摘要
目的肿瘤抑制基因异常甲基化状态与白血病发生关系密切,发展白血病甲基化标志物成为研究热点。但白血病类型复杂,利用单个基因甲基化模式作为肿瘤标志物具有一定局限性。本研究通过探讨基于肿瘤抑制基因p73和死亡相关蛋白激酶(death-associated protein kinase gene,DAPK)异常甲基化模式对白血病诊断分型的价值和意义,寻找一种联合多基因异常甲基化模式作为白血病肿瘤标志物的方法。方法应用亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP),分析正常人白细胞、单核细胞系白血病细胞株U937、粒细胞系HL-60及淋巴细胞系Jurkat基因组中,p73和DAPK基因启动子区CpG岛甲基化状态,将测序结果汇集到Office Excel 2010文件中,并计算各CpG位点甲基化率,绘制2个基因在4种细胞来源基因组中的CpG岛甲基化模式图,从而筛选特异甲基化位点组合。通过甲基化特异性PCR法(methylation specific PCR,MSP),在白血病细胞株、30例正常对照和104例白血病患者外周血标本中,验证基因异常甲基化模式的诊断效能。结果成功测序约107个含BSP产物转化质粒的菌株,并绘制出p73和DAPK基因CpG岛甲基化模式图。正常细胞基因组两者去甲基化程度远高于白血病细胞株。p73基因在正常细胞与白血病细胞株中,甲基化状态完全相反。DAPK基因在HL-60中与其他白血病细胞株甲基化状态存在明显差异。p73基因MSP甲基化检测可以鉴别正常细胞和白血病细胞,在临床白血病标本诊断中的灵敏度、特异度和准确度分别是21.5%、100.0%和43.1%,DAPK基因甲基化检测可以鉴别HL-60与其他白血病细胞株,其诊断急性非淋巴细胞白血病的灵敏度、特异度和准确性分别是59.1%、100.0%和77.2%。结论 p73和DAPK基因启动子区CpG岛在不同类型白血病细胞基因组中存在特异的甲基化位点,将其作为白血病初步诊断分型的潜在肿瘤标志物具有一定临床意义,也为今后发现白血病等肿瘤的甲基化标志物提供方法并奠定实验研究基础。
OBJECTIVE There is close relationship between the abnormal methylation status of tumor suppressor gene and the occurrence of leukemia. Development of methylation biomarkers of leukemia has become a research hotspot. But the type of leukemia is complex, and there are some limitations using the methylation pattern of single gene as tumor bionlarker. The objective of this study was to explore the value and significance with aberrant methylation patterns of tumor suppressor p73 and death-associated protein kinase gene for diagnosis and classification of leukemia, and find the method of combining multiple gene methylation patterns as the tumor biomarkers of leukemia. METHODS The methyla- tion status and patterns of p73 and DAPK gene promoter＇s CpG island were analyzed in the genome of normal human white blood cells, U937 （derived from mononuclear cells）, HL-60（derived from granulocyte）and Jurkat（derived from lymphocyte）cell lines by bisulfite sequencing PCR. The sketch of methylation pattern of p73 and DAPK gene in different cell genome was drawn by collecting the sequencing results in the office excel 2010 file and calculating the methylation rate of each CpG site. The effectiveness was verified using differential and multiple methylation sites of p73 and DAPK gene by methylation specific PCR in the leukemia cell lines, peripheral blood samples of normal control（30 cases） and leukemia patients（104 cases）. RESULTS The sequences of 107 of bacterial strains which contained transformation plasmid fromBSP product were successfully detected. The sketch of methylation pattern of p73 and DAPK gene in different cell genome dis- played that the degree of unmethylation in normal cell genome was higher than that of leukemia cell lines. The methylation status was completely opposite in normal cells and leukemic cell lines for p73 gene. There was significant difference in HL-60 and other leukemia cell lines for DAPK gene. The differential CpG sites in p73 gene could be used to identify normal and leukemia cells lines by MSP. The sensitivity, specificity and accuracy of the diagnosis of leukemia were 21.5%, 100. 0% and 43.1%. Meanwhile, the differential CpG sites in DAPK gene could be used to differentiate HL-60 and the other three cell lines. The diagnostic sensi- tivity, specificity and accuracy of acute non lymphocytic leukemia were 59. l%, 100. 0% and 77.2% respectively. CONCLU- SIONS The differential methylation patterns could be found in p73 and DAPK gene promoter＇s CpG island. It has clinical signi icance as potential tumor biomarker for diagnosis and classification of leukemia, and could also provide ideas and establish the ex- perimental basis for finding leukemia and other tumor＇s DNA methylation biomarkers in the future.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2016年第11期716-721,共6页
Chinese Journal of Cancer Prevention and Treatment
