去泛素化酶UCH37促进人肝癌复发的分子机制研究

The abnormal balance of protein ubiquitination and protein deubiquitination in the cells plays a crucial role in numerous biological processes and oncogenesis. Ubiquitin C-terminal hydrolase 37 (UCH37), a member of the ubiquitin C-terminal hydrolase (UCH) which is a subfamily of deubiquitinating enzymes (DUBs), is involved in such balance. In our preliminary work, clinicopathologic data showed that UCH37 was over-expressed in HCC cancerous tissues and was associated with HCC recurrence after curative resection. In vitro, we discovered that UCH37 could promote cell migration and invasion. Subsequently, co-immunoprecipitation (co-IP), GST pull-down assays and proteomic approaches were performed to screen UCH37-interacting proteins in HCC. Consequently, GRP78, a molecular chaperone that facilitate proper protein folding, and PRP19, an essential RNA splicing factor, were identified as two interacting with UCH37. In the current study, the large-scale prospective trial will be performed to validate the role of UCH37 over-expression in HCC recurrence and metastasis after curative resection, and to elucidate its mechanisms. We will focus on the relationship between UCH37, GRP78 and PRP19, and gain an insight into mechanisms that UCH37, interacting with GRP78, can deubiquitinate PRP19 which leads to the accumulation of PRP19 in the cells and influences the mature of miRNAs, resulting in promoting HCC recurrence and metastasis.

细胞内蛋白质泛素化和去泛素化调节的动态平衡异常在肿瘤发生、发展中起关键作用。UCH37是一种去泛素化酶，是调节上述平衡的关键因子之一。课题组前期工作发现UCH37在肝癌组织中表达上调，且与肝癌术后复发相关；同时发现UCH37能促进肿瘤细胞迁移和侵袭；通过免疫共沉淀等技术，筛选到与UCH37相互作用的两个蛋白质――GRP78和PRP19。GRP78在蛋白质折叠过程中起分子伴侣作用；PRP19参与mRNA剪接和DNA损失修复。本课题将在既往工作的基础上，进一步扩大临床样本，明确UCH37高表达在肝癌术后转移复发中的作用，探索UCH37作用的关键靶分子。重点研究UCH37、GRP78与PRP19三者相互作用关系，希望能证实肝癌组织中UCH37高表达后，在GRP78的辅助下，影响PRP19去泛素化，使其泛素化降解减少，导致PRP19细胞内积累，从而进一步影响miRNAs的成熟、促进肝癌转移复发。

去泛素化酶异常表达与肿瘤发生密切相关。本课题主要探讨去泛素化酶UCH37在肝癌中的作用并进一步探讨其分子机制。该课题表明UCH37在肝癌组织中呈高表达，且与肝癌复发密切相关，是影响肝癌患者根治术后TTR的独立危险因素。通过构建UCH37过表达和下调的稳定的单克隆细胞株以及细胞功能实验，我们发现UCH37能显著增强肝癌细胞的迁移和侵袭能力；具有促进细胞增殖和肿瘤形成的能力；对肝癌细胞株的细胞周期和细胞凋亡没有影响。具体分子机制探讨上，一方面通过GST pull-down 分析筛选到了同一个与UCH37特异性结合的蛋白质，即GRP78，并通过免疫共沉淀和激光共聚焦技术证实了UCH37和GRP78之间存在相互作用。另一方面，应用蛋白质定量分析技术，筛选到了一系列与UCH37相关的差异表达蛋白，并通过免疫共沉淀、激光共聚焦、siRNA等技术，证实了UCH37通过与PRP19相互作用，抑制其泛素化降解，导致PRP19持续高表达，从而增强了肝癌细胞的迁移和侵袭能力。同时，这一部分研究筛选到了许多与UCH37相互作用或者受UCH37调控的蛋白质，为进一步探讨UCH37在肝癌术后复发中的作用机制提供了新的线索。综上所述，本研究阐明了去泛素化酶UCH37通过与Prp19相互作用使其去泛素化，促进肝癌细胞迁移和侵袭。此外该工作首次表明UCH37在肝癌患者根治术后复发的预测中临床应用前景，可能成为肝癌复发的诊断和治疗新靶点，具有进一步深入探讨的价值。