去泛素化酶USP7通过稳定KRAS促进非小细胞肺癌发生发展的分子机制研究

去泛素化酶USP7是去泛素化超大家族一员，通过与癌蛋白，抑癌蛋白等特定底物相互作用，在肿瘤的发生发展中发挥重要作用。越来越多的证据表明，USP7在多种肿瘤组织中高表达，且这种高表达与肿瘤的发生发展呈正相关。为了更完整地了解USP7在非小细胞肺癌中相互作用蛋白，项目组前期在非小细胞肺癌中进行了免疫共沉淀和质谱联用，鉴定出USP7的结合靶标-KRAS。KRAS亦为参与肿瘤发生发展的癌基因，然而KRAS的去泛素化酶和分子机制尚不明确。因此申请人推测，USP7是调控KRAS多聚泛素化的首个去泛素化酶，且调控分子机制可能是非小细胞肺癌发生发展的关键。为证实以上假说，本项目拟在非小细胞肺癌中，采用多种体内，体外实验包括免疫印迹，免疫共沉淀，去泛素化，ITC，复合物晶体衍射，细胞增殖及裸鼠皮下成瘤等技术明确USP7去泛素化KRAS促进非小细胞肺癌的分子机制，为抗肿瘤药物研发及癌症治疗策略提供新思路。

Ubiquitin-specific protease USP7 is a member of the large family of deubiquitinases. It plays an important role in the occurrence and development of tumors by interacting with specific substrates. Accumulating evidence indicated that USP7 was overexpressed in many tumors, which is positively correlated with the occurrence and development of tumors. To gain a more complete profile of USP7 interactions in cancer cells, we performed affinity purification coupled to mass spectrometry to identify USP7 binding targets in non-small cell lung cancer (NSCLC), and identified a novel interaction with KRAS. KRAS is a well-known oncogene, but its deubiquitinating enzyme is rarely reported. Therefore, applicant speculates USP7 is the first de-ubiquitinating enzyme that regulates the polyubiquitination of KRAS, and the molecular regulation may be the key to the occurrence and development of non-small cell lung cancer. To confirm the hypothesis, this project intends to use a variety of in vivo and in vitro experiments in non-small cell lung cancer, including immunoblotting, immunoprecipitation, deubiquitination, ITC, complex crystal, cell proliferation, and nude mouse model. In conclusion, we hope to clarify the molecular mechanism of USP7 deubiquitination of KRAS to promote non-small cell lung cancer, and provide new ideas for the development of anti-tumor drugs and cancer treatment strategies.