E3泛素连接酶NEDD4-1调控肝癌细胞能量代谢和生长的机制研究

E3 ubiquitin ligase NEDD4-1 plays an important role in tumorigenesis and progression in various types of cancers. However, the role and mechanism of NEDD4-1 in the regulation of energy metabolism and growth of hepatocellular carcinoma (HCC) remains unclear. In our preliminary study, we found that NEDD4-1 is up-regulated in HCC tissues and its high expression correlated with poor prognosis of HCC patients. Gene expression profile and Gene Ontology analyses revealed that NEDD4-1 is associated with cellular metabolism process. Knockdown of NEDD4-1 in HCC cells resulted in decreased glucose uptake, glucose consumption, lactate production and reduced ATP level, and inhibition of cell growth. Based on these lines of evidence, we hypothesize that NEDD4-1 acts as a key regulator of energy metabolism and tumor growth in HCC. To verify this hypothesis, this project is designed to explore the function of NEDD4-1 in energy metabolism, biosynthesis and tumor growth. Further, we will screen and validate the downstream pathway and target genes of NEDD4-1, by using mRNA-Seq, genechip and mass-spectrometry, and subsequent bioimformatics analysis. Moreover, the clincal relevance of NEDD4-1 and its downstream target genes will aslo be explored by immunohistochemistry. Our findings will provide further understanding of the molecular basis of regulation of energy metabolism of HCC, and also provide new theoretical and experimental basis for seeking for novel therapeutic target in HCC.

E3泛素连接酶NEDD4-1与多种恶性肿瘤的发生发展有密切关系。NEDD4-1在肝癌细胞能量代谢中的作用迄今未见报道。我们前期研究发现NEDD4-1在肝癌组织中高表达且与不良预后相关。基因表达谱及功能注释显示NEDD4-1与细胞代谢密切相关。干扰其表达后肝癌细胞葡萄糖摄取、葡萄糖消耗、乳酸生成和ATP生成显著减少，同时肝癌细胞生长减慢。提示NEDD4-1可能通过调控能量代谢从而影响肿瘤生长。基于以上初步研究，本项目拟在体外、体内系统研究NEDD4-1在肝癌细胞能量代谢、生物合成和肿瘤生长中的作用；采用mRNA-Seq、基因表达谱芯片和质谱分析等方法，筛选和验证NEDD4-1下游通路和基因；并进一步分析NEDD4-1及其下游基因表达与肝癌预后的关系。从分子、细胞、动物和临床不同层次，深入探讨NEDD4-1在肝癌能量代谢中的作用和机制，为靶向能量代谢治疗肝癌寻找新的分子靶点提供理论和实验依据。

热消融是一种潜在的治疗早期肝细胞癌（HCC）的方法。然而，不全消融在已有的研究中被证明会促进HCC进展。E3泛素连接酶是在恶性肿瘤中发现的一类影响因子，促进蛋白质降解、影响蛋白质功能的分子。E3泛素连接酶是否参与了不全消融引起的HCC进展仍不清楚。在本研究中，我们使用RNA测序联合体外功能缺失库筛选，确定E3泛素连接酶NEDD4-1在不全消融的HCC组织中上调。此外，NEDD4 -1缺失显著抑制了HCC细胞的迁移。进一步结果表明，抑制NEDD4-1表达在HCC细胞中可抑制转化生长因子β (TGF-β)触发的信号转导，从而抑制HCC的生长和转移。在机制上，NEDD4-1通过直接结合TGF-β I型受体(TGFBR1)，并在赖氨酸391位点形成k27连接的泛素，显著增强TGF-β信号转导。此外，在HCC细胞中，经亚致死热应激证实NEDD4-1表达上调。热应激促进肝癌进展效应在HCC中被证明是NEDD4-1依赖的。临床上NEDD4-1高表达与HCC患者侵袭性肿瘤表型及预后不良呈正相关。肝癌患者人源异种移植(HCC-PDX)模型中靶向干预NEDD4-1证实了上述结论。总的来说，本研究发现，不全消融诱导的NEDD4-1在促进HCC进展中发挥了关键作用，并为HCC提供了一个新的治疗靶点。