泛素化酶TNFAIP3调控肝癌糖代谢与病理进展的作用机制研究

The metabolism in hepatocellular carcinoma (HCC) cells is different from that of normal hepatocytes. Ubiquitin-proteasome system (UPS) can regulate the metabolism of HCC. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is one of the most important ubiquitinases in UPS. Here, we found an aberrant expression level of TNFAIP3 in HCC tissues and cells, when compared with the nonmalignant ones. Preliminary study showed TNFAIP3 is closely related to glycometabolism, suggesting its regulatory role in HCC pathological development. Further study showed TNFAIP3 promotes the ubiquitination of liver phosphofructokinase (PFKL), and induces its degradation via UPS. Notably, PFKL is the key rate-limiting enzyme in glycolysis. Thus in this project, we propose the scientific hypothesis: TNFAIP3 regulates HCC glycometabolism and pathological progress by promoting the degradation of PFKL via ubiquitination-proteasome pathway. We plan to clarify the function of TNFAIP3 in HCC glycometabolism and pathological progression by using cell models and nude mice models; to investigate the regulation mechanism of TNFAIP3 in PFKL ubiquitination and degradation; and to elucidate the relationship between TNFAIP3, PFKL and prognosis of HCC patients from clinical data. This project will reveal the important role of ubiquitination in HCC glycometabolism, and provide a new precise target for HCC treatment.

代谢异常是肝癌的重要特征之一，泛素-蛋白酶体系统可调控肝癌代谢。申请人前期研究发现，泛素化调控酶肿瘤坏死因子α诱导蛋白3（TNFAIP3）在肝癌组织和肝癌细胞中异常表达，并且其与肝癌细胞的糖代谢密切相关。进一步研究发现TNFAIP3可负向调控糖酵解限速酶——肝磷酸果糖激酶（PFKL）的蛋白水平。因此，我们提出“TNFAIP3通过促使PFKL泛素-蛋白酶体途径降解，从而参与调控肝癌的糖代谢与病理进展”这一假说。本项目拟利用肝癌细胞模型、裸鼠皮下成瘤模型，观察TNFAIP3对肝癌糖代谢与病理进展的影响；阐明TNFAIP3促进PFKL泛素化降解的分子调控机制；结合临床数据探讨TNFAIP3、PFKL与肝癌病人预后的关系。本项目成果将有助于揭示泛素化调控系统在肝癌糖代谢过程中的重要功能，为肝癌的精准防治提供新靶点。

蛋白质的泛素化修饰参与调控肝癌的发生发展，因此泛素化调控酶可成为肝癌临床治疗的重要靶点。A20是一种急性期反应蛋白质，具有E3泛素连接酶的活性，并在肝脏保护中发挥关键作用。肝细胞癌发生时常伴有A20蛋白质的表达异常，通过体内和体外基因敲低和过表达的方法，我们发现A20对HCC细胞的增殖、转移及糖酵解发挥抑制功能。利用质谱和免疫沉淀等方法，我们鉴定了磷酸果糖激酶，肝型（phosphofructokinase, liver type，PFKL）是A20相互作用的蛋白质，并确认PFKL是A20发挥泛素化作用的底物蛋白，经泛素-蛋白酶体途径降解。进一步研究发现，下调A20的水平促进HCC细胞增殖、迁移及糖酵解这一现象可被敲低PFKL所抑制，表明A20是通过调控PFKL的蛋白质水平在肝细胞癌恶性进展中发挥重要作用。最后，我们发现A20在晚期HCC组织中低表达，并与PFKL的表达成负相关。本项目研究表明，TNFAIP3-PFKL-糖代谢的相互作用网络，可解释代谢与肝癌进展的关联；此外，临床样本数据表明，TNFAIP3及其PFKL有潜力作为肝癌发展过程中的生物学标志物。