白血病相关基因zo-1参与白血病发病过程及其机制的初步研究

Preliminary Study on Leukemia Related Gene zo-1 Involved in Pathogenesis of Leukemia

本研究探讨新的白血病相关基因zo-1在白血病细胞中的作用及其初步机制。应用甲基化PCR方法验证THP-1等白血病细胞中基因zo-1甲基化与白血病的关系,用甲基化PCR及RT-PCR方法验证THP-1等白血病细胞系中基因zo-1甲基化与基因表达的关系。选择基因zo-1高表达的THP-1细胞,用脂质体Oligofectamine将针对基因zo-1的小干扰RNA转染THP-1细胞,RT-PCR方法验证抑制效率,AnnexinV和PI观察细胞凋亡率,PI检测细胞周期,CCK-8检测细胞增殖。结果表明:急性白血病人中基因zo-1呈高甲基化状态,健康人群中基因zo-1不发生甲基化。基因zo-1未甲基化的THP-1细胞系高表达基因zo-1;基因zo-1高甲基化的Molt4及HL-60细胞系中不表达基因zo-1。脂质体Oligofectamine转染针对zo-1的小干扰RNA成功地抑制了THP-1细胞基因zo-1的表达,而不影响THP-1细胞增殖、细胞周期及细胞凋亡。结论:基因zo-1为白血病相关基因,急性白血病中基因zo-1高甲基化,基因zo-1的甲基化状态调控基因zo-1表达。THP-1细胞中基因zo-1表达缺失不影响细胞增殖、细胞周期及细胞凋亡。

The study was aimed to identify a new leukemia related gene zo-1 from leukemia and to explore its mechanism in leukemia. Methylation specific PCR（MSP） was used for testing gene zo-1 methylation in leukemia cells. The gene zo-1 specific siRNA was designed according to its sequence, and transfected into THP-1 cell, and the cells were cultured for 48 hours before harvesting. The effect of zo-1 siRNA was monitored by RT-PCR. The cellular proliferation activity was assayed by CCK-8, the apoptosis was detected by Annexin-V- fl isothiocyanate （FITC） assay, and cell cycle was observed by propidium iodide （PI）. The results indicated that the gene zo-1 in patients with acute leukemia was hypermethylated, while the gene zo-1 in healthy persons was unmethylated. The THP-1 cells with unmethylation of zo-1 gene promoter overexpressed the gene zo-1, while the Molt4 and HL-60 cells with hypermethylation of gene zo-1 promoter did not express the gene zo-1. The silenced zo-1 gene in Molt4 and HL-60 leukemia cell lines could be reactivated by demethylation treatment with 5-AZA-dC. The oligofectamine-transfected siRNA for zo-1 gene successfully inhibited the expression of gene zo-1 in THP-I cells, but did not interfere with cell proliferation, cell cycle and apoptosis. It is concluded that gene zo-1 is a leukemia-related gene. Gene zo-1 in acute leukemia was hypermethylated, the methylation status of gene zo-1 regulates the expression of gene zo-l. Lack of gene zo-1 expression in THP-1 cells does not infuence the cell proliferation, apoptosis and cell cycle, which suggests that the methylation of gene zo-1 may be involved in the genesis of acute leukemia, its mechanism is worthy to be studied.