E3泛素连接酶RNF114促进肝癌发生发展的作用及分子机制研究

The E3 ubiquitin ligase RNF114 plays a critical role in the maintenance of immune tolerance and the regulation of interferon responses. Recent studies suggest that RNF114 is involved in the development and progression of certain types of cancers, but the molecular mechanisms are still unclear. Our preliminary results demonstrated that RNF114 expression was significantly upregulated in hepatocellular carcinoma (HCC) tissues compared to normal liver tissues. RNF114 was mainly located in cytoplasmic compartment of non-tumorous hepatocytes, whereas the protein was predominantly distributed in nucleus of HCC cells. Moreover, RNF114 knockdown inhibited the proliferation of HCC cells independently of p21 induction. Based on our preliminary results, we will further explore the role of RNF114 in the development and progression of HCC, including 1) to explore the molecular details accounting for the dramatic disparities in the subcellular location of RNF114 in hepatocytes and HCC cells; 2) to address how RNF114 functions as a nuclear protein in HCC cells; 3) to find the proteins targeted by RNF114 and their contributions to the function of RNF114. We will further investigate the correlation between the expression levels of RNF114 and the clinicopathological parameters of HCC patients. The study will provide evidence regarding whether and how RNF114 regulates the development and progression of HCC as well as the potential of RNF114 being a target for HCC treatment.

E3泛素连接酶RNF114在维持免疫耐受和控制干扰素反应中发挥关键作用。虽近来研究显示，RNF114与肿瘤的发生发展相关，但其作用分子机制仍不清楚。我们预实验结果表明：相对癌旁组织，肝癌组织中RNF114的表达水平显著增高；癌旁组织中RNF114主要分布于胞浆，而肝癌组织中RNF114定位于细胞核；敲降RNF114抑制肝癌细胞的生长增殖，此效应并不依赖p21的功能。课题将以前期工作为基础，进一步探讨RNF114在肝癌发生发展中的作用；研究正常肝及肝癌细胞中RNF114蛋白在细胞亚定位上呈现差异的分子机制；明确肝癌细胞中核RNF114的作用靶蛋白及其相关生物学效应；利用肝癌临床样本研究RNF114表达水平与肝癌患者预后等临床指标的相关性。本研究将为认识RNF114在肝癌的发生发展中的作用及探讨其作为潜在治疗靶点的可能性，提供研究数据。

泛素、去泛素化是重要的蛋白翻译后修饰方式，精细调控多种重要生物进程，对维持体细胞稳态、抑制恶性转化具有十分重要的意义；然而，泛素化事件在肝癌发生发展中的作用的仍不清楚。.在这项研究中，我们利用Nras G12V/Myr-AKT以及Nras G12V/c-Myc诱导的原位小鼠肝癌模型发现，癌基因诱导的肝癌细胞中RNF114表达上调；在Hras G12V、Myr-AKT和c-Myc癌基因转化的肝始祖细胞中，RNF114蛋白水平升高。此外，RNF114表达在人肝癌组织相对癌旁组织上调，且RNF114的表达与不良预后相关。在肝癌细胞系中，敲降RNF114可促进细胞焦亡，从而抑制肿瘤细胞体内外增殖。利用RNA-seq和质谱鉴定方法筛选RNF114的下游靶蛋白，我们发现RNF114作为一种E3泛素连接酶，能够与Mitochondial antiviral signaling protein(MAVS)结合。机制上，RNF114可上调MAVS泛素化修饰，从而对其进行蛋白酶体依赖的降解。敲除RNF114后，MAVS蛋白上调，活化GSDMD，促进MAVS介导的细胞焦亡，从而影响肝癌的发生及发展。.以上发现表明了RNF114在Hras G12V、Myr-AKT和c-Myc等癌基因驱动的肝癌发生及发展的重要性，并揭示了RNF114在肿瘤发展过程中通过抑制MAVS介导的细胞焦亡作用从而促进肿瘤生长的机制。为寻找新的肿瘤标记物和治疗靶点，对提高肝癌的诊疗效果具有重要意义。