条带,非甲基化扩增阴性;第一外显子甲基化PCR扩增阴性,而非甲基化PCR扩增出阳性条带;药物处理后启动子及第一外显子甲基化PCR均不能扩增出阳性条带,而非甲基化扩增阳性。2.Zeb与5-AzaCdR处理前后,HL-60细胞WWOX基因启动子及第一外显子甲基化PCR扩增均阴性,非甲基化均扩增出阳性条带。3.经Zeb与5-Aza-CdR处理后,K562细胞WWOX基因mRNA表达升高,处理前后比较差异有统计学意义(P<0.05),不同浓度的Zeb处理组间比较差异有统计学意义(P<0.05)。4.Zeb与5-Aza-CdR处理前后,HL-60细胞WWOX基因mRNA表达差异无统计学意义(P>0.05)。结论:Zeb能够发挥去甲基化作用,上调K562细胞WWOX基因mRNA表达,具有浓度依赖性,对HL-60细胞WWOX基因mRNA表达无明显影响。
OBJECTIVE: To investigate the effects of demethylation drug 1 (β-D-ribofuranosyl) -1,2-dihydropyrimidin-2-one (Zebularine, Zeb) on WW-60 and K562 cells Methylation changes in the analysis of the significance of Zeb in the treatment of leukemia. Methods: The HL-60 and K562 cells were treated with Zeb and 5-Aza-CdR (decitabine) as positive control. The WWOX gene promoter and exon 1 The methylation status of WWOX mRNA was detected by QRT-PCR. The relationship between methylation status and mRNA expression of drug-treated and pre-treated groups was analyzed. Before drug treatment, WWOX gene promoter K562 cells amplified by methylation PCR positive bands, non-methylation amplification negative; first exon methylation PCR negative, rather than methyl The positive bands were amplified by PCR. The positive bands could not be amplified by the promoter and the first exon methylated PCR, while the non-methylated amplification was positive. Before and afterZeb and 5-AzaCdR treatment, WWOX gene promoter and first exon methylation PCR of HL-60 cells were negative, and non-methylation amplified positive bands. The WWOX gene mRNA expression of K562 cells increased after treatment with Zeb and 5-Aza-CdR, the difference was statistically significant before and after treatment (P <0.05), and there was significant difference between different concentrations of Zeb treatment group P <0.05). The mRNA expression of WWOX in HL-60 cells had no significant difference before and afterZeb and 5-Aza-CdR treatment (P> 0.05). Conclusion: Zeb can demethylate and up-regulate the expression of WWOX mRNA in K562 cells in a concentration-dependent manner, which has no significant effect on WWOX mRNA expression in HL-60 cells.