三基序蛋白TRIM44泛素化降解生长分化因子GDF15抑制肝癌发展的机制研究

Inflammatory microenvironment is closely related to the development of HCC and its mechanism is complex and worthy of further study. The E3 ubiquitin ligase function as a bridge to connect E2 ubiquitin-conjugating enzyme and protein, which was confirmed to be an oncogene or tumor suppressor gene. We have proved several ubiquitin-related proteins were associated with the progression of HCC. In the investigation of the effect about inflammatory cytokines on HCC cells, we detected the tripartite motif protein 44 ( TRIM44 ) protein, an E3 ubiquitin ligases, was significantly down-regulated in HCC cells treated by IL-6. The further data from Co-immunoprecipitation and mass spectrometry showed the growth differentiation factor 15 ( GDF15 ), a member from TGF-β family, was related to and might be the substrate of TRIM44. It is reported that there is a potential connection between inflammatory microenvironment, ubiquitination enzymes and TGF-β family proteins. Thus, we hypothesized that there might be a new approach which TRIM44 suppressed HCC progression induced by IL-6 through ubiquitin degrading GDF15. In this proposal, we will assess and verify the expression of TRIM44 and GDF15 in HCC tissues, and explore the effect of these two interacting proteins in clinicopathological features and prognosis of patients with HCC. We will also investigate the biological functions of TRIM44 in HCC cells, then further address and confirm the regulation and mechanisms in vivo. Our study aims to elaborate the role of ubiquitination and the effect of IL-6 in development of HCC to provide a novel therapeutic target.

炎症微环境与肝癌发生发展密切相关，其作用机制错综复杂，值得深入研究。泛素-蛋白酶体系统中E3连接酶特异性识别底物蛋白，在泛素化和生理过程中地位重要。我们既往的研究已证实多种泛素相关蛋白参与调控肝癌进展，近期在检测炎症因子IL-6对肝癌细胞的影响中发现泛素连接酶TRIM44在IL-6处理的细胞中显著下调，应用免疫共沉淀结合质谱分析技术筛选TGF-β家族成员GDF15可能为其潜在作用底物。文献报道炎症微环境，泛素化关键酶及TGF-β家族蛋白之间存在潜在联系，因此我们推测TRIM44可能通过泛素化降解GDF15抑制肝癌细胞侵袭转移。本课题拟在肝癌组织中研究TRIM44和GDF15表达及与患者预后的关系，细胞中检测TRIM44对生物学功能的影响，进而在分子水平解析其作用机制并经体内实验验证。阐述泛素化修饰在炎症微环境诱导肝癌进展中的作用，探讨IL-6影响肝癌发展的机制，为肝癌的靶向治疗提供思路。

原发性肝癌是我国常见恶性肿瘤之一，以肝细胞肝癌为主。我国肝炎患者基数大，寻找肝炎相关肝癌治疗靶点符合我国国情。病情进展一般经历“正常肝细胞—炎性损伤—增生结节—硬化改变—肿瘤形成”这一动态演化过程，其高转移率和高复发率一直是治疗的难点，总体生存率较低。对肝癌的发展和侵袭转移过程的机制研究，寻找有效的干预肿瘤演进和预测肿瘤预后的靶点是延长肝癌患者生存的重要途径，也是当前研究的重点和热点。. 细胞维持稳态，发挥正常功能需要胞内蛋白质的生成与降解保持动态平衡。作为真核细胞内特异的蛋白质选择性降解的重要途径，泛素-蛋白酶体系统主要通过泛素激活酶、结合酶和连接酶与靶蛋白结合，将底物蛋白泛素化后被蛋白酶体识别降解。泛素化过程异常与多种肿瘤的发生发展紧密关联，其内在作用机制值得深入研究。. 白介素6作为一种细胞因子，通过自分泌或旁分泌的方式参与免疫应答，诱导并促进肝脏炎症反应发生发展。我们应用IL-6诱导低转移潜能的肝癌细胞，通过RNA-seq技术分析并筛选IL-6作用后细胞内表达改变明显的泛素化相关蛋白，检测得到差异基因共145个，进一步分析筛选得到表达明显下调的TRIM44蛋白。. 我们进一步检测TRIM44在肝癌肿瘤组织和对应癌旁组织中的表达情况及其对肝癌细胞生物学功能的影响，探讨其与肝癌发展的关系。证实TRIM44表达与肝癌细胞增殖、裸鼠体内转移等生物学功能呈负相关。iTRAQ技术结合免疫共沉淀及液相质谱分析鉴定肝癌细胞中与TRIM44结合的相互作用蛋白，筛选得到的TRIM44表达相关差异蛋白与细胞增殖、分裂、移动等功能相关，发现GDF15是TRIM44相互作用蛋白中的一个重要分子，并进一步证实TRIM44通过泛素化降解GDF15途径调控MAPK信号通路，影响肝癌细胞增殖。在肝癌组织中检测GDF15的表达及其与TRIM44的关系，发现GDF15在肝癌组织中表达较高，与TRIM44表达呈负相关，得到结论：TRIM44是影响肝癌患者术后生存和肿瘤复发的独立预后因素，低表达TRIM44结合高表达GDF15的患者预后较差，结合TRIM44和GDF15表达有望作为预测肝癌患者术后生存和肿瘤复发的指标。