甲基化寡核苷酸灭活死亡相关蛋白激酶1基因对白血病K562细胞增殖的影响

Inactivation of DAPK1 gene by methylated oligonucleotides and its effect on the proliferation ofleukemia cell line K562

目的探讨互补甲基化寡核苷酸诱导灭活K562白血病细胞死亡相关蛋白激酶1基因（DAPKl）及对其增殖的影响。方法应用Lip02000将与DAPKl基因启动子序列互补的甲基化寡核苷酸转染进K562白血病细胞，分别应用甲基化特异性聚合酶链反应（MSP）和反转录PCR（RT-PCR）检测转染前后DAPKl基因启动子甲基化状态和mRNA表达改变。应用噻唑蓝（MTT）法检测转染前后细胞增殖变化。结果正常组K562细胞的DAPKl基因启动子表现为未甲基化状态，可检测到相应mRNA表达；对照组寡核苷酸转染后，DAPKl基因启动子表现为未甲基化状态，mRNA表达和细胞增殖速度与正常组无明显差异；互补甲基化寡核苷酸转染后，DAPKl基因启动子呈甲基化状态，mRNA呈低表达状态，细胞增殖速度较正常组、甲基化对照寡核苷酸转染组显著增加。结论互补甲基化寡核苷酸可诱导灭活K562白血病细胞DAPKl基因并抑制其mRNA表达，促进细胞增殖。

Objective To inactivate Death-associated protein kinase 1 gene （DAPK1） by transfecting complementary methylated oligonucleotides and studies its effect on the proliferation of myelogenous leukemia cell line K562. Methods Methylated oligonucleotides complementary to DAPK1 gene promoter were transfected into K562 cells by Lipo2000. Methylation specific PCR （MSP） and Reverse transcription PCR （RTPCR） were applied to detect DAPK1 gene promoter methylation status and its mRNA expressions respectively. MT]＂ was used to detect the proliferation of K562 cells pre- and post- oligonucleotides transfection. Results DAPK1 gene promoter in non-treated and control groups were unmethylated with detectable mRNA expressions, but it became methylated with inhibited mRNA expressions after methylated oligonucleotide transfection. Proliferation in methylated oligonucleotide treatment group was significantly higher than that in non-treated and control groups. Conclusion Complementary methylated oligonucleotides could inactivate DAPK1 gene and inhibit its expression in K562 cells, which could promote its proliferation.