去泛素化酶USP7稳定EZH2促进胰腺癌发生发展及耐药的机制研究

去泛素化酶USP7及组蛋白甲基化酶EZH2的表达失调在肿瘤增殖及侵袭转移过程中扮演重要角色，但二者的作用关系在胰腺癌中未见报道。我们的前期研究发现：EZH2在胰腺癌中上调表达；质谱分析及IP实验证实USP7是EZH2的互作蛋白，可稳定EZH2的蛋白水平；削减USP7的表达可以降低EZH2的蛋白水平，且抑制胰腺癌细胞的侵袭、迁移；USP7抑制剂可克服EZH2高表达细胞株对EZH2i的耐药。这些结果提示在肿瘤细胞中，USP7的高表达降低了EZH2的泛素化水平，稳定了EZH2，促进了细胞的增殖及侵袭迁移，同时对EZH2抑制剂更加耐受。本课题将进一步在分子、细胞、个体及临床样本等水平，系统分析USP7及EZH2在胰腺癌中的表达特征和临床意义，研究USP7稳定EZH2在胰腺癌发生发展及耐药过程中的作用及机制，探讨USP7与EZH2小分子抑制剂联用的抑癌效果，为胰腺癌的精准治疗提供新的靶点和思路。

The disordered expression of deubiquitination enzyme USP7 and histone demethylase EZH2 play important roles in the proliferation, invasion and metastasis of malignant tumors, but the function of USP7 in pancreatic cancer progress and its relationship with EZH2 are not reported. In preliminary experiments, we found that EZH2 expression is significantly up-regulated in pancreatic cancer. Tandem affinity purification and immunoprecipitation experiments verified that USP7 is a bona fide binding protein of EZH2 and, it can specifically stabilizes the protein level of EZH2. Oppositely, knocking down USP7 by shRNA decreases the protein level of EZH2 and inhibits the invasion and migration of pancreatic cancer cells. Moreover, USP7 inhibitor can overcome the resistance of EZH2 highly expressed cell lines to EZH2 inhibitors. These results indicate that USP7 is up-regulated, which decreases the ubiquitination of EZH2, stabilizes its protein level and, promotes the proliferation,invasion, migration and also EZH2i resistance in tumor cells. However, when tumor cells were combinationally treated with USP7 and EZH2 inhibitors, the ubiquitination level of EZH2 is increased and the protein is easy to be degraded, which overcomes the drug resistance of tumor cells caused by the increase of EZH2 protein level. This project will further study the correlation between clinical indicators and the expression of USP7 and EZH2 in pancreatic cancer, verify the function of USP7 on pancreatic cancer progression, ascertain the molecular mechanisms of USP7 in regulating the stability of EZH2 and also analyse the inhibition effect by combination of USP7 and EZH2 inhibitors. In brief, this study will provide new ideas on exploring the molecular targets in precise treatment of pancreatic cancer.