CUL4B调控Wnt/β-catenin信号通路促进结肠癌细胞上皮间质转化的功能及机制研究

In our previous study, the expression of CUL4B was up-regulated in colon cancer tissues and correlated with lymph node metastasis and distant metastasis in the patients with colon cancer. Epithelial mesenchymal transition(EMT) is a critical phase that involves tumor metastasis and the negative correlation between CUL4B/E3-ubiquitin ligase expression and E-cadherin was observed in colon cancer tissue microarray(TMA) by using immunohistochemistry studies. Our preliminary data of anti-FLAG Immunoaffinity chromatography purification demonstrated that GSK-3β, a key component of Wnt/β-catenin signaling pathway, was screened for CUL4B-interacting protein. It is reported that aberrant activation of Wnt/β-catenin signaling pathway plays important role in tumor EMT. Our preliminary data showed that down-regulation of CUL4B could enhance GSK-3β activity and suppress the level of β-catenin. Based on the research work above, we inferred that CUL4B might perform its ubiquitination effect by turn the Wnt/β-catenin pathway on through suppressing the activity of GSK-3β and subsequent accumulation and translocate into nucleus of β-catenin. CUL4B could promote EMT through CUL4B-Wnt/β-catenin-EMT novel molecular pathway and ultimately promote metastasis in colon cancer. So as to verify the above hypothesis, this study first deeply investigate the influence of the expression of CUL4B on the invasion and migration of colon cancer cells in vitro and in vivo mainly using RNA interference, gene transfection and animals living imaging technology. To make it clear that the regulation mechanism of CUL4B, Wnt/β-catenin signaling pathway and EMT, combining the use of co-immunoprecipitation, confocal immunofluorescence and intervention study by using inhibitor of Wnt/β-catenin signaling pathway. The aim of this study is to clarify a novel regulation mechanism in colon cancer and provide potential target for diagnosis and therapy of colon cancer.

课题组前期研究发现CUL4B基因在结肠癌组织中表达上调并与转移相关。上皮间质转化(EMT)是肿瘤转移的关键步骤，我们发现E3泛素连接酶CUL4B与EMT标志物E-cad表达呈负相关，是肿瘤EMT潜在新调控分子。免疫亲和纯化提示Wnt通路关键组份GSK-3β为CUL4B作用蛋白。文献报道Wnt通路异常活化是调控EMT的重要分子事件。我们沉默CUL4B后GSK-3β活性增强，β-catenin水平降低。据此分析：CUL4B通过泛素化抑制GSK-3β活性，促进β-catenin集聚并核转位导致EMT，结肠癌可能存在CUL4B-Wnt/β-catenin-EMT新型肿瘤转移分子途径 。本项目拟正负向干预CUL4B表达，通过体内、体外功能实验证实其促进结肠癌转移的作用；应用免疫共沉淀、激光共聚焦、Wnt通路抑制剂等方法，明确CUL4B调控Wnt通路促进EMT的分子机制，为结肠癌诊疗提供新靶点。

课题组前期研究发现CUL4B基因在结肠癌组织中表达上调并与转移相关。上皮间质转化(EMT)是肿瘤转移的关键步骤，我们发现E3泛素连接酶CUL4B与EMT标志物E-cad表达呈负相关，是肿瘤EMT潜在新调控分子。免疫亲和纯化提示Wnt通路关键组份GSK-3β为CUL4B作用蛋白。文献报道Wnt通路异常活化是调控EMT的重要分子事件。我们沉默CUL4B后GSK-3β活性增强，β-catenin水平降低。据此分析：CUL4B通过泛素化抑制GSK-3β活性，促进β-catenin集聚并核转位导致EMT，结肠癌可能存在CUL4B-Wnt/β-catenin-EMT新型肿瘤转移分子途径 。本项目拟正负向干预CUL4B表达，通过体内、体外功能实验证实其促进结肠癌转移的作用；应用免疫共沉淀、激光共聚焦、Wnt通路抑制剂等方法，明确CUL4B调控Wnt通路促进EMT的分子机制，为结肠癌诊疗提供新靶点。