利用二代深度测序研究48个靶基因甲基化水平与乳腺癌的关系

Breast cancer is the most commonly diagnosed cancer in women worldwide. Early breast cancer detection in patients could significantly increase their survival rate and require less severe treatment. Aberrant DNA methylation patterns usually occur at an early stage of cancer development, which makes them ideal biomarkers for early detection in breast cancer for high-risk identification, disease prognosis，treatment response prediction, and monitoring therapy. .We will use candidate-gene approach to investigate the methylation of 48 target genes in breast cancer and evaluate a gene panel for breast cancer early detection using DNA methylation. A novel microfluidic Access Array system combined with bisulfite DNA treatment will be applied for next generation sequencing to detect DNA methylation. The tumor tissue and circulating cell-free DNA from over 200 breast cancer patients and normal controls will be bisulfite treated and amplified with Access Array then sequenced on Illumina GAII next-gen sequencer. The association of DNA methylation level of the target genes with breast cancer disease risk, ER/PR/Her-2 status, lymph node metastasis, treatment response will be evaluated..In addition, the gene expression level of the 48 candidate genes in the breast cancer tumor /normal tissues will be analyzed using real-time RT-PCR and correlated with their DNA methylation level to study the regulation of methylation on gene expression in breast cancer. The regulation mechanism will be further evaluated in breast cancer cell lines using demethylation agent 5-azacytidine. We hope to develop a critical methylation gene panel that would be useful tool for breast cancer early detection, high-risk identification and therapy prediction.

DNA异常甲基化是乳腺癌肿瘤早期发生的重要发病机制之一。本研究从大量文献搜索中筛选了48个在乳腺癌发病通路中重要的候选靶基因，应用Access Array纳米微流体PCR芯片和第二代深度测序技术结合亚硫酸氢钠测序法，将在200例以上的乳腺癌患者外周血及肿瘤组织中分析48个靶基因的DNA甲基化水平，研究它们与乳腺癌发病风险的关联，并分析它们与患者病理类型、细胞学分级、ER/PR/Her-2状况和腋淋巴结转移等的相关性。同时检测乳腺癌肿瘤组织中48个靶基因的基因表达水平，并在乳腺癌细胞株中观察5-氮杂胞苷逆转甲基化对基因表达的影响，验证DNA甲基化对基因表达的调控机制，并观察基因甲基化对癌细胞增殖、转移及入侵等功能的调控。从多层次、多角度深入的研究DNA甲基化与乳腺癌发病机制的相关性，发现与乳腺癌相关的关键甲基化靶基因组合，并探讨其甲基化谱作为乳腺癌早期诊断及病理分型的生物标记的可能性。

DNA异常甲基化是乳腺癌肿瘤早期发生的重要发病机制之一。本研究从大量文献搜索中筛选了48个在乳腺癌发病通路中重要的候选靶基因，应用Access Array纳米微流体PCR芯片和第二代深度测序技术结合亚硫酸氢钠测序法，在89例乳腺癌患者肿瘤组织中检测了靶基因甲基化水平，成功分析45个靶基因的DNA甲基化水平，发现29个基因的甲基化变化与乳腺癌发病相关。我们分析了这些基因甲基化与患者病理分型、激素状况和腋淋巴结转移等的相关性，没有发现明显相关性。我们探索检测了41例乳腺癌患者外周血游离DNA40个基因的甲基化水平，发现游离DNA中基因甲基化水平明显高于乳腺癌肿瘤及正常组织。我们观察乳腺癌细胞株中5-氮杂胞苷对癌细胞增殖、转移及入侵等功能的调控，并检测其对基因表达的影响，发现5-氮杂胞苷对不同细胞株作用不同。表达水平有显著升高的基因在乳腺癌组织中也有明显的表达升高。本研究从多层次、多角度深入的研究DNA甲基化与乳腺癌发病机制的相关性，发现29个与乳腺癌相关的关键甲基化靶基因组合，为探索甲基化谱作为乳腺癌早期诊断及病理分型的生物标记的提供了新思路。