急性白血病细胞系中SFRP基因启动子甲基化及去甲基化诱导表达的研究

 目的：探讨分泌型卷曲相关蛋白（SFRP）基因家族启动子CpG岛异常甲基化状态与急性白血病（AL）发生发展的关系，以及DNA甲基转移酶（DNMT）抑制剂5-氮杂-2-脱氧胞苷酸（5-Aza-CdR）去甲基化诱导SFRP基因表达作用的可能机制。方法：采用甲基化特异性PCR检测不同AL细胞系（Molt-4、Jurkat、HL-60和NB4）和不同浓度5-Aza-CdR作用下Jurkat细胞系中SFRP1、SFRP2、SFRP4和SFRP5基因启动子区的甲基化状态，采用实时荧光定量RT-PCR检测SFRP1、SFRP2、SFRP4和SFRP5 mRNA表达，采用半定量RT-PCR检测DNMT1、DNMT3A和DNMT3B mRNA表达。结果：在正常人细胞中不存在SFRP基因的甲基化。SFRP1、SFRP2和SFRP5基因在HL-60、NB4、Molt-4和Jurkat细胞系中均呈完全甲基化状态；SFRP4基因在NB4、Molt-4和Jurkat细胞系中完全甲基化，在HL-60细胞系中则部分甲基化。5-Aza-CdR可逆转SFRP1、SFRP2、SFRP4和SFRP5基因的高甲基化状态，并能够下调DNMT1、DNMT3A和DNMT3B mRNA水平，诱导SFRP基因恢复表达。结论：在AL细胞系中，SFRP1、SFRP2、SFRP4和SFRP5基因出现高甲基化，与AL的发生密切相关，可能成为AL新的基因标志物。5-Aza-CdR通过抑制DNMT表达，逆转SFRP基因的甲基化状态，恢复其表达。

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL)，and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.