TBC1D31基因在肝癌中的作用和分子机制研究

Hepatocellular carcinoma (HCC) is a malignant tumor prevalent in China, with high morality and poor prognosis. The discovery of new candidate genes related to HCC may help to clarify its pathogenesis and develop novel prevention, diagnosis and therapy strategies for HCC. Our previous bioinformation analyses have identified the amplification of 8q24.13 was significantly associated with shorter overall survival time (P<0.05) and disease-free survival time (P<0.05) of HCC patients. Then, we performed high content screening experiments and found that TBC1D31 gene may be the key HCC-related genes within 8q24.13 amplification. Furthermore, in vitro functional assays showed that knockdown of TBC1D31 suppressed the proliferation, migration and invasion of liver cancer cell line SMMC-7721. In addition, we found that TBC1D31 can activate epidermal growth factor receptor (EGFR) signaling pathway. Moreover, TBC1D31 was shown to be interacted with EGFR by co-immunoprecipitation (co-IP) assay. And, the expression levels of TBC1D31 mRNA in HCC tumors are higher than those in adjacent-tumor tissues. Together, these results indicate that TBC1D31 is a novel candidate HCC-related oncogene. However, there is no published study on the biological function and molecular mechanisms of TBC1D31 in tumorigenesis and development of HCC. Based on these previous results, this project aims to confirm the tumorigenicity of TBC1D31 by both in vivo and in vitro assays, and then determine the mechanisms, verify whether TBC1D31 plays an oncogenic role through activating EGFR signaling pathway in HCC. Finally, we will address whether TBC1D31 is abnormally expressed in HCC tissues, and analyze the correlation between the TBC1D31 expression and the course and prognosis of HCC patients. Hope that through this study, we can clarify the molecular mechanism of TBC1D31 in the development of HCC, and contribute to the future treatment of HCC.

肝癌是高发于我国的恶性肿瘤，死亡率高、预后差。发现新的肝癌相关基因有助于解析其致病机制和研发新的防诊治措施。本实验室前期利用生物信息学方法发现8q24.13区域的扩增状态与肝癌的预后相关，高内涵筛选实验显示该区域的TBC1D31基因是一个新的肝癌相关基因候选者。初步的功能研究显示敲低TBC1D31能抑制肝癌细胞的生长、迁移和侵袭；TBC1D31可激活EGFR通路，并与EGFR相互作用；肝癌组织中其mRNA水平显著高于对应癌旁组织。目前，TBC1D31在肝癌中的生物学功能和分子机制均未见报道。本项目拟首先通过在体和体外实验确证TBC1D31的致瘤性，确定其作用机制，验证其是否通过EGFR信号通路促进肝癌的发生发展，最后评价其在肝癌组织中的异常表达情况，分析其表达水平与肝癌病人的病程和预后等的相关性。期望阐明TBC1D31在肝癌发生发展中的分子机理，为未来基于TBC1D31的应用研究奠定基础。

肝细胞癌（HCC）具有广泛的体细胞拷贝数变化（CNA）。但是，它们的功能相关性很大程度上未被揭晓。我们通过基于814例肝癌患者的转录组数据的整合分析，鉴定发现8q24.13基因组区域的拷贝数扩增与患者的不良预后显著相关。我们进一步证明8q24.13扩增驱动的Rab GTPase激活蛋白TBC1D31的过表达通过激活EGFR信号在体内、外促进了肝癌细胞的的生长和转移。从机理上讲，高表达的TBC1D31通过水解Rab22A，然后减少Rab22A介导的EGFR的从早期胞内体向溶酶体转运及降解而发挥促癌作用，从而延长了下游级联通路的激活。值得注意的是，TBC1D31的基因组扩增可以导致肝癌细胞对MEK抑制剂的耐药性，而敲低TBC1D31显著逆转这种耐药表型。总的来说，我们的发现证实了8q24.13扩增区域中的TBC1D31是HCC相关的新的癌基因，并提示通过靶向EGFR/MEK途径治疗肝癌的潜力。