Aberrant promoter methylation and tumor suppressive activity of the DFNA5 gene in colorectal carcinoma
Aberrant promoter methylation and tumor suppressive activity of the DFNA5 gene in colorectal carcinoma
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DOI：
10.1038/sj.onc.1211021
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发表时间：
2008-06-05
期刊：
影响因子：
8
通讯作者：
Sidransky, D.
中科院分区：
文献类型：
--
作者：
Kim, M. S.;Chang, X.;Sidransky, D.
为了鉴定新的甲基化基因启动子，我们比较了经过或未经 5-aza-2'-脱氧胞苷 (5-aza-dC) 处理的结直肠癌 (CRC) 细胞系的差异 RNA 表达谱。通过基因表达阵列分析，在最初“缺失（即沉默）”的 1776 个基因中，我们选择了 163 个基因，这些基因在三种 CRC 细胞系中的至少两种中经 5-aza-dC 处理后增加。微阵列结果通过逆转录-PCR 得到证实，并对基因启动子的 CpG 岛进行扩增和测序，以检查癌症特异性甲基化。在鉴定的基因中，耳聋常染色体显性 5 基因 DFNA5 启动子在原发性肿瘤组织中甲基化频率较高（65%，65/100）。 DFNA5 的定量甲基化特异性 PCR 可以清楚地区分原发性 CRC 组织和正常结肠组织 (3%, 3/100)。与正常组织相比，五分之四的结肠癌组织中 DFNA5 的 mRNA 表达显着下调。此外，在CRC细胞系中强制表达全长DFNA5显着降低了细胞生长和集落形成能力，而DFNA5的敲低则增加了培养物中的细胞生长。我们的数据表明 DFNA5 是 CRC 中的一种新型抑癌基因，也是人类癌症的一种有价值的分子标记。
To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription-PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.