The Role And Mechanism Of UCH37on HCC Recurrence

Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes such as transcriptional regulation, growth and differentiation, and oncogenesis. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37and BRCA1-associated protein-1(BAP1). Although there is growing evidence that UCH enzymes and human malignancies are closely correlated, there have been few studies on UCH37. The aim of this study is to investigate the biological roles of UCH37in human hepatocellular carcinoma (HCC) and to elucidate its mechanisms. This study includes three parts:Part Ⅰ:The association of UCH37expression with clinicopathologic characteristics and prognosis of HCC patients.Immunostaining for UCH37was assessed in the cancerous tissues and the adjacent non-cancerous liver tissues in90cases of human HCC. The expression of UCH37in the cancerous tissues was significantly higher than that in the adjacent para-cancerous tissues. Among61recurrence patients, we found out the similar results. Subsequently, univariate analysis showed that BCLC stage, TNM stage, vascular invasion, tumor size, encapsulation and UCH37expression were unfavorable predictors for the time to recurrence (TTR). Multivariate analysis indicated that UCH37expression was an independent prognosticator for TTR. The patient, whose extra part from the score of the HCC tissue minus that of the adjacent para-cancerous tissue was no less than2, had a higher cumulative risk of developing HCC recurrence after resection. These data indicated that UCH37could be a predictive marker of HCC recurrence in HCC patients after curative resection.Part Ⅱ:The biological roles of UCH37in HCC cells.Stable monoclonal cell lines by altering the UCH37expression level were established to explore the biological roles of UCH37in HCC. Compared with the control cells, UCH37down-regulation inhibited cell proliferation, while UCH37over-expression did not affect cell proliferation. UCH37did not affect cell apoptosis and cell cycle distribution in HCC cells. Intriguingly, UCH37was found to promote cell migration and invasion in HCC cells. Furthermore, UCH37over-expression was shown to promote tumorigenecity in nude mice. These data indicated that UCH37promoted cell migratory and invasive abilities in vitro, and demonstrated that reducing UCH37expression level could attenuate HCC cell agressiveness.Part III:Mechanisms of UCH37influence on migration and invasion in HCC cells.In this part, on one hand, recombinant GST-UCH37fusion protein was purified, and GST pull-down assays were performed using such protein with whole Huh7cell lysates. The precipitates were further identified by mass spectrometer assay, and showed that63proteins were found in the precipitates, but not in the control. Meanwhile, total proteins were extracted from one human HCC tissue, and then co-immunoprecipitated with anti-UCH37antibody. In the co-immunoprecipitates,22candidate UCH37-interacting proteins were identified using two-dimensional gel electrophoresis and mass spectrometer assay. Comparing the two groups of results, we found glucose-regulated protein78(GRP78) as a common protein. The interaction of UCH37with GRP78with both exogenously over-expressed UCH37and endogenous proteins was confirmed via co-immunoprecipitation (co-IP) and confocal laser scanning microscopy analysis.On the other hand, the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), was utilized to identify differentially expressed proteins. Consequently,19proteins were found to be up-regulated and21down-regulated in UCH37over-expressing cells, compared with the control cells. The differentially expressed proteins were further analyzed using the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database. The data showed that these proteins were widely distributed in the cell, and demonstrated that they played a role in all aspects of cell biology, indicating that UCH37was involved in almost all biological processes. PRP19, an essential RNA splicing factor, was chosen for further study. The data showed that UCH37formed a complex with PRP19and regulated PRP19via deubiquitination. Moreover, using small interference RNA (siRNA), knock-down of PRP19in UCH37over-expressing cells reduced the capability of cell migration and invasion, compare with the control cells. These data indicated that UCH37could promote cell migration and invasion in HCC cell lines through interacting and deubiquitinating PRP19.Furthermore, we screened a lot of proteins interacting with UCH37in HCC and provided a new insight into the association of UCH37with oncogenesis.Taken together, this study provided evidence for the first time that UCH37could promote cell migration and invasion in HCC cell lines through interacting and deubiquitinating PRP19, and this is the first study showing UCH37as a predictive marker of HCC recurrence in HCC patients after curative resection. We believe that UCH37could be a good diagnostic and therapeutic target for controlling HCC recurrence, and further investigations in this field are needed.