Division of Gastroenterology, Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea.
BMC Cancer. 2010 Oct 22;10:577. doi: 10.1186/1471-2407-10-577.
NORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated.
Expression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays.
Expression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress.
Our data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.
NORE1（RASSF5）是 Ras 效应器功能的 RASSF 家族的新成员。NORE1 的表达在许多人类癌症中经常因启动子异常高甲基化而失活，这表明它可能是一种潜在的肿瘤抑制因子。然而，NORE1 的表达和突变状态及其在结直肠肿瘤发生中的意义尚未得到评估。
采用定量 PCR、SSCP 和亚硫酸氢盐 DNA 测序分析，研究了 10 种癌细胞系和 80 例原发性肿瘤中 NORE1A 和 NORE1B 的表达、突变和甲基化状态。通过细胞计数、流式细胞术和集落形成实验评估了 NORE1A 和 NORE1B 表达对肿瘤细胞生长的影响。
所有正常结肠上皮组织中均能检测到 NORE1A 和 NORE1B 转录本的表达，但在 10 种癌细胞系中的 7 种（70%）和 4 种（40%）以及 80 例原发性癌组织中的 31 种（38.8%）和 25 种（31.3%）中，表达量显著降低。此外，80 对匹配组织中分别有 46 对（57.6%）和 38 对（47.5%）显示肿瘤特异性的 NORE1A 和 NORE1B 降低。与早期和低级别肿瘤相比，高级别和晚期肿瘤中异常降低的情况更为常见。虽然未发现该基因的体细胞突变，但在用去甲基化剂 5-aza-dC 处理后，所有低表达细胞的表达均被重新激活。对启动子内 31 个 CpG 位点的亚硫酸氢盐 DNA 测序分析表明，NORE1A 的异常降低与启动子 CpG 位点高甲基化密切相关。此外，瞬时表达和 siRNA 介导的敲低实验表明，NORE1A 和 NORE1B 均可降低肿瘤细胞的细胞生长和集落形成能力，并增强肿瘤细胞对凋亡应激的反应。
我们的数据表明，由于启动子异常高甲基化导致的 NORE1 的表观遗传失活是结直肠肿瘤发生中的一个常见事件，可能与结直肠肿瘤的恶性进展有关。
