TRAIL协同IER3调节NF-κB信号通路介导肝癌细胞凋亡的相关机制研究

Primary liver cancer is a common malignancy, with the characteristics of occult onset, rapid onset, poor treatment efficacy and poor prognosis, and the new therapeutic targets have become clinical cancer research consensus. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced tumor cell apoptosis selectively without damaging normal cells, but the mechanism is unclear. We obtained the interaction proteins between TRAIL and IER3 in our previous research, and put forward the hypothesis that the protein interaction between TRAIL and IER3 promotes the apoptosis of liver cancer cells mediated by NF-κB signaling pathway. This program is verified by the following experiments. 1) To identify the interaction and subcellular localization of TRAIL and IER3, GST pull-down, Co-IP, confocal imaging, deletion mutants and other methods were analyzed; 2) To explore the influence on the apoptosis of liver cancer cells by TRAIL and IER3 co-expression, Western blot, flow cytometry and nude mice tumor-bearing experiments were identified with the in vitro and in vivo experiments; 3) To discuss the mechanism of IER3 and TRAIL in promoting the apoptosis of liver cancer cells, NF-κB signaling pathway was analyzed, which provides new theoretical and experimental basis for prevention and treatment of liver cancer.

原发性肝癌是常见的恶性肿瘤，具有起病隐匿、发病迅速、治疗疗效不佳和预后较差的特点，寻找新的治疗性靶标已成为临床肿瘤研究共识。肿瘤坏死因子相关凋亡诱导配体（TRAIL）能选择性诱导肿瘤细胞凋亡而不损伤正常细胞，在肿瘤凋亡研究中备受关注，然机理不清。本课题组在前期工作中获得了与TRAIL相互作用的蛋白质IER3，据此提出假说：TRAIL与IER3相互作用促进肝癌细胞凋亡，其生物学效应是介导NF-κB信号通路实现的。本课题拟进行实验验证1）GST pull-down、Co-IP、激光共聚焦、缺失突变体等验证TRAIL与IER3的相互作用及其亚细胞定位2）利用Western blot、流式细胞术、裸鼠成瘤等体内外实验，探讨TRAIL与IER3共表达对肝癌细胞凋亡的影响3）通过NF-κB信号通路分析，研究TRAIL与IER3共表达促进肝癌细胞凋亡的作用机理，为肝癌的预防治疗提供新的理论和实验依据。

以肝癌为研究对象，采用Real time PCR、Western blot、FACS、过表达和RNA干扰等手段，从分子、细胞、组织以及动物水平探讨TRAIL与IER3介导的肝癌细胞凋亡中的重要作用，为肝癌的预防治疗提供新的理论和实验依据。.miR-429在肝癌组织中的表达明显下调，提示miR-429在肝癌中可能作为抑癌因子存在；TRAF6在肝癌组织中的表达水平明显高于在肝癌旁组织中的表达。TRAF6是miR-429的直接靶基因；体外实验表明，miR-429可以明显抑制肝癌细胞的增殖和迁移，机理研究表明miR-429通过下调NF-kB信号通路抑制了肝癌细胞的增殖与迁移，并在裸鼠体内实验进一步证明了miR-429明显抑制了肝癌细胞的增殖，该研究结果阐明了miR-429在调节肝癌发生、发展中的关系。