肿瘤相关成纤维细胞中RAB31调控HGF的分泌促肠癌肝转移的机制研究

Liver metastasis is the leading cause of death in colorectal cancer, hepatocyte growth factor（HGF）plays and important role in the process of metastasis. RAB31 is a G protein implicated in the trafficking of cell membrane proteins. In our previous study, we found that RAB31 expression was significantly increased in stage IV and CMS4 (stromal type) colorectal cancers. High RAB31 expression in the tumor stroma was associated with poor prognosis. Preliminary experiment results found that culture medium obtained from RAB31 overexpressing primary cancer associated fibroblasts promoted the migration ability and activation of HGF-Met signaling in colorectal cancer cell lines. We detected increased levels of HGF in the culture medium of CAFs described above, the underlying molecular mechanism is unclear . RAB31 is localized in the golgi complex and endosomes and colocalizes with HGF. We propose that RAB31 can regulate the secretion of HGF by CAFs through interaction with HGF, which subsequently promotes metastasis of colorectal cancer. In this project, we will be using immunofluorescence histology and ELISA to study the specificity of HGF regulation by RAB31; Immunofluorescence colocalization assay will be used for live tracking of RAB31 and HGF, while immuno-coprecipitation and WB will be used to further identify the mode of interaction and the key domains required for RAB31 and HGF interaction; Finally, hemi-spleen liver metastasis mouse model and human colorectal cancer PDX mouse models will be used to further support the above findings. The aim of this project is to elucidate the role of CAF expressed RAB31 in the biological process of colorectal cancer metastasis.

肝转移是肠癌的主要致死原因，肝细胞生长因子（HGF）在转移过程中扮演重要角色。RAB31为参与细胞膜蛋白运输的G蛋白。本团队前期研究发现RAB31在IV期和CMS4型（间质型）肠癌中表达显著增高，其在肠癌间质中高表达者预后差。预实验发现过表达RAB31的肿瘤相关成纤维细胞（CAF）上清能促进肠癌细胞系的迁移并激活其HGF-Met通路。该上清中HGF蛋白水平显著升高，其分子机制不明。RAB31在高尔基体和囊泡中与HGF共定位。我们假设：CAF中RAB31能结合HGF，调控其分泌，促进肠癌转移。本课题拟使用免疫荧光染色、ELISA法进一步明确RAB31对HGF的调控；使用免疫荧光共聚焦实时追踪RAB31和HGF在细胞内的动态变化并用免疫共沉淀明确两者的作用方式和关键结构域；最后用半脾肝转移和小鼠PDX肠癌模型进行功能验证，以阐明CAF中RAB31调控HGF分泌促肠癌肝转移的分子机制。

结直肠癌是世界高发癌种，其发生发展和转移的分子机制不明。我们前期研究发现RAB31在晚期肠癌中显著升高。本研究中我们对RAB31在肠癌中的功能和相关分子生物学机制进行进一步探究。我们使用免疫组化和免疫荧光化学对RAB31在肠癌中的表达和定位进行分析，并使用肠癌组织芯片和免疫组化染色探究RAB31表达和肠癌预后的关系。为了进一步探究RAB31在肠癌中的生物学功能，我们建立过表达或敲低细胞系，用小室穿透试验和瘢痕愈合试验研究RAB31对肠癌细胞迁移能力的影响，使用小动物成像技术探究RAB31表达水平对肠癌肝转移的影响。我们进而使用抗体芯片，药物阻断试验和免疫印迹实验探究RAB31调控肠癌转移的分子机制。结果显示，RAB31可表达于肠癌细胞和肠癌间质细胞中，而仅间质RAB31表达高的肠癌患者预后较差，提示间质中RAB31的表达能够促进肠癌的转移。在肠癌间质的CAF细胞中过表达RAB31后，其上清能够显著促进肠癌细胞系的迁移能力。且RAB31过表达CAF和肠癌细胞系的混合液接种小鼠脾脏后，其肝转移能力较WT CAF和肠癌细胞混合液接种者显著增强，提示CAF中RAB31的高表达能够促进肠癌细胞的迁移和转移能力。细胞因子抗体芯片结果显示，RAB31过表达CAF上清中多种细胞因子水平增高。其中HGF对肠癌细胞有最强的促迁移能力。而RAB31过表达CAF上清能够增强肠癌细胞中MET通路的活化水平。RAB31过表达CAF上清对肠癌细胞的促迁移能力可以通过敲低肠癌细胞中中MET的表达或药物阻断MET活化以及HGF中和抗体来消除影响。提示RAB31可能通过调控CAF中HGF的分泌水平，间接调控肠癌中MET信号通路，从而促进肠癌的肝转移。本研究为肠癌肝转移的分子机制提供了新的理论基础，具有重要科学意义和潜在转化价值。