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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 investigate the DNA methylation level of WT1 gene promoter region in leukemia cell lines by polymerase chain reaction (PCR) and its relationship with WT1 gene expression. Methods ① The expression of WT1 gene mRNA in 8226, HL-60, Jurkat, KG-1 and Raji hematological malignant tumor cell lines was detected by RT-PCR and Methylation-specific PCR (MSP) The methylation status of its promoter region; ② demethylation of U937 cell line with 5-aza-deoxycytidine (5-aza-CdR), and observed WT1 gene expression levels change. Results ① The expression of WT1 was high in HL-60, K562, KG-1, NB4 and SHI-1 cell lines, but WT1 in 8226, Jurkat, Raji, U266 and U937 cell lines was extremely low. , Raji, U266 and U937 cells had hypermethylation in the promoter region of WT1 gene. ② After demethylation, the expression of WT1 gene in U937 cell line increased compared with that in the untreated group, accompanied by the WT1 promoter Regional DNA methylation levels decreased and unmethylated levels increased. Conclusion DNA hypermethylation in the promoter region of WT1 is one of the mechanisms that inhibits its expression.