造血系统肿瘤WT1基因启动子区域DNA甲基化及其调控的研究

Study on DNA methylation status of WT1 gene in its promoter region in hematologic malignancy cell lines

目的应用聚合酶链反应(PCR)的实验方法研究白血病细胞系中WT1基因启动子区域的DNA甲基化水平,及其与WT1基因表达的关系。方法①采用RT-PCR技术及甲基化特异性PCR(Methylation-specific PCR,MSP)技术检测8226、HL-60、Jurkat、KG-1及Raji等血液系统肿瘤细胞系中WT1基因mRNA表达水平及其启动子区域的DNA甲基化状态;②以5-杂氮脱氧胞嘧啶(5-aza-CdR)对U937细胞系进行去甲基化处理,并观察WT1基因表达水平的改变。结果①HL-60、K562、KG-1、NB4及SHI-1细胞系中WT1表达水平高,而8226、Jurkat、Raji、U266和U937细胞系WT1表达水平则极低,同时检测到在8226、Jurkat、Raji、U266和U937这5个细胞系存在WT1基因启动子区域DNA高甲基化;②经去甲基化处理后,U937细胞系的WT1基因表达水平较未处理者上升,同时伴随着WT1启动子区域DNA甲基化水平的下降和未甲基化水平的升高。结论WT1基因启动子区域DNA高甲基化是抑制其表达的机制之一。

Objective To study the DNA methylation status of WT1 gene promoter region in hematologic malignancy cell lines and its correlatiou with WT1 gene expression. Methods ①RT-PCR and methvlation-specific PCR were performed for detecting WT1 gene expression and DNA methylation status in its prorooter region in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines. ②Treatment of U937 cells with 5-aza-CdR, a demethvlation inducing agent and the changes in WTI gene expression level and its promoter regioti methylation status were determined. Results ①HL-60, K562, KG-1, NB4 and SHI-1 cells showed higher levels while 8226, Jurkat, Raji, U266 and U937 cells showed extremely low levels of WT1 expression. DNA hypermethylatil,n in WT1 gene promoter region was ideutified in 8226, Jurkat, Rajl, U266 and U937 cells. ②The WT1 gene expression in U937 was enhanced after treatmeut with 5- aza-CdR accompanied with the decrease of methylated and the increase of unmethylated levels in its promoter region. Conclusion Modulation of the DNA methytation status in WT1 promoter region is one of the epigenetic mechanisms for regulating its expression.