HOX11＋急性T淋巴细胞白血病RUNX2基因启动子区域CpG岛甲基化及其表达的研究

Methylation of CpG Island in Promoter Region of RUNX2 Gene and Its Expression in HOX11^+ Acute T Lymphoblastic Leukemia

本研究检测RUNX2基因在HOX11+急性T淋巴细胞白血病(T-ALL)细胞株和T-ALL患者骨髓细胞中的甲基化状态和表达水平,探讨其表达水平与启动子区CpG岛甲基化的关系。以3株急性T淋巴细胞白血病细胞株sil-ALL、DND41和RPMI及38例T-ALL患者、29例治疗后完全缓解T-ALL患者骨髓和8例正常人骨髓为样本,采用RT-PCR检测RUNX2 mRNA表达水平,采用重硫酸盐测序法、甲基化DNA免疫沉淀技术和启动子寡核苷酸微阵列芯片分析基因启动子区CpG岛甲基化状态和基因功能。结果表明,高表达HOX11的T-ALL患者样本中会出现RUNX2基因启动子区域的甲基化。RUNX2基因启动子区域在HOX11+T-ALL的甲基化比率为78.9%,明显高于HOX11-ALL(36.8%),两组差异有统计学意义(P<0.01)。RUNX2在HOX11+的细胞株中的表达明显低于HOX11-的细胞株,RUNX2在HOX11+的T-ALL患者中的表达水平(0.581±0.257)明显低于HOX11-的T-ALL患者(0.835±0.317),并且RUNX2 mRNA的相对表达程度与HOX11 mRNA的相对表达程度成负相关(rs=-0.378,P<0.01)。RUNX2基因在T-ALL中的表达水平与其甲基化呈负相关(rs=-0.419,P<0.01)。结论:HOX11能负调控RUNX2的表达,RUNX2基因启动子甲基化是导致其在T-ALL中表达下调或基因沉默的原因,这种HOX11对RUNX2基因的抑制作用有可能就是临床上HOX11高表达T-ALL患者具有更好的无事件生存率和更长的总生存率的原因。RUNX2基因的表达和甲基化水平对判断T-ALL患者的疗效有一定意义,并有望成为一个诊断T-ALL的分子标志。

This study was purposed to detect the methylation status in promoter region of RUNX2 gene and its expression in cell lines and patients with HOX11＋ T-cell acute lymphoblasfic leukemia （T-ALL） and to explore the relationship between the expression level of RUNX2 gene and methylation of CpG island in its promoter region. The methylation pattern in promoter region of RUNX2 gene was detected with bisulfite sequencing PCR, DNA methylation immunoprecipitation technique and promoter oligonucleotide microarray analysis and the expression levels of RUNX2 mRNA was detected with RT-PCR in 3 T-ALL cell lines （ sil-ALL, DND41 and RPMI）, as well as in 75 clinic bone marrow samples including 38 de novo T-ALL patients, 29 complete remission T-ALL patients and 8 normal samples. The results showed that there were hypermethylation of CpG island in promoter region of RUNX2 gene in patients with highly expressing HOX11＋ T-ALL. The methylation rate of the promoter CpG islands of RUNX2 gene in HOX11＋ T- ALL （78.9%） was significantly higher than that in HOX11 - T-ALL （36.8%） （P 〈 0.01 ）. The expression of RUNX2 in HOX11＋ cell lines was significantly lower than that in HOX11 - cell lines, and the expression level of RUNX2 in the HOX11＋ T-ALL patients （0. 581 ± 0.257） was significantly lower than that in HOXll - T-ALL patients （0.835 ± 0. 317）. The relationship between RUNX2 and HOX11 mRNA expression level showed a negative correlation（ r_s =-0. 378 ,P 〈 0.01 ）. The expression levels of RUNX2 gene negatively correlated with the methylation of CpG island in its promoter region （ r_s = - 0. 419, P 〈 0.01 ）. It is concluded that HOX11 is a negative regulator of RUNX2 gene and the expression of RUNX2 is downregnlated or even lost by promoter methylation in T-ALL, which demonstrate a better event-free survival and a marked trend for longer overall survival for HOX11-high T-ALLs. The expression and methylation level of RUNX2 gene may have some significance in evaluating the curative effect of T-ALL. The abnomal expression of RUNX2 may be a prognostic marker in T-ALL patients.