人食管鳞癌中CCNYL1启动子区甲基化的研究.docVIP

人食管鳞癌中CCNYL1启动子区甲基化的研究 　　[摘要] 目的 筛选在食管鳞状细胞癌（ESCC）发病过程中异常甲基化基因，为治疗提供潜在靶点。 方法 采用简化的、具有代表性的DNA甲基化测序（RRBS）及基因表达谱芯片分析7例ESCC患者癌组织及其癌旁DNA甲基化水平和mRNA表达水平。将甲基化差异基因及表达差异基因用DAVID工具进行功能富集分析，筛出甲基化与表达负相关基因。对候选基因进行硫化测序PCR（BSP）验证获得DMR序列，采用QUMA分析软件对17对癌和癌旁甲基化差异测序结果进行分析，并对其DMR单个CpG位点的甲基化水平进行统计学分析。最后采用实时定量PCR（RT-PCR）检测候选基因表达水平。 结果 RRBS测序得到癌和癌旁差异甲基化区基因共5268个，其中689个差异甲基化区位于Promoter及CDS上。这些富集得到15条显著信号通路，包括Notch信号通路、Wnt信号通路及黏着斑信号通路等肿瘤中常见信号通路。结合7例ESCC组织表达谱芯片，筛选出启动子区高甲基化且表达降低基因Cyclin Y-like 1（CCNYL1），在17对ESCC组织中用BSP验证，CCNYL1基因DMR处CpG位点13、CpG位点16、CpG位点17、CpG位点18和CpG位点19呈现高甲基化。此外，在10对冰冻ESCC组织中进行RT-PCR检测，结果与RRBS测序和表达谱芯片一致，在ESCC中CCNYL1基因启动子区甲基化水平整体高于癌旁组织，并且表达水平低于癌旁组织。 结论 CCNYL1基因甲基化水平可能作为ESCC生物标志物。 　　[关键词] DNA甲基化；差异甲基化区；食管鳞状细胞癌；CCNYL1 　　[中图分类号] R735.1 [文献标识码] A [文章编号] 1673-7210（2016）05（c）-0004-05 　　[Abstract] Objective To screen candidate genes to characterize the alterations of DNA methylation in esophageal squamous cell carcinoma （ESCC）， in order to provide targets for cancer therapy. Methods Genome-scaled DNA methylation and mRNA expression profiles between 7 ESCCs and their adjacent normal tissues were analyzed by reduced representation bisulfite sequencing （RRBS） and digital gene expression profiling. After analyzing the annotation and pathways of these differential genes by DAVID tool， screened the genes which had a negative correlation between methylation and expression. Bisulfite sequencing PCR （BSP） was used to obtain the DMR sequence of these candidate genes. Then QUMA software was used to analyze the methylation difference between 17 pairs of carcinoma and adjacent normal tissues. Finally， real-time reverse transcription PCR （RT-PCR） was used to measure the expression of these candidate genes. Results RRBS indicated many differentially methylated regions （DMRs） in 5628 genes， of which 689 genes were differentially methylated in Promoter and CDS regions. These genes were included in 15 signaling pathways， such as Notch signaling pathway， Wnt signaling pathway and Focal adhesion， w