5-氮杂胞苷对HL60细胞P16、DAPK及MGMT基因甲基化状态影响

Effects of 5-azacytidine on methylation state of P16，DAPK and MGMT genes in HL60 cells

摘要:

目的：通过检测5-氮杂胞苷作用于急性早幼粒细胞白血病细胞系（HL60）细胞前后P16、死亡相关蛋白激酶（Death associated protein kinase，DAPK）及6-氧-甲基鸟嘌呤-DNA甲基转移酶（O-6-methylguanine-DNA methyltransferase，MGMT）基因甲基化状态的改变及基因表达的变化情况，探讨5-氮杂胞苷对髓系白血病细胞系作用的相关机制。方法：采用常规细胞培养，加入5-氮杂胞苷培养48 h后，以甲基化特异性PCR（Methylation specific PCR，MSP）检测基因甲基化状态，以RT-PCR检测药物作用前后基因表达水平。结果：MSP显示用药前P16基因、DAPK基因及MGMT基因呈高甲基化状态，应用5-氮杂胞苷处理后P16及DAPK基因发生了完全去甲基化，MGMT基因在用药后发生了部分去甲基化；应用5-氮杂胞苷处理前后P16、DAPK及MGMT基因表达强度分别是（0.22±0.04）与（0.64±0.12）、（0.36±0.08）与（1.27±0.09）及（0.75±0.1）与（1.37±0.1），３个基因前后表达强度均有统计学差异（P<0.05）。结论：5-氮杂胞苷通过对P16、DAPK及MGMT甲基化基因的去甲基化作用，使这些基因恢复表达，从而抑制髓系白血病细胞系的增殖，发挥肿瘤细胞增殖抑制作用。

Abstract:

Objective：To explore the effect of 5-azacytidine on myeloid leukemia cell lines through detecting methylation state and expression changes of P16，death associated protein kinase（DAPK） and O-6-methylguanine-DNA methyltransferase（MGMT） after 5-azacytidine treatment for acute promyelocytic leukemia cell lines（HL60 cells）. Methods：HL60 cells were cultured with 5-azacyti－dine by routine method for 48 h. Then methylation-specific PCR（MSP） and RT-PCR were respectively used to detect gene methyla－tion status and expression changes of P16，DAPKand MGMT before and after being treated with 5-azacytidine. Results：MSP showed hypermethylated P16，DAPK and MGMT genes before 5-azacytidine treatment. Complete demethylation was observed in P16 and DAPK genes and partial demethylation was observed in MGMT gene after HL60 cells being co-cultured with 5-azacytidine. Expression intensities of P16，DAPK and MGMT genes in HL60 cells before and after treatment of 5-azacytidine were （0.22±0.04） vs. （0.64±0.12），（0.36±0.08） vs. （1.27±0.09） and （0.75±0.1） vs. （1.37±0.1） respectively（P<0.05）. Conclusions：The CpG islands of P16，DAPK and MGMT genes are hypermethylated in HL60 cells. 5-azacytidine inhibits proliferation of leukemic cells by demethyla－tion of some related genes.