白血病相关基因DNA甲基化模式分析及去甲基化研究

DNA hypermethylation in the promoter regions of genes is recognized as an important mechanism for inactivation of tumor suppressor genes in cancer. To identify the clinical significance of DNA methylation in leukemia we analyzed DNA methylation patterns in leukemia patients with molecular techniques. The study consists of the three parts: (1). Restriction landmark genome scanning (RLGS) that is an effective technique for detecting hypermethylation of CpG islands in the promoter regions of leukemia associated genes was performed on three bone marrow (BM) samples collected from an acute myeloid leukemia (AML) patient at diagnosis, remission and relapse. The rare-cutting methylation sensitive restriction enzyme NotI is used as a landmark on RLGS profile. A total of 1284 NotI DNA fragments were analyzed in each RLGS profile. Two known genes (Wit-1 and DRD4) and an un-known methylated NotI DNA fragments (LRP15) that were absent only in the relapse sample, were cloned from an arrayed library of NotI/EcoRV fragments and sequenced. Expressed sequence tags (ESTs) were obtained from a database search using LRP15 NotI/EcoRV clone sequence. A novel 1.718kb full-length cDNA was cloned by rapid amplification of cDNA end based on a contig of the ESTs and named as LRP15 ( GenBank accession number: nm-052953). (2). The DNA methylation patterns of the three genes as well as another tumor suppressor gene P15 were analyzed in bone marrow samples from acute leukemia patients. The frequency of DNA methylation is higher in the samples from relapse and refractory leukemia patients than that from the diagnosis patients. DNA methylation analysis is helpful in monitoring relapse and estimating prognosis of leukemia in clinic. (3). To identify correlation between promoter methylation and expression of the genes, DNA demethylation treatment was performed on leukemia cell line K562 and Molt4 with 5'-aza-2'-deoxycitidine and traditional chinese medicine arsenic. Both of the two genes were re-activated after demethylation treatment. Demethylation effect of arsenic was confirmed in leukemia cell lines. The results underline the potential importance of DNA methylation in pathogenesis of leukemia.

我们用分子生物学技术（RLGS）在一例急性白血病中首次发现三个DNA甲基化导致异常的基騑IT-1、DRD4及RP15。在此基础上，本研究将对这三个基因在不同类型白血病中DNA甲基化模式进行全面细致的研究，明确其与白血闰的相关性以及在白血病发生、发展中的作用，为白血病在基因水平进行去甲基化治疗提供理论依据。.