Cytoglobin, the newest member of the globin family, functions as a tumor suppressor gene.
Cytoglobin, the newest member of the globin family, functions as a tumor suppressor gene.
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DOI：
10.1158/0008-5472.can-08-0565
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发表时间：
2008-09-15
期刊：
影响因子：
11.2
通讯作者：
Gazdar AF
中科院分区：
文献类型：
--
作者：
Shivapurkar N;Stastny V;Okumura N;Girard L;Xie Y;Prinsen C;Thunnissen FB;Wistuba II;Czerniak B;Frenkel E;Roth JA;Liloglou T;Xinarianos G;Field JK;Minna JD;Gazdar AF
细胞珠蛋白（CYGB）是最近发现的一种脊椎动物珠蛋白，与肌红蛋白有远亲关系，但功能未知。 CYGB 位于染色体区域 17q25，该区域在多种恶性肿瘤中经常丢失。之前的研究未能发现 CYGB 基因突变的证据。最近的研究为肺癌中该基因甲基化增加提供了初步证据。我们的研究旨在调查 CYGB 作为抑癌基因的作用。通过对肺癌和乳腺癌细胞系以及支气管和乳腺上皮细胞系进行巢式甲基化特异性 DNA 测序分析，我们发现 CYGB 启动子的 110 bp 富含 CpG 片段的甲基化与基因沉默相关。我们专门针对该序列开发了一种定量甲基化特异性 PCR 检测方法，适用于高通量分析。我们使用活检和痰样本证明了 CYGB 甲基化在区分肺癌患者和非肺癌患者方面的肿瘤特异性。我们进一步证明了该测定对多种其他上皮和血液恶性肿瘤的肿瘤特异性。为了显示 CYGB 的肿瘤抑制活性，我们进行了以下操作：（a）RNA 干扰介导的 CYGB 基因敲低对 CYGB 表达阳性肺癌细胞系中集落形成的影响，导致集落形成增加； (b) 在 CYGB 表达阴性的肺癌和乳腺癌细胞系中强化基因表达，减少集落形成； (c) 鉴定 CYGB 基因下游的潜在邻近靶标。我们的数据构成了珠蛋白家族最新成员 CYGB 作为肿瘤抑制基因的第一个直接功能证据。
Cytoglobin (CYGB) is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. CYGB is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the CYGB gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of CYGB as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the CYGB promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of CYGB methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of CYGB, we performed the following: (a) RNA interference–mediated knockdown of CYGB gene on colony formation in a CYGB expression–positive lung cancer cell line, resulting in increased colony formation; (b) enforced gene expression in CYGB expression–negative lung and breast cancer cell lines, reducing colony formation; and (c) identification of potential proximate targets down-stream of the CYGB genes. Our data constitute the first direct functional evidence for CYGB, the newest member of the globin family, as a tumor suppressor gene.