去泛素化酶USP1调控YAP影响肝癌发生与索拉非尼耐药的机制研究

肝癌是一种致死率很高的恶性肿瘤，其靶向药索拉非尼存在着严重的耐药问题。然而去泛素化酶在肝癌发生与耐药中的作用机制尚不清楚。课题组初步研究显示去泛素化酶USP1在TCGA肝癌组织中高表达并与患者预后呈显著负相关，敲低USP1抑制YAP蛋白表达，提示YAP是USP1潜在的下游靶基因。本项目拟在此基础上，通过肝癌组织芯片进一步确认USP1与YAP蛋白表达与肝癌临床病理指标的相关性。运用过表达和RNAi深入研究USP1对YAP的调控作用。运用Co-IP和GST pull-down探究USP1结合靶蛋白YAP并调控其泛素化水平的分子机制。深入研究YAP转录调控USP1启动子活性并上调其表达的作用机制。在细胞水平和动物模型上，研究USP1/YAP轴促进肝癌细胞增殖侵袭和索拉非尼耐药的生物学功能。本研究将有助于阐明USP1/YAP调控肝癌发生和耐药的分子机制，为USP1作为潜在药物靶点提供理论依据。

Hepatocellular cancer (HCC) is a deadly malignant tumor worldwide. Sorafenib is a target drug for advanced HCC, whose resistance significantly limits its therapeutic efficacy. However, the mechanisms by which deubiquitinating enzymes promote initiation and progression of liver cancer and drug resistance are poorly understood. Our previous results showed that the USP1 expression was increased in HCC tissues from TCGA database. The elevated USP1 expression was negatively correlated with tumor recurrence and survival. Moreover, we found that USP1 deprivation inhibited YAP protein expression, suggesting that YAP may be a potential downstream target of USP1. In this project, we will investigate the expression of USP1 and YAP and correlation between them and clinicopathological features in HCC tissue array. We will determine YAP expression using overexpression and silence of USP1. Moreover, we will clarify that how USP1 binds with YAP protein and regulates its stability by reducing its ubiquitin levels using Co-IP and GST pull-down. Then we will identify the USP1 promoter and determine which regulatory element of USP1 promoter is responsible for increased USP1 expression by YAP. Finally, we will reveal the roles of USP1/YAP loop in promoting cell proliferation and invasion and sorafenib resistance in HCC in vitro and in vivo. Taken together, this study will provide the insight into the mechanism of HCC carcinogenesis and drug resistance mediated by USP1/YAP, which makes USP1 as a potential therapeutic target in HCC.