驱动结直肠癌转移的关键基因甲基化的机制研究

The incidence of colorectal cancer (CRC) in China is currently keeping on increasing. During the clinical treatment against cancer, the patients of phase III or IV often present to be a poor prognosis due to lymph node or distant metastasis. Even the patients, who are all in the stage of phase II, always come with this event within a different prognosis. It is very important to study the molecular mechanism of metastasis of colorectal cancer in sophisticated manner, to explore and discover the early molecules and colorectal cancer metastatic bio-markers for the precise diagnosis and individualized medication. The increased expression of DNA methyltransferase 1 (DNMT1) have been identified that could promote colorectal cancer metastasis which was caused by the abnormal regulation of miR-342. Further investigation revealed that certain metastasis-associated tumor suppressor genes are silenced due to promoter region of the hypermethylation. Therefore, we hypothesis that abnormal genome methylation level is an important biological phenomena for colorectal cancer progression and metastasis which is regulated by the key switch molecules or genes. This project intends to focus on the methylation and metabolism of colorectal cancer progression and metastasis-associated genes by sophisticated research on the metastasis-associated genes methylation levels, how and when non-coding RNA (ncRNA) participate in the regulation with advanced molecular biology techniques. We expect to explore the important molecular markers' life cycles in the colorectal cancer metastatic process and find feasible group of molecular markers for early diagnosis of the colorectal carcinoma, as well as personalized treatment of colorectal cancer as a scientific experimental evidence.

结直肠癌的转移预后较差。深入研究结直肠癌转移的分子机制对于早期诊断及治疗具有重要意义。基因组中甲基化水平异常是促进结直肠癌进展和转移的重要因素。甲基化水平异常是由关键开关分子参与并调控的。DNA甲基转移酶DNMT1就是开关分子之一。研究证明在结直肠癌中DNMT1活性升高，促使相关抑癌基因的启动子区甲基化而表达沉默，从而促进结直肠癌的转移。本项目拟从DNMT1介导的基因组甲基化水平异常入手，利用芯片筛选由甲基化异常引发的功能基因及miRNA的表达谱变化，得到全景信息。继而以重要候选基因为切入点，研究其在直结肠癌进展和转移中所扮演的角色，得到特异性信息。然后再回到候选基因的甲基化研究，并在临床大样本中评价候选基因的功能及甲基化改变与结直肠癌进展和转移的关系。本课题的顺利实施，有利于阐明甲基化改变促进直结肠癌进展及转移的分子机制，有望为结直肠癌的早期诊断及个体化治疗提供科学依据。

本项目通过对结直肠癌病人组织标本进行全外显子组测序、基因表达谱分析、启动子甲基化分析，发现了两个在结直肠癌中高度甲基化基因：OLFM1和PCDHB3；两个在结直肠癌中表达上调的基因：WDR5和SUV39H2。对该4个基因表达变化的机制及其与临床预后的关系以及对结直肠癌细胞生物学功能的影响进行了分析和研究，取得了阶段性进展。. 通过pubmed数据库查询、在线工具Meth-Primer序列分析，我们发现OLFM1和PCDHB3转录起始位点上游存在CpG岛。经过甲基化特异性PCR（MSP）和重亚硫酸盐测序实验（BSP）证实，在肠癌细胞系和肠癌组织中OLFM1和PCDHB3启动子普遍高度甲基化，甲基化程度在要明显高于它们的正常对照。OLFM1和PCDHB3均能抑制CRC细胞的增殖和迁移，qPCR及western blot实验证实它们在CRC组织及细胞系中低表达。免疫组化（IHC）分析证实OLFM1在CRC组织中的低表达为不良预后因子。而且仅胞浆表达而无核膜表达PCDHB3预示CRC晚期病人不良预后。. 我们发现在肠癌组织中WDR5和SUV39H2比在癌旁组织中表达上调。统计分析表明， WDR5和SUV39H2高表达与远处转移和TNM分期相关，并预示着CRC患者较短的总存活时间和无进展生存期。体外和体内测定均证明过表达WDR5和SUV39H2增强了CRC增殖和转移。PI3K/AKT通路的活化正调控WDR5的表达，是WDR5在结直肠癌组织及细胞中异常高表达的机制；WDR5通过结合到ZNF407的启动子区，调控其转录，最终影响EMT进程，促进结直肠癌细胞迁移。我们通过靶标分子筛选发现SUV39H2负调节SLIT1的表达，挽救实验表明SLIT1可拮抗CRC中SUV39H2的功能。进一步研究证实SUV39H2可以直接结合到SLIT1启动子，通过催化组蛋白H3K9三甲基化而抑制SLIT1转录，从而促进CRC恶性表型。综上，我们建议OLFM1和PCDHB3、WDR5和SUV39H2可以作为CRC的治疗靶点和预测CRC患者预后的指标。