白血病相关基因RP16的克隆及生物学意义研究

To identify DNA methylation pattern in acute myeloid leukemia (AML), restriction landmark genome scanning (RLGS) analysis was performed on three bone marrow (BM) samples collected from an 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. Altered NotI DNA fragments were identified by comparing the RLGS profiles of the diagnosis, remission and relapse BM samples. LRP16, one of eight methylated NotI DNA fragments that were absent only in the relapse sample, was cloned from an arrayed library of NotI/EcoRV fragments and sequenced. The LRP16 NotI/EcoRV DNA clone was mapped by BLAST search to chromosome 11q12.1. Expressed sequence tags (ESTs) were obtained from a database search using LRP16 NotI/EcoRV clone sequence. A novel 1.225kb full-length cDNA was cloned by rapid amplification of cDNA end based on a contig of the ESTs and named as LRP16 (GenBank accession number: AF202922). The protein of LRP16 gene was identified at nucleus of cell by fluorescence confocol analysis. It was confirmed by Southern Blot analysis that the DNA methylation occurred at a CpG island on intron 9 of the gene. The frequency of DNA methylation in relapse or refractory AML patients is higher than that in the diagnosis patients. DNA methylation analysis of LRP16 gene is useful in monitoring leukemia relapse and estimating prognosis of AML patients. Northern Blot and multiple tissue expression array analysis showed that LRP15 gene is highly expressed in most of normal tissues. RT-PCR showed that expression of LRP16 gene is higher in bone marrow samples from leukemia patient than in normal bone marrow donor but can not be detected in cord blood mononuclear cells. The results suggest LRP16 gene is involved in pathogenesis of leukemia. We are working on functional study of LRP16 gene with gene transfection technique.

我们在研究白血病基因甲基化时得到一个晰的基因片段，该基因在睾丸及卵巢中表达很强，故推测其功能与细胞增殖及分化有关，与白血病发展有关。本课题拟用RACE技术扩增并测定其序列，克隆到载体；研究该基因在白血病不同类型、不同阶段的表达水平及其与白血病发生、发展及预后的关系；研究其启动子区甲基化与其表达调控的关系。