甲基化FOXK1通过Snail促进结直肠癌转移的作用及机制

Metastasis of colorectal cancer (CRC) greatly decreases the survival rate of CRC patients, the mechanism of which is not clear. Our previous study found that, the expression of FOXK1 was dysregulated and increased in CRC metastases which may regulate stability of Snail protein, epithelial-mesenchymal transition (EMT) and metastasis of CRC. This project intends to confirm the enhancing effect of FOXK1 on metastasis of CRC through cellular and metastatic animal models and elucidate the involved mechanism. The methods including the point mutation, co-immunoprecipitation , half-life and ubiquitination assays are performed to validate that FOXK1 reduces the FBXO11 mediated ubiquitination and degradation of Snail, protects and increases Snail to induce EMT process and promotes the metastasis of colorectal cancer after its modification on at site of R127 arginine by PRMT5 methyltransferase for activation. Finally, immunohistochemistry assay is used to detect and validate the relationship between FOXK1 and Snail, markers of EMT, clinico-pathological parameters and prognosis of CRC patients. Statistical analysis is used to assess the prognostic value of FOXK1. Accordingly, the molecular mechanism and clinical significance of enhancing effect of FOXK1 on metastasis of colorectal cancer will be elucidated. This study will contribute to elucidating the mechanism of CRC metastasis and providing a novel tumor biomarker and potential therapeutic target for metastatic CRC.

结直肠癌（CRC）转移极大降低患者生存率，转移机制尚不明确。我们前期研究发现：CRC转移组织中， FOXK1蛋白表达失调升高，可能调节Snail稳定性、上皮-间质转化（EMT）和CRC转移。本项目拟通过细胞和动物转移模型进一步证实FOXK1促进CRC转移的作用；并采用位点突变、免疫共沉淀、半衰期和泛素化等实验明确调控机制为：FOXK1在R127位精氨酸被甲基转移酶PRMT5甲基化修饰和激活后，降低FBXO11介导的Snail泛素化降解，稳定、升高Snail而诱导EMT，促进CRC转移；最后，免疫组化方法在CRC临床标本检测验证FOXK1与Snail、EMT标志物、患者临床病理指标及预后的相关性，统计评估FOXK1的预后预测价值 。从而阐明FOXK1在结直肠癌转移中的作用、分子机制及临床意义。本项目的开展将有助于探明CRC发生转移的机制，为转移性CRC提供新的肿瘤标志物和潜在的治疗靶点。

消化系统肿瘤包括胃癌、结直肠癌、肝癌和食管癌等，其发病和死亡人数占所有恶性肿瘤的50%以上，是严重威胁我国国民健康的恶性肿瘤。消化系统肿瘤存在的主要问题是早诊率和预后预测准确率低，缺乏分子分型指导下的个体化治疗。.在胃癌组织标本中，我们通过qRT-PCR和western blot方法检测发现：ZBED1基因在胃癌组织和胃癌细胞系中表达显著升高。通过siRNA敲低或基因克隆过表达ZBED1后，western blot、MTT、克隆形成以及流式细胞检测等功能学实验进一步证明ZBED1可以促进胃癌细胞的增殖和克隆形成，ZBED1拮抗化疗药5-FU诱导的胃癌细胞凋亡，同时通过分析公共基因芯片大数据库发现ZBED1高表达胃癌患者预后差，我们的研究结果提示ZBED1可能为胃癌新的预后指标和潜在治疗靶点。.同时，在食管鳞状细胞癌组织中，我们通过qRT-PCR，western blot和免疫组织化学方法检测发现：与相应配对的癌旁组织相比，IGBP1基因在食管鳞状细胞癌癌标本组织中mRNA和蛋白明显上调。统计学分析结果显示，IGBP1高表达与ESCC患者T分期以及患者生存状态明显相关， IGBP1高表达的食管鳞状细胞癌患者预后差，生存期短，Cox回归模型进行单因素和多因素分析提示IGBP1表达可以作为食管鳞状细胞癌的一个独立预后指标。IGBP1可能为食管鳞状细胞癌一个新的预后指标和潜在治疗靶点。. 我们的研究结果为消化系统肿瘤的早期诊断和预后预测提供了新的分子标志物，为消化系统肿瘤的分子分型和个体化治疗提供了一定的理论基础。