Restriction Landmark Genomic Analysis of Cancer

描述（由申请人提供）： 本研究的总体目标是使用限制性标志基因组扫描（RLGS）来识别与人类结肠肿瘤发生相关的CpG岛改变的靶点。使用NotI或具有富含GC的识别序列作为限制性标志的其他限制性酶，RLGS靶向富含基因的CpG岛区域，并主要检测DNA甲基化状态的改变，尽管也可以检测DNA缺失和扩增。将使用基于计算机模拟的虚拟RLGS计算方法克隆表现出与人结肠肿瘤发生相关的频繁改变的RLGS基因座。"虚拟的" RLGS序列和"真实的" RLGS序列之间同一性的确认将通过使用衍生自虚拟序列的引物对RLGS斑点DNA进行PCR扩增来完成。将对RLGS分析的相同肿瘤进行阵列比较基因组杂交（CGH），以整合与人结肠癌相关的表观遗传和遗传畸变。这将使我们能够确定是否有一类具有高表观遗传不稳定性的肿瘤可以与具有高遗传不稳定性的肿瘤区分开来，以及表观遗传和遗传畸变之间是否存在协同相互作用。将通过甲基化敏感性PCR和亚硫酸氢盐测序评价选定RLGS基因座的甲基化状态，以确定DNA甲基化的边界和密度。定量表达试验将用于评估原发性肿瘤中推定靶基因的表达，以确定CpG岛改变是否与基因表达变化相关。识别CpG岛改变的新靶点可能会揭示肿瘤发生的新途径，并进一步了解表观遗传改变如何影响恶性表型。

DESCRIPTION (provided by applicant): The overall aim of this research is to identify targets of CpG island alterations associated with human colon tumorigenesis using Restriction Landmark Genomic Scanning (RLGS). Using Notl or other restriction enzymes that have GC rich recognition sequences as the restriction landmark, RLGS targets gene rich CpG island regions and primarily detects alterations in DNA methylation status although DNA deletions and amplifications can also be detected. RLGS loci exhibiting frequent alterations associated with human colon tumorigenesis will be cloned using in silico based, virtual RLGS computational methods. The confirmation of identity between the "virtual" RLGS sequence and the "real" RLGS sequence will be accomplished by PCR amplification of the RLGS spot DNA using primers derived from the virtual sequence. Array Comparative Genomic Hybridization (CGH) will be performed on the same tumors analyzed by RLGS to integrate epigenetic and genetic aberrations associated with human colon cancer. This will allow us to establish whether there is a class of tumors with high epigenetic instability that can be distinguished from those with high genetic instability and whether there is cooperative interaction between epigenetic and genetic aberrations. The methylation status of selected RLGS loci will be evaluated by methylation sensitive PCR and bisulfite sequencing to determine the boundaries and density of DNA methylation. Quantitative expression assays will be used to assess the expression of putative target genes in primary tumors to determine whether the CpG island alteration was associated with a change in gene expression. The identification of novel targets of CpG island alteration may uncover new pathways of tumorigenesis and further our understanding of how epigenetic alterations impact on the malignant phenotype.