Identification of molecular target for diagnosis and therapy of esophageal squamous-cell carcinoma based on microarray technology.

我们构建了基于阵列的基因组DNA拷贝数、mRNA表达和蛋白表达的定量和定性分析系统。为了鉴定一组对食管鳞状细胞癌（ESCC）分子靶向诊断和治疗有用的基因，我们应用我们的系统在全基因组范围内筛选假定的ESCC相关基因。1)利用阵列-比较基因组杂交（array-CGH）技术分析基因组拷贝数畸变和靶基因鉴定我们利用阵列-比较基因组杂交（array-CGH）技术分析ESCC细胞系和通过显微解剖分离的原发肿瘤细胞的基因组DNA。从表现出隐而显著的基因组拷贝数变化的区域，如高水平扩增和纯合缺失，我们试图确定这些畸变的靶基因。此外，我们通过比较基因组拷贝数变化模式和临床病理参数，探索与特定表型（如淋巴结转移和预后）相关的分子标记。同样的mRNA和蛋白质表达分析方法也被应用于其他癌症，从而鉴定出许多癌症相关基因，包括与预后相关的基因。2)利用BAC-array-based methylated CpG-island amplification （BAMCA）技术鉴定DNA甲基化沉默的肿瘤抑制基因我们将MCA方法与array-CGH （BAMCA）技术相结合，扩增甲基化序列，筛选肿瘤中异常甲基化序列。通过BAMCA数据库搜索和有药理学修饰和没有药理学修饰的表达分析，我们成功地鉴定了ESC和其他肿瘤中被甲基化沉默的几个基因。其中，一个基因在ESC中经常被CpG岛甲基化沉默，并对ESC细胞表现出生长抑制作用，表明该基因是ESC的候选肿瘤抑制基因（未发表数据）。

We have constructed array-based system to analyze genomic DNA copy-number, mRNA expression, and protein expression quantitatively and qualitatively. In order to identify a set of genes useful for the molecular-targeted diagnosis and therapy of esophageal squamous-cell carcinoma (ESCC), we applied our system to screen putative ESCC-associate genes in a genome-wide manner.1) Analysis of genomic copy-number aberration using array-comparative genomic hybridization (array-CGH) and identification of target geneWe analyzed genomic DNA from ESCC cell lines as well as primary tumor cells specifically isolated by microdissection using in-house array-based CGH (array-CGH). From the regions showing cryptic but remarkable genomic copy-number changes, such as high-level amplification and homozygous deletion, we tried to identify target genes for those aberrations. In addition, we explored molecular markers associated with specific phenotypes, such as lymph node metastasis and prognosis, by comparing pattern of genomic copy-number changes and clinicopathological parameters. The same approach with mRNA and protein expression analyses was applied to other cancers, resulting in the identification of many cancer-related genes, including genes correlated with prognosis.2) Identification of tumor-suppressor genes silenced by DNA methylation using BAC-array-based methylated CpG-island amplification (BAMCA)We combined MCA method to amplify methylated sequences with array-CGH (BAMCA), and screened aberrantly methylated sequences in cancers. Using BAMCA with following database search and expression analyses with and without pharmacological modifications, we successfully identified several genes silenced by methylation in ESC and other tumors. Among them, one gene was frequently silenced by CpG island methylation in ESC and showed growth suppressive effect on ESC cells, suggesting that this gene is a candidate tumor-suppressor for ESC (unpublished data)