AIMS技术高通量筛选鼻咽癌低甲基化谱

项目名称： AIMS技术高通量筛选鼻咽癌低甲基化谱

项目编号： No.30860311

项目类型： 地区科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 黄光武

作者单位： 广西医科大学

项目金额： 24万元

中文摘要： 肿瘤细胞的表观遗传学改变主要体现在DNA甲基化模式的改变，包括肿瘤抑制基因启动子区CpG岛的高甲基化和整体基因组的DNA低甲基化，前者一直是多年来的研究热点，而后者少人问津，尤其在鼻咽癌中尚无人涉足。系统探索鼻咽癌中低甲基化基因靶点并建立鼻咽癌低甲基化谱，对深入了解鼻咽癌的表观遗传学发病机制并开发早期分子诊断技术具有重要意义。本课题小组利用甲基化间区位点扩增技术，在21例鼻咽癌原发癌组织及相应对照中筛选基因组差异甲基化片段，通过提交BLAST与人类基因组序列比对分析，结合RepeatMasker Web Server比对，共发现31个肿瘤特异性高甲基化条带，和106个低甲基化条带。从中获得2个重复序列和10个特异序列为肿瘤特异性低甲基化基因。我们对GLRX3和CLPTM1L等基因开展了验证工作，通过RT-PCR及甲基化CpG结合域蛋白捕获SOLiD高通量测序、亚硫酸氢盐测序和甲基化特异性PCR等方法验证这些基因在鼻咽癌中的表达及甲基化状态，发现这些基因的启动子区是呈现低甲基化，初步建立了鼻咽癌的低甲基化谱。我们还对高甲基化的基因也开展了部分验证工作，报道了一些高甲基化改变的基因。

中文关键词： 鼻咽癌；AIMS；甲基化；低甲基化谱

英文摘要： DNA methylation, including promoter hypermethylation of tumor suppressor genes and global hypomethylation, were involved in tumorigenesis and cancer development.Although hypermethylation aternation in cancer cells is well studied, the mechanism and consequences of global hypomethylation in cancer cells remains unclear,especially in nasopharyngeal carcinoma (NPC). Identifying hypomethylated targets and hypomethylatoin profile of NPC is critical for better understanding the epigenomic pathogenesis and also improve molecular diagnosis of NPC. In this study, we compared the genome methylation changes in 21 NPC biopsies tissues and their normal controls by the DNA fingerprints generated by amplification of inter-methylated sites (AIMS).31 and 106 sequences were identified to be tumumor specifically hypermethylated and hypomethylated in NPC. The 31 hypomethylated sequences belongs to 2 repetitive sequences and 10 sepecific genes. By further validate the expression and methylation status of these genes by RT-PCR, enrichment-based technique using methyl-CpG binding domain protein capture by SOLiD high-throughput sequencing (MethylCap-seq), bisulphite sequencing and methylation-specific PCR, a profile of hypomethylated genes was established. We also indentified several candidate oncogenes such as GLRX3 and CLPTM1L, were downregulated and promoter-hypomethylated in NPC cell lines and tumor biopsies. We also identified several candidate tumor suppressor genes which were hypermethylated in this work.

英文关键词： Nasopharyngeal carcinoma; AIMS; methylation；hypomethylation profile.