结直肠癌特异性甲基化诊断标志物筛选验证及机制研究

结直肠癌诊断金标准为组织病理，具有侵入性。而外周血中标志物检测微创、依从性高。基因甲基化在癌症中广泛存在且具有肿瘤特异性。cfDNA甲基化可反映原发病灶情况，有巨大诊断潜力。然而现有研究发表的甲基化标志物均不具有肿瘤特异性。据此，本研究拟使用全基因组甲基化测序检测结直肠癌、肺癌、乳腺癌、胃癌和肝癌五种常见肿瘤及健康对照cfDNA甲基化水平，以TCGA等数据库分析构建的肿瘤特异性甲基化谱为辅助，筛选结直肠癌特异性甲基化标志物；进一步采用病例和非病例的诊断试验设计，用dPCR及qMSP等检测方法，在cfDNA中对所筛选特异甲基化基因进行评价验证，最终提出cfDNA中用于特异性诊断结直肠癌的基因甲基化标志物，以提高对已知和未知原发灶结直肠癌的诊断效率。并对结直肠癌有肿瘤特异性诊断作用但其机制不清楚的基因，研究其甲基化介导的基因表达异常在肿瘤中的作用，揭示其在结直肠癌中的表观遗传发生机制。

The current gold standard for colorectal cancer diagnosis is histopathology, which is invasive. Whereas, the detection of markers in peripheral blood is minimally invasive and highly compliant. Gene methylation is widely existed in cancer, with tumor-specific. cfDNA methylation can not only reflect the primary lesion, but also has great potential application value in the diagnosis of malignant tumors including colorectal cancer. However, all the methylation markers reported in published studies are not tumor-specific. Therefore, this study intends to use Whole-Genome Bisulfite Sequencing to detect the cfDNA methylation status of five common tumors of colorectal cancer, lung cancer, breast cancer, gastric cancer and liver cancer, and healthy controls; combining with the tumor-specific methyation spectrum constructed by public databases such as TCGA, to screen for colorectal cancer-specific methylation markers. We will further adopt case and non-case diagnostic test design, using digital PCR and qMSP detection methods to evaluate and verify the selected specific methylation genes in cfDNA. Finally, our study will propose specific gene methylation markers used in cfDNA diagnosis of colorectal cancer, to achieve the aim that improve the diagnosis efficiency of known and unknown primary colorectal cancer. For tumor-specific genes for diagnosis but with unclear mechanism in colorectal cancer, the role of methylation-mediated gene expression abnormalities in tumors will be studied to reveal their epigenetic mechanism in colorectal cancer.