setdb1与Tiam1相互作用通过调控EMT促进肝癌侵袭转移

Tiam1 is a metastasis-promoting protein for hepatocellular carinoma we have screened and verified. Using the Tiam1-C1199 as a bait, We identified a novel Tiam1-interacting protein setdb1. Preliminary study showed that setdb1 expression in hepatocellular carcinoma cell lines is consistent with Tiam1. Overexpression of setdb1 promoted HCC cells proliferation and invasion ability in vitro while knockdown of setdb1 resulted in the reverse. Moreover, setdb1 induced EMT and affected cancer stemness characteristics. Based on those findings, we hypothesized that setdb1 cooperates with Tiam1 to promote hepatocellular carcinoma metastasis by regulating epithelial-mesenchymal transition. To test this hypothesis, the role of setdb1 in HCC will be firstly explored, while setdb1 overexpression and knockdown HCC cell lines are constructed to investigate its biological function. The interaction between the two should be verified by GST pull down, co-immunoprecipitation and fluorescence colocalization analyses. Their binding domain and regulatory mechanism will be further investigated. Finally, the molecular mechanisms that setdb1 cooperates with Tiam1 to promote hepatocellular carcinoma metastasis by regulating epithelial-mesenchymal transition will be discovered. All of the work are aim to provide a new target for HCC metastasis and treatment strategies.

Tiam1是我们前期筛选并验证的肝癌转移促进蛋白。我们应用酵母双杂交技术获得一个全新的Tiam1相互作用蛋白setdb1。初步研究结果显示setdb1可以促进肝癌细胞体外增殖和侵袭能力，其在肝癌细胞株的表达趋势与Tiam1一致，并且setdb1可以诱导肝癌细胞EMT、影响肿瘤干细胞特征。基于前期研究结果，我们提出setdb1与Tiam1相互作用通过调控EMT促进肝癌侵袭转移的假说。为验证这一假说，本项目首先探讨setdb1在肝癌中的作用，过表达或沉默setdb1，利用肿瘤体内、外实验探讨其生物学功能。通过GST pull down、免疫共沉淀、荧光共定位等方法，验证两者的相互作用，研究Tiam1与setdb1之间的结合区域，分析相互作用的位点。然后解析setdb1/Tiam1蛋白质相互作用对EMT发生及肝癌侵袭转移的影响和分子机制，以期为临床拮抗肝癌转移提供新的靶点和治疗策略。

Tiam1是本研究前期筛选并验证的肝癌转移促进蛋白。我们应用酵母双杂交技术筛选获得一个全新的Tiam1相互作用蛋白SETDB1。初期研究结果提示SETDB1可以促进肝癌细胞体外增殖和侵袭能力，其在肝癌细胞株的表达趋势与Tiam1一致，提示SETDB1可能参与肝癌转移过程。基于这些前期研究结果，本项目研究首先通过免疫组化方法证明SETDB1在肝癌组织中高频扩增表达，表达水平显著高于癌旁组织。SETDB1与Tiam1在肝癌组织的表达水平呈正相关关系。功能学实验证明SETDB1促进肝癌细胞的恶性生物学行为，调控肝癌细胞EMT。回复实验证明，敲低Tiam1表达，可以逆转SETDB1的促癌作用。接着，通过 GST pull down、交联免疫共沉淀和荧光共定位实验，确认了SETDB1与Tiam1存在蛋白质相互作用。并且，通过生物信息学方法预测、染色质免疫共沉淀和双荧光素酶报告实验，发现SETDB1可以与Tiam1启动子结合，调控Tiam1转录翻译，特异性表达SETDB1不仅能上调Tiam1表达，而且能够促进H3K9me3。研究结果解析了SETDB1/Tiam1相互作用对肝癌侵袭转移的影响，有望为临床拮抗肝癌转移提供新的靶点和治疗策略。