EZH2的去泛素化调控在乳腺癌发生发展中的作用研究

EZH2 (Enhancer of Zeste Homolog 2) is an important histone methyltransferase which plays a role of catalyzing the trimethylation of histone 3 lysine 27 (H3K27) on the promoter of its target genes, thus repressing their expression. EZH2 is related with various malignant diseases including breast cancer. However, regulation of EZH2 protein expression is still not well studied. Till now, two E3 ubiquitin ligases of EZH2 have been found, that is, Smurf2 and β-TrCP, which catalyze EZH2 ubiquitination and play important roles in EZH2-related diseases. However, the ubiquitination process is a dynamic equilibrium process. So we need to further find deubiquitination enzymes of EZH2, and reveal its regulatory mechanism in order to make more specific treatment for EZH2-related diseases. Firstly, we used the internationally accepted DUBs (deubiquitination enzymes) expression library for screening of EZH2 deubiquitinase. Our preliminary results showed that of all the DUBs we screened, USP7 is able to up-regulate the expression of EZH2 obviously. Further experiments confirmed that USP7 can up-regulate EZH2 in a dose-dependent manner. This project aims to identify the specific DUB of EZH2, and to explore the regulatory mechanism of EZH2 by protein ubiquitination and its relationship with EZH2-related diseases, providing potential new drug targets for clinical treatment of EZH2-related diseases including breast cancer.

EZH2是一种重要的组蛋白甲基转移酶，它可以催化组蛋白H3第27位赖氨酸发生三甲基化，抑制靶基因的转录。EZH2与乳腺癌等多种恶性疾病相关。然而，关于EZH2自身调控机制的研究还不是很完善。两种泛素连接酶Smurf2和β-TrCP可催化EZH2发生泛素化，并在EZH2相关疾病中起着重要作用。然而，泛素化过程是一种动态平衡过程，只有找到EZH2的去泛素化酶，揭示其调控机制，才能更有针对性得治疗EZH2相关疾病。我们首先在293T细胞中利用国际公认DUBs（去泛素化酶）表达文库进行筛选。初步结果表明，去泛素化酶USPs家族成员USP7可明显使EZH2蛋白表达水平增加，并且具有剂量依赖效应。本项目通过鉴定EZH2的特异性DUB，探索蛋白去泛素化调控对EZH2功能的调节机制及其与疾病之间的关系，旨在为临床治疗乳腺癌等EZH2高表达相关的疾病提供潜在靶点。

表观遗传调控在肿瘤等恶性疾病的发生发展中起着重要的作用。Ezh2作为一种PcG蛋白，可以与SUZ12，EED等分子组成PRC2复合体，参与染色质结构的形成，基因表达调控及生长控制。Ezh2的过表达，过度激活及体细胞突变，与多种肿瘤的发病有关。Ezh2在肿瘤和其他恶性疾病中如何被调控，如何影响疾病进程，也成为亟待研究的问题。蛋白质泛素化调控是国际公认的调节细胞蛋白表达水平及信号通路的重要机制。本项目前期通过在293T细胞中利用国际公认的DUB（去泛素化酶）文库，与EZH2质粒共转染，筛选出能够上调EZH2表达的去泛素化酶USP7，并通过一系列分子实验，确定了1）在293T及Hela细胞中， USP7可以梯度上调EZH2的表达; 2）过表达USP7不能影响EZH2的mRNA转录水平；3）USP7能够增强EZH2蛋白的稳定性；4）USP7能够与EZH2相互作用；5）过表达USP7使EZH2蛋白的泛素化水平升高；6）过表达USP7可通过上调EZH2使其靶基因表达下降。