Trip1在肝癌细胞中的表达调控及其在控制蛋白质翻译过程中的作用机制研究

Protein synthesis is a key step in gene expression regualtion, which plays an important role in cell growth, proliferation and metabolism. Translation initiation is the rate limited step in protein synthesis, which is determined by the activity and expression level of various translation initiation factors. Most tumor cells often over-express eukaryotic translation initiation factors (EIFs) to support their ability to unlimited proliferate and resist to apoptosis. However, the expression regulation of EIFs and their functions in tumor cells remains largely unknown. In previous study, we investigated the expression of an EIF, trip1, in various clinical tissue samples by tissue microarray and found the express level of trip1 is much higher in cancer tissues,including hepatocyte carcinoma, than that in normal tissues. And the expression of trip1 is positively regulated by multiple oncogenic signaling such as mTOR, hif1a and c-myc.Silencing trip1 gene in hepatocarcinoma cell, HepG2, leads to reduced proliferation and angiogenesis, and increased sensitivity to apoptosis induction. We also found that trip1 is highly expressed during zebrafish embryonic development. And trip1 mutation in zebrafish embryos inhibited cell proliferation, promoted apoptosis and lead to angiogenesis defects. In agreement with these observations, we demonstrated that trip1 is required for the translation initiation mediated by IRES and production of hif1a, c-myc and vegfa proteins. Based on these resutls, we concluded that trip1 is critical for carcinoma genesis. In the following study, we planed to further investigate the regulation machenisms of trip1 expression and to study the functions of trip1 signaling network in the protein translation program in cancer cells, so as to discover novel targets and mechanisms for cancer treatment.

蛋白质合成是基因表达调控的主要环节，在细胞增殖和代谢中具有重要调节作用。翻译起始是蛋白合成的限速步骤，多数肿瘤细胞常过表达翻译起始因子以支持其抵抗凋亡、无限增殖等表型,但目前对起始因子过表达的调控机制和意义等方面还知之甚少。前期我们筛查了翻译起始因子trip1在临床标本中的表达情况，发现trip1在肝癌等多种肿瘤高表达；mTOR、hif1a和c-myc正向调控trip1的表达；敲降/突变trip1抑制细胞增殖、存活和血管生成；trip1选择性地控制hif1a、c-myc和vegfa的蛋白水平；因此trip1和c-myc和hif1a等通路之间存在正反馈环路。本项目将在前期结果的基础上，进一步系统研究trip1的表达调控机制，揭示以trip为节点的基因表达调控环路，通过解析trip1的下游靶基因探索该环路在控制肿瘤细胞蛋白质翻译过程中的作用，以期为基于翻译调控的肿瘤治疗提供新的靶点和理论基础。

翻译是基因表达调控的重要环节，在细胞增殖、分化和代谢等方面中具有重要作用。翻译起始是蛋白合成的限速步骤，多数肿瘤常过表达翻译起始因子以支持其抵抗凋亡、无限增殖等表型,但目前对起始因子过表达的调控机制和意义还知之甚少。本项目围绕一个翻译起始因子Trip1/eIF3i开展（后统称eIF3i）。eIF3i是项目组在斑马鱼中鉴定到早期发育必须因子。利用FLK1、FLABP等转基因鱼，我们发现缺失eIF3i的斑马鱼出现血管发育障碍，细胞增殖减少凋亡增加，肝脏发育障碍等表型。通过原位杂交，我们发现eIF3i在早期胚胎发育时高表达于新生血管内皮细胞和肝脏等组织；我们在临床肿瘤标本中发现 eIF3i在肝癌等多种肿瘤高表达，与肿瘤的恶性程度相关；表明eIF3i的表达是接受调控的。在此基础上，我们利用体内外模型深入地研究了eIF3i在肿瘤中的上游表达调控及其下游靶基因等机制。通过信息学分析，我们发现eIF3i启动子中含有hif1a、NF-KB 和 c-myc 结合位点，进一步的实验表明hif1a和c-myc的确可正向调控eIF3i的表达，而NF-KB对eIF3i的表达无显著影响；报告基因分析、染色质免疫共沉淀实验证明了hif1a对eIF3i的直接调控作用。接下来，我们利用细胞和斑马鱼体内模型，系统地研究了eIF3i的细胞生物学作用，我们发现敲降/突变 eIF3i抑制细胞增殖、存活，促进细胞自噬； eIF3i缺失导致胚胎血管发育障碍、抑制肿瘤新生血管生成。通过免疫印迹、核糖体分析等实验，我们发现eIF3i 选择性地控制肝脏肿瘤细胞VEGFA翻译，控制内皮细胞VEGFR、ERK蛋白的翻译；我们通过定量蛋白组学手段，在肿瘤细胞中鉴定到一系列eIF3i靶基因，包括YAP1、SCD1等；通过代谢组学分析，我们发现eIF3i参与肿瘤细胞的脂类、氨基酸代谢调控。总之，通过本项目的实施，我们初步探究了的 eIF3i 的基本生物学功能， 揭示了 以eIF3i 为节点的调控网络以及该网络对肿瘤新生血管、肿瘤细胞代谢、自噬中的作用，为进一步理解以 eIF3i 为节点的调控网络在肿瘤发生、发展中的作用奠定基础。