Up-regulation of DNA-methyltransferase 3A expression is associated with hypomethylation of intron 25 in human testicular germ cell tumors
Up-regulation of DNA-methyltransferase 3A expression is associated with hypomethylation of intron 25 in human testicular germ cell tumors
复制标题
DOI：
10.1620/tjem.212.177
复制
发表时间：
2007-06-01
影响因子：
2.2
通讯作者：
Arai, Yoichi
中科院分区：
文献类型：
--
作者：
Ishii, Tomohiko;Kohu, Kazuyoshi;Arai, Yoichi
从发育的角度来看，人类睾丸生殖细胞肿瘤（TGCT）可以追溯到胚胎中的原始生殖细胞，这些原始生殖细胞在转化后成为腺瘤或非腺瘤。因此，TGCT提供了一个有用的模型系统的研究基因调控参与肿瘤的发生以及发展。本研究旨在分析TGCT组织中DNA甲基转移酶（DNMT）基因的表达和表观遗传学改变。所检测的基因包括DNMT 1、DNMT 3A和DNW 3B，其功能是维持或产生基因组DNA的甲基化状态。使用半定量逆转录-聚合酶链反应，我们发现，DNMT 3A的表达，而不是DNMT 1或DNMT 3B，在TGCT标本相比，非肿瘤睾丸组织明显上调。为了探索DNMT 3A上调的机制，我们检测了该基因中CpG的甲基化状态。DNW 3A的远端和近端启动子区在TGCT和非肿瘤组织中均未甲基化。相比之下，非肿瘤睾丸组织表现出DNMT 3A的内含子25中甲基化和非甲基化CpG的混合物，而TGCT标本中内含子25中的大多数CpG是去甲基化的。Southern印迹分析证实了这种甲基化程度的差异，其中内含子25中的EcoR 1位点仅在CpG未甲基化时才能被消化。因此，内含子25朝向去甲基化的表观遗传改变与TGCT中DNMT 3A的表达增加相关。内含子25可以代表DNMT 3A中的差异甲基化区域，其在生殖细胞的发育和/或肿瘤发生期间被调节。
From a developmental point of view, human testicular germ cell tumor (TGCT) can be traced back to the primordial germ cells in the embryo, which, upon tranformation, become either seminoma or non-seminoma. Thus, TGCT provides a useful model system for the study of gene regulation involved in oncogenesis as well as development. In this study, we focused and analyzed the expression and epigenetic alteration of DNA -methyltransferase (DNMT) genes in TGCT tissues. The examined genes included DNMT1, DNMT3A and DNW3B that function to maintain or generate a methylation status of genomic DNA. Using semi-quantitative reverse transcription-polymerase chain reaction, we found that the expression of DNMT3A, but not DNMT1 or DNMT3B, was up-regulated markedly in TGCT specimens compared to non-tumor testicular tissues. To explore mechanisms involved in the up-regulation of DNMT3A, we examined the methylation status of CpGs in the gene. The distal and proximal promoter regions of DNW3A were non-methylated in both TGCT and non-tumor tissues. In contrast, non-tumor testicular tissues exhibited a mixture of methylated and non-methylated CpGs in intron 25 of DNMT3A, whereas most CpGs in intron 25 were demethylated in TGCT specimens. This difference in the degree of methylation was confirmed by Southern blot analysis, in which an EcoR1 site in intron 25 could be digested only when the CpG was non-methylated. Thus, epigenetic alteration of intron 25 toward de-methylatiolf is associated with increased expression of DNMT3A in TGCT. The intron 25 may represent a differentially-methylated region in DNMT3A that is modulated during development and/or tumorigenesis of germ cells.