泛素折叠修饰蛋白UFM1修饰系统关键蛋白UFBP1调控胃癌细胞增殖与侵袭的机制研究

Gastric cancer (GC) is the second most common malignant tumor in China. At present, the main treatments for patients with middle or terminal stage of GC are chemotherapy and targeted therapy. However, there is no evident breakthrough. Therefore, it is urgent to find new pathogenesis of GC for targeted treatments. Ubiquitin-fold modifier 1 (UFM1) is the most recently identified ubiquitin-like protein. UFM1 has an ubiquitin-like tertiary structure and substrate modification function. UFM1-binding and PCI domain-containing protein 1 (UFBP1) is the first protein identified as a UFM1 substrate while the ufmylation of activating signal cointegrator 1 (ASC1), the second validated substrate, also requires ufmylation of UFBP1, suggesting its important role in the ufmylation. Our preliminary experiments discovered that UFBP1 was expressed significantly higher in the GC tissue than normal gastric tissue and the high expression reduced the overall patient survival rate. We also found that UFBP1 could regulate the progression of cell cycle and upregulate the protein level of elongation factor 2 (EF2), a protein involved in cell proliferation and invasion. Therefore, we hypothesize that UFBP1 regulates the GC development through its downstream target, EF2. In order to test this hypothesis and to elucidate the molecular mechanism by which UFBP1 regulates the progression of GC cells, we will first use MTT, Transwell and xenograft mouse model to examine whether UFBP1 influences the proliferation and invasion of GC cells at the cellular and animal levels. Then, we will elucidate the underlying mechanism by quantitative proteomic approach and biochemical techniques such as protein expression, gene silencing, affinity purification, co-immunoprecipitation, immunoblotting, immunohistochemistry and xenograft mouse model. We will further examine the molecular mechanism by which UFM1 conjugation system regulates these processes. We expect this study will elucidate the role of UFBP1 and ufmylation in the progression of GC and provide potential molecular targets for the treatment of this cancer.

胃癌是我国第二大肿瘤，临床上对中晚期胃癌患者的治疗仍无明显突破，因此亟需发现新的发病机制。近年来发现的泛素折叠修饰蛋白UFM1有类似于泛素的三级结构和修饰底物蛋白的功能。UFM1结合蛋白1（UFBP1）是第一个发现的UFM1修饰底物，在UFM1修饰系统中起着重要的作用。我们预实验发现UFBP1在胃癌组织中高表达，且与患者的生存期相关；并发现UFBP1参与细胞周期的调控，上调参与细胞增殖与侵袭的蛋白EF2的表达。因此，我们推测UFBP1是通过下游蛋白EF2调控胃癌细胞增殖与侵袭的。为证明这一假说，我们将用MTT、Transwell、小鼠荷瘤等实验在细胞和动物水平上验证UFBP1促进胃癌细胞增殖与侵袭，并用定量蛋白质组学、蛋白表达、基因沉默、亲和纯化、免疫印迹、免疫组化、小鼠荷瘤等实验阐明其作用的分子机制。本研究预期揭示UFM1修饰系统调控胃癌发生发展的分子机制，为胃癌治疗提供潜在药物靶点。

胃癌是世界上常见的恶性肿瘤之一，其病死率在肿瘤相关死亡率中排第三位。铂类药物作为中晚期胃癌患者的基础化疗药物之一，广泛应用于临床。然而这类患者的5年生存期仍然低于30%，其主要原因是肿瘤细胞耐药及药物不良反应。犹素（Ubiquitin-fold modifier 1，UFM1）是一个类泛素蛋白，可以修饰底物，使底物发生犹素化修饰。UFM1结合蛋白1（UFM1-binding and PCI domain-containing protein 1，UFBP1）是第一个被发现的犹素修饰底物，在犹素修饰系统中起着关键的作用。研究发现UFBP1能促进或抑制与其发生相互作用的蛋白质的泛素化降解，提示UFBP1在泛素修饰系统也发挥着重要作用。近期有文献报道，UFBP1高表达能显著延长胃癌患者的生存期，但具体机制尚不明确。因此，本研究将从探索UFBP1是如何调控胃癌细胞增殖及是否影响胃癌细胞对化疗药物的敏感性入手，通过定量蛋白质组学和多种生化实验逐步揭示UFBP1增加胃癌细胞对铂类化疗药物敏感性的作用机制，并在动物和临床标本中证实该分子机制。结果显示UFBP1不影响胃癌细胞的增殖和细胞周期的进展，但却能显著增加胃癌细胞对顺铂和奥沙利铂的药物敏感性，增加顺铂诱导的胃癌细胞凋亡。UFBP1的这一新功能是通过与氧化应激反应关键核转录因子Nrf2发生相互作用，增加其泛素化修饰，促进其通过泛素蛋白酶体途径降解，进而下调了其下游靶基因AKR1Cs的转录和蛋白表达而实现的。本研究从分子、细胞、动物和临床样本水平系统地阐明了犹素修饰系统关键蛋白UFBP1与胃癌细胞化疗药物敏感性的关系，并揭示了其具体的分子机制，提示UFBP1是胃癌患者使用铂类化疗药物预后的分子标志物，为临床合理用药提供了理论依据，并将肿瘤对化疗药物的敏感性与犹素修饰系统紧密联系起来，为该领域的基础研究提供了新思路。