E2F1/USP11正反馈环路介导Snail去泛素化促进肝癌转移的机制研究

Hepatocellular carcinoma (HCC) has a high morbidity and poor prognosis. Tumor invasion and metastasis are responsible for the main causes of death in patients. However, the nature of HCC invasion and metastasis remains elusive. Our preliminary data showed that the deubiquitinating enzyme USP11 is highly expressed in HCC tissues which suggests the poor prognosis . The in vitro experiments showed that the protein stability of E2F1 and Snail was enhanced by USP11 and USP11 promoted the proliferation and migration of HCC cells. In addition, after silenced E2F1, the expression of USP11 was down-regulated significantly. This project intends to use a series of in vitro and in vivo experiments in human specimens, multiple cell lines and animal models to clarify the expression and clinical significance of USP11 and its effector molecules in HCC tissues, to reveal the mechanism of USP11 deubiquitinating E2F1 and Snail, to explore the exact mechanism of E2F1 transcriptional regulating USP11, to explain the role of USP11-E2F1 positive feedback pathway in HCC invasion and metastasis, and to analyze its oncological significance. Our study is to confirm the scientific hypothesis: E2F1-USP11-Snail signal pathway promoting the malignant progression of HCC, and further, provide an experimental basis for interpreting the development mechanism of HCC and finding therapeutic targets.

肝细胞癌（HCC）发病率高，预后差。肿瘤侵袭转移是患者死亡的重要原因，但机理尚未明了。我们前期发现去泛素化酶USP11在HCC组织中高表达，并与预后差相关；体外实验结果显示USP11增强转录因子E2F1和Snail的蛋白稳定性，促进HCC细胞增殖迁移；此外，沉默E2F1显著下调USP11。本项目拟在人体标本、多个细胞和动物模型中，采用一系列体内外实验，明确USP11及其效应分子在HCC组织中的表达和临床意义，揭示USP11去泛素化E2F1和Snail的作用机制，探明E2F1转录调控USP11的确切机理，深入阐述USP11-E2F1正反馈传导通路在HCC侵袭转移中的作用并解析其肿瘤学意义，从而确证E2F1-USP11-Snail信号轴促进HCC恶性进展的科学假设，为诠释HCC发生发展机制和寻找治疗靶点提供实验基础。

肝细胞癌（HCC）是世界上最常见的恶性肿瘤之一，其发病率高，预后差。肿瘤侵袭转移是导致其预后不良的重要原因，但机理尚未明确。前期发现去泛素化酶USP11在肝癌组织中高表达，并与侵袭转移和预后不良密切相关。本项目拟在人体标本采用免疫组化等实验明确USP11及其靶分子在HCC组织中的表达及临床意义。通过增殖、克隆形成和转移实验，研究 USP11在体外和体内肝癌细胞中的功能作用。实时荧光定量，蛋白免疫印迹，ChIP，免疫荧光和Co-IP等实验来探索USP11调控靶分子的具体机制。免疫组化等实验表明USP11在肝癌中高表达且与预后密切相关。敲低USP11显著抑制肝癌细胞的增殖和转移能力。敲低USP11后可抑肝癌细胞的自噬，过表达USP11促进肝癌细胞自噬。IP及体内泛素化实验表明USP11可与E2F1相互作用，USP11去泛素化并稳定E2F1蛋白。回补实验证明E2F1参与USP11介导的肝癌细胞增殖转移和自噬。有趣的是，E2F1在转录水平调控 USP11的表达，因此，E2F1/USP11形成正反馈环路促进肝癌细胞增殖转移。E2F1/USP11通过激活ERK/mTOR通路抑制自噬。此外，联合抑制 USP11和自噬对小鼠肝癌细胞凋亡和肿瘤生长的抑制效果优于单独治疗。综上所述，E2F1/USP11信号轴促进肝癌增殖和转移，并抑制自噬，为肝癌的治疗提供了实验依据。另外，本项目通过分析敲低USP11的RNA-seq数据发现，USP11可能还调控HIF1α通路。首先我们敲低USP11，RT-qPCR实验检测HIF-1信号通路的几个关键基因HIF-1α、LDHA和PDK1等的变化，发现敲低USP11可以降低这几个基因的表达。敲低USP11降低HIF-1α的表达，且过表达USP11上调HIF-1α的表达，但过表达USP11不影响HIF-1α的mRNA水平。IP及体内泛素化实验表明USP11与HIF-1α及其降解HIF-1α的VHL和EGLN1-3互作，且USP11去除HIF-1α的泛素化并保持其稳定性。另外USP11通过LDHA影响乳酸的产生。最后研究还发现在临床标本上，USP11与HIF-1α的表达呈正相关性。综上所述，USP11可作为治疗肝癌的潜在的靶标。本项目目前已经发表2篇SCI文章，目前还有2篇SCI论文在投稿。