Exploration of molecular targets for diagnosis and therapy of oral cancer through array-CGH based analysis of cancer genome.

1)收集口腔癌及其癌前病变的原始标本和临床病理资料，收集口腔癌及其癌前病变的细胞系和有临床病理资料的口腔癌前病变的原始标本，用于口腔癌基因组分析。2)比较基因组杂交(ARRAY-CGH)分析基因组拷贝数变异及目的基因的确定我们使用基于内部阵列的CGH(MCG全基因组阵列-4500和MCG癌症阵列-800)分析口腔和食道细胞系的基因组DNA。从隐蔽但显著的基因组拷贝数变化的区域，如高水平扩增和纯合子缺失，我们试图确定这些异常的靶基因。3)利用基于BAC阵列的甲基化CpG岛扩增(BAMCA)和染色质免疫沉淀(ChIP)芯片方法鉴定DNA甲基化沉默的抑癌基因。我们结合MCA方法对阵列CGH甲基化序列进行扩增，筛选出肿瘤基因组中异常甲基化的序列。我们还将CHIP和ARRAY-CGH(芯片上芯片)相结合用于甲基化序列的筛选。使用这些筛选方法和后续的数据库搜索和表达分析，在有和没有药物修饰的情况下，我们成功地在食道癌和口腔癌中发现了几个甲基化沉默的基因。其中，口腔癌和食道癌中频繁甲基化的候选抑癌基因已被成功识别(未发表数据)

1) Banking of primary samples of oral cancer and its premalignant lesions with clinicopathological dataWe made collections of cell lines as well as primary samples of oral cancer and its premalignant lesions with clinicopathological data for analyses of cancer genome of this disease.2) Analysis of genomic copy-number aberration using array-comparative genomic hybridization (array-CGH) and identification of target geneWe analyzed genomic DNA from oral and esophageal cell lines using in-house array-based CGH (MCG Whole Genome Array-4500 and MCG Cancer Array-800). 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. Together with further analyses using primary tumor samples, we successfully identified many cancer-related genes, including genes correlated with clinicopathological as well as biological significances.3) Identification of tumor-suppressor genes silenced by DNA methylation using BAC-array-based methylated CpG-island amplification (BAMCA) and Chromatin immunoprecipitation (ChIP) -on-chip methodWe combined MCA method to amplify methylated sequences with array-CGH (BAMCA), and screened aberrantly methylated sequences in cancer genome. We also combined ChIP with array-CGH (ChIP-on-chip) for screening of methylated sequences. Using those screening methods with following database search and expression analyses with and without pharmacological modifications, we successfully identified several genes silenced by methylation in esophageal and oral cancers. Among them, candidate tumor-suppressors frequently methylated in oral and esophageal cancers have been successfully identified (unpublished data)