去泛素化酶USP11/EZH2/NAKED1轴介导肝癌细胞侵袭转移能力的机制研究

Deubiquitinating enzymes USP11 is closely related to the development and progression of many tumors. However, the expression pattern and biological function of USP11 in hepatocellular carcinoma (HCC) still needs to be further addressed. Our previous study has found out that USP11 was abnormal higher expression in HCC tissues and cells, and associated with patients’ poor prognosis. USP11 promoted HCC cell invasion and migration potency via NAKED1. RNA-seq and mass spectrometry analysis showed that USP11 interacted with and stabilized EZH2 by promoting its deubiquitination, which further inhibited the expression of NAKED1. Based on the above results, we suppose that USP11/EZH2/NAKED1 axis promotes HCC cell invasion and migration ability. In order to verify this hypothesis, we will uncover the combination region and regulation mechanism of ubiquitination between USP11 and EZH2 via designing of mutants and co-Ip. What’s more, through the luciferase report gene and functional recovery experiment, we plan to investigate the mechanism of USP11 negatively regulating NAKED1 transcription via EZH2, which further remove the inhibition of classic Wnt signaling pathways and ultimately promote the invasion and metastasis of HCC cells. Finally, we suppose to confirm our results through the nude mouse tumorigenicity assay and clinical samples in vitro. In this study, we study the mechanism of this axis on regulating HCC cell invasion and migration ability for the first time. The goal of this study is expected to provide a theoretical basis for clinical diagnosis and treatment of HCC.

去泛素化酶USP11与多种肿瘤的发生发展密切相关。然而，其在肝癌中的表达及生物学功能依然不清。前期预实验发现：USP11在肝癌组织和细胞中异常高表达并与患者不良预后相关。USP11通过NAKED1促进肝癌细胞侵袭转移能力。RNA-seq结合质谱分析发现，USP11通过与EZH2结合，减少其泛素化从而稳定其表达，进而抑制NAKED1的表达。基于以上结果提出假说：USP11/EZH2/NAKED1轴介导肝癌细胞的侵袭转移能力。为验证此假说，本项目拟通过设计突变体和免疫共沉淀等明确USP11与EZH2的结合区域及泛素化调控机制；通过荧光素酶报告基因和功能恢复实验明确USP11通过EZH2负向调控NAKED1的转录，从而解除其对下游经典Wnt信号通路的抑制，最终促进肝癌细胞侵袭转移；最后通过动物模型和临床样本进行验证。本项目首次阐述该调控轴在肝癌侵袭转移中的作用机制，为肝癌诊断和治疗提供理论依据。

去泛素化酶USP11与多种肿瘤的发生发展密切相关。然而，其在肝癌中的表达及生物学功能依然不清。.我们通过肝癌患者临床组织样本中的检测发现，USP11在肝癌组织中相对高表达并与患者的不良预后密切相关。构建USP11"敲低"的稳定转染肝癌细胞株，体内和体外实验均证实了USP11正向调控肝癌细胞的侵袭转移能力。同时，CCK-8、克隆形成和体内裸鼠皮下成瘤实验也表明USP11能促进肝癌细胞的增殖能力。进一步的机制探究首次发现，USP11与NF90蛋白相互结合，通过调控NF90的蛋白泛素化程度进而稳定其在肝癌细胞中的表达水平。功能恢复实验证实USP11调控肝癌细胞的侵袭和增殖能力依赖于NF90。临床样本中也进一步证实USP11与NF90之间的正相关。.另外，我们通过测序和功能分析验证，首次确定了VEGF为USP11的候选下游靶基因，Real-time PCR实验验证了芯片的结果，"敲低"USP11后，VEGF的mRNA表达水平发生了明显的下调。另外，ELISA实验结果发现，"敲低"USP11后，肝癌细胞分泌VEGF的量也明显减少。"敲低"USP11的肝癌细胞上清处理血管内皮细胞HUVECs，结果发现，USP11"敲低"组，血管内皮细胞的成管能力被明显削弱；同时，将"敲低"USP11的肝癌细胞和血管内皮细胞共培养，基质胶实验发现，"敲低"USP11后，血管内皮细胞的迁移能力也减弱；CCK-8实验也表明，"敲低"USP11后，血管内皮细胞的增殖能力也降低。.本项目成果对阐明肝癌发生发展的分子机制和挖掘潜在治疗策略提供重要的理论依据，USP11可能成为肝癌复发转移潜在的治疗靶点。