TACC3介导Wnt/β-catenin通路影响肝癌干性及其机制研究

TACC3 is a member of the TACCs family, which have the centrosomes/microtubules-associated proteins characterized by a highly conserved C-terminal coiled-coil "TACC domain". The function of TACCs is interacting with spindle, regulating of gene transcription, maintaining the stability of microtubule protein and keeping centrosome integrity during mitosis.TACC3, located in 4p16.3, is associated with tumor and neurodegeneration diseases. Whether the expression of TACC3 is disregulated, and whether it is involved in the hepatocellular carcinoma (HCC) development and metastasis are still remain elusive. Our preliminary results showed that TACC3 is high expressed in HCC compared to matched normal tissues, and the high expression of TACC3 is correlated with poor outcome. In vitro studies, we found that the downregulation of TACC3 inhibited tumor proliferation and tumorigenisis capacity. Meanwhile，as TACC3 inhibitor, KHS101 also inhibited tumor proliferation and tumorigenisis capacity. IP suggested that TACC3 protein was associated with β-catenin protein. In this project, in addition to investigate the potential target gene identified by Luciferase assay, we will further investigate the alternative mechanisms of how TACC3 is involved in HCC through ChIP-seq, microarray, immunoprecipitation and mass spectrum analysis method etc., providing new insight into TACC3 as a potential target gene in HCC, revealing molecular mechanisms and new therapy for HCC.

TACC3是TACCs家族蛋白成员之一，其共有特征是含有高度保守的TACC结构域，在调节纺锤体形成，调控基因转录活性，维持微管蛋白稳定和中心体完整等作用。TACC3位于人染色体4pl6.3，该区域与肿瘤和神经退行性疾病有关。肝癌发生发展中是否存在TACC3的表达异常？其可能的作用和机制如何？我们前期研究发现TACC3在肝癌中的表达上调，且高表达的TACC3与肝癌的不良预后相关。在体外实验中我们发现下调TACC3抑制肝癌增殖、克隆形成及成球能力，并引起干性相关因子的改变。应用TACC3抑制剂KHS101可显著降低肝癌细胞的成球能力。初步机制研究发现TACC3表达上调后可同时引起β-catenin蛋白的过表达。本项目拟进一步探讨TACC3影响肝癌干性的机制，通过表达谱芯片、ChIP-seq、免疫沉淀和质谱分析等方法深入探讨TACC3促进肝癌发生发展的机制，为寻找肝癌治疗新手段提供理论依据。

我们已经证实了TACC3是促进肝癌发生的驱动基因之一，TACC3表达水平的异常上调与肝癌的恶性进程相关联，但是TACC3调控肝细胞肝癌的具体进程仍有待发现，我们的研究发现TACC3激活了Wnt/β-catenin通路，在敲低TACC3的肝癌细胞中，我们同时使用影响Wnt/β-catenin通路激动剂Wnt agonist 1处理细胞，肝癌的恶性表型得到了恢复。我们同时还发现了TACC3能够激活TGF-β通路。同时，干扰TACC3抑制了肝癌细胞细胞周期由G1向S期表达，加速了肝癌细胞的凋亡。我们接下来详细的研究了TACC3通过何种机制来影响肝癌进展，并且对TACC3在肝癌中的生物学行为做了详细的探索。我们通过生物信息学分析，TACC3 的表达与CDCA5的表达有着极其高的相关性，因此，我们接下来通过双荧光素酶报告实验R技术，验证了TACC3的确对于CDCA5有着正向的转录调控作用。以上结果支持TACC3通过调控CDCA5来促进肝癌细胞周期进展，TACC3的转录调控功能介导的肝癌细胞周期异常事件在肝癌发生和疾病进展过程中的作用，为肝癌的治疗提供了潜在的作用靶标。