Yue Wen, Dacic Sanja, Sun Quanhong, Landreneau Rodney, Guo Mingzhou, Zhou Wei, Siegfried Jill M, Yu Jian, Zhang Lin
Department of Pharmacology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Clin Cancer Res. 2007 Aug 1;13(15 Pt 1):4336-44. doi: 10.1158/1078-0432.CCR-07-0015.
The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer.
Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth.
Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2'-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor-containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation.
Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.
本研究的目的是鉴定在肺癌中因启动子高甲基化而频繁沉默的新基因。
进行生物信息学分析以鉴定在肺癌中显著下调的候选基因。确定DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷对候选基因表达的影响。使用亚硫酸氢盐DNA测序鉴定候选基因启动子中的甲基化CpG位点。开发甲基化特异性PCR并用于分析细胞系和临床标本中的DNA甲基化。进行病理和功能分析以研究一种候选基因，即受体活性修饰蛋白2（RAMP2）在抑制肺癌细胞生长中的作用。
在肺癌中下调的54个候选基因中，发现31个在其启动子中含有CpG岛。在分析的六个肺癌细胞系中的至少四个中，这31个基因中的六个可以被5-氮杂-2'-脱氧胞苷重新激活。在22个肺癌细胞系的36%至77%以及32个原发性肺肿瘤的38%至50%中检测到RAMP2、含表皮生长因子的纤连蛋白样细胞外基质蛋白1和U Twenty Twenty细胞中缺失基因的启动子高甲基化，而在匹配的正常样本中很少发现这些基因的高甲基化。这些基因在肺癌中的甲基化频率与常用的甲基化标志物，如RAS关联结构域家族蛋白1A、p16和甲基鸟嘌呤-DNA甲基转移酶相似。免疫组织化学显示，RAMP2在大多数肺肿瘤中下调，并且RAMP2下调与高肿瘤分级相关。RAMP2的异位表达抑制肺癌细胞生长并导致凋亡性细胞死亡。通过RNA干扰敲低RAMP2刺激细胞增殖。
研究新鉴定的基因可能为肺癌发生提供新的见解。这些基因可能作为肺癌的分子标志物有用。
