Liu Huaying, Zhang Liming, Niu Zhaoxia, Zhou Ming, Peng Cong, Li Xiayu, Deng Tan, Shi Lei, Tan Yixin, Li Guiyuan
Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan, PR China.
BMC Cancer. 2008 Sep 8;8:253. doi: 10.1186/1471-2407-8-253.
Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription.
The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter.
We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals.
BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.
鼻咽癌（NPC）是一种在东南亚和中国南方高发的头颈部恶性肿瘤。最近的研究结果表明，多个肿瘤抑制基因的表观遗传失活在鼻咽癌的肿瘤发生中起重要作用。BRD7是一个与鼻咽癌相关的溴结构域基因，在正常组织中比在鼻咽癌活检组织和细胞系中表现出更高水平的mRNA表达。在本研究中，我们探讨了DNA甲基化在BRD7转录调控中的作用。
分别通过EMBOSS CpGplot和Softberry CpGFinder预测BRD7启动子内CpG岛的存在。采用巢式甲基化特异性PCR和RT-PCR检测肿瘤细胞系以及临床样本中BRD7启动子的甲基化状态和BRD7基因的mRNA表达。电泳迁移率变动分析（EMSA）和荧光素酶分析用于检测胞嘧啶甲基化对与BRD7启动子结合的核蛋白作用。
我们发现DNA甲基化抑制NPC细胞中BRD7的表达。NPC细胞中的体外DNA甲基化使BRD7启动子活性沉默，并抑制核蛋白（可能是Sp1）与BRD7启动子中Sp1结合位点的结合。相反，DNA甲基化的抑制增强了NPC细胞中内源性BRD7 mRNA的诱导。我们还发现，鼻咽癌患者的肿瘤及配对血液样本中BRD7启动子的甲基化频率远高于正常个体的血液样本。
BRD7启动子去甲基化是BRD7基因高水平诱导表达必不可少的条件。BRD7启动子的DNA甲基化可能作为鼻咽癌的诊断标志物。
