白血病细胞HOX A基因亚群启动子区域异常甲基化的研究

Aberrant methylation at promoter region of HOX A gene cluster in leukemia cells

目的研究HOXA基因启动子区域CpG岛在多种白血病细胞系和白血病患者骨髓细胞中的甲基化特点和全反式维甲酸（ATRA）对其甲基化的影响。方法选取11种白血病细胞系、3种白血病45例患者治疗前后骨髓细胞以及2名正常成人外周血白细胞，常规提取DNA并用重亚硫酸盐法处理，然后用PCR法扩增目标序列，采用焦磷酸测序法测定其中CpG位点的甲基化，以5-杂氮-2’-脱氧胞苷（DAC）或ATRA处理HL-60和K562细胞后测定以上片段的甲基化。结果所检测的HOXA基因亚群在正常白细胞均无甲基化，HOXA1在所有标本均无甲基化，HOXA4、A6、A7、A9、A10和A11均存在多个CpG位点的甲基化甚至高甲基化，其中T细胞白血病细胞系和B细胞白血病细胞系CpG位点甲基化比例（分别为71．4％和85．7％）明显高于其他组细胞；HOXA4在所有细胞系中均存在甲基化，HOXA6和HOXA7在绝大多数细胞系中存在甲基化，HOXA10和HOXA11在K562和HL-60细胞中存在甲基化（38％～86％），HOXA9在髓系白血病细胞系中多表现为低甲基化，HOXA11在B细胞白血病细胞系中为高甲基化（均〉50％）。治疗缓解后的急性髓系白血病及急性淋巴细胞白血病患者比治疗前HOXA4、A6甲基化水平明显减低，而慢性粒细胞白血病患者HOXA6和A9甲基化水平明显减低。用ATRA处理后HL-60细胞HOXA4、A6和A10甲基化水平明显减低，而K562细胞仅HOXA6甲基化减低。结论白血病细胞在HOXA基因亚群启动子区域多呈现异常甲基化，且部分基因亚群表现为不同种类白血病特异性高甲基化；ATRA可以通过多种途径降低白血病细胞系，尤其是HL-60细胞部分HOXA基因亚群CpG甲基化水平。

Objective To explore the characteristics of CpG islands methylation at promoter region of HOX A gene cluster in leukemia cells before and after all-trans retinoic acid（ATRA） treatment. Methods Eleven human leukmia cell lines, bone marrow cells from leukemia patients before and after therapy and white blood cells from normal subjects were collected. HL-60 and K562 cells were treated by 2-deoxy-5-azacytidine （DAC） or ATRA respectively. Bisulfite modified DNA of these cells were amplified with PCR and quantitatively analyzed by pyrosequencing for methylation of CpG islands. Results In normal ceils, CpGs at all loci of HOX A cluster were unmethylated. In HOX A4,A6,A7, A9,A10 and A11, many CpG sites were methylated （ 〉 20% ） or hypermethylated （ 〉 50% ） in leukemia cell lines. Percentages of methylated CpGs were higher in T-cell leukemia （71.4%） and B-cell leukemia （85.7%） than in others. For individual CpGs methylations there were HOX A4 in all leukemia cells, HOX A6 and HOX A7 in most of the leukemia samples and HOX A10 and HOX All in K562 and HL-60 cells （38% -86% ）. HOX A9 CpGs showed hypomethylation in most of myeloid leukemia cells, whereas HOX A11 CpGs were hypermethylated in B-cell leukemia （ 〉 50% ）. Methylation levels of HOX A4 and A6 in AML and ALL patients after complete remission were decreased obviously, and so did HOX A6 and A9 in CML patients. Methylation levels of HOX A4, A6 and A10 in HL-60 ceils and of HOX A6 in K562 ceils were reduced by ATRA treatment. Conclusions In all leukemia cell lines, aberrant methylation of CpGs was observed at promoter regions of 6 HOX A cluster genes, and some of these genes showed leukemia-type-specific hypermethylation. CpGs methylation of some HOX A genes in leukemia cell lines, especially in HL-60 cells, were down-regulated by ATRA.