UCHL3 promotes hepatocellular carcinoma cell migration by de-ubiquitinating and stabilizing Vimentin

Background Hepatocellular carcinoma (HCC) is a common malignant tumor associated with a poor prognosis. Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) has been reported to promote diverse tumors, but little is known about its role in HCC. Methods Expression levels of UCHL3 in Huh7 and Hep3B cells were measured by qRT-PCR. UCHL3, Vimentin protein levels, and ubiquitination levels were determined by Western blot assay. co-immunoprecipitation, Immunofluorescence, and IHC were used to detect the interaction and expression association between UCHL3 and Vimentin in the cells. Wound healing and Transwell assays were used to measure cell migration. Spheroid formation assay were used to assess stem-like properties. Results UCHL3 expression was found to be significantly elevated in HCC and associated with poor prognosis. UCHL3 promoted migration and stem-like properties of HCC cells. Vimentin was identified as a potential de-ubiquitination substrate of UCHL3 and UCHL3 interacted with and promoted the de-ubiquitination of Vimentin, enhancing its stability. Moreover, the suppression of UCHL3 by siRNA or the inhibition by TCID upregulated ubiquitinated Vimentin. Vimentin attenuated the suppression of cell migration caused by knockdown of UCHL3. Conclusion UCHL3 was highly expressed in HCC and functioned as an oncogene. Vimentin is a novel substrate of UCHL3 and its stabilization and de-ubiquitination enhanced HCC cell migration.


Introduction
Liver cancer is the third most common cause of global cancerrelated death (1). Over 90% of liver cancer cases are hepatocellular carcinoma (HCC), of which only 5%-15% are candidates for earlystage surgical resection. Chemotherapy and immunotherapy are used to treat advanced HCC, but metastasis leads to unsatisfactory long-term survival rates (2-4). The identification of novel therapeutic targets is thus an urgent matter.
Epithelial cells lose polarity and acquire mesenchymal characteristics during the EMT, a process that contributes to tumor cell migration (26). The involvement of Vimentin in the initiation of the EMT has recently been demonstrated (27, 28), and Vimentin is known to be regulated by post-translational modifications, including ubiquitination, phosphorylation, O-linked glycosylation, sumoylation, and ADP-ribosylation (29)(30)(31). The ubiquitin ligases, Trim16 (32) and Trim56 (33), have been found to ubiquitinate Vimentin, but less is known about its de-ubiquitination, other than by USP14 (34).
The current study found that UCHL3 was upregulated in HCC patients, indicating a poor prognosis. UCHL3 promoted the migration and stem-like properties of HCC cells. UCHL3 was identified as a DUB, which targets Vimentin, and the consequent de-ubiquitination may be responsible for the promotion of cell migration.

Clinical tissues
Clinical specimens were obtained from patients with hepatocellular carcinoma at Zhejiang Provincial People's Hospital.
Freshly excised HCC tissues were fixed using formalin and embedded in paraffin for immunohistochemical (IHC) staining.

Wound healing assay
Cells were cultured in six-well plates until a monolayer formed and a wound created in the middle of the well; cells were washed with 1× phosphate-buffered saline (PBS) to remove debris, and serum-free medium was added. Cell migration was assessed by inspecting the wound area under an inverted microscope (Nikon, DS-Ri2). Images were analyzed by ImageJ software.

Transwell assay
A total of 600 µl of complete medium was added to the lower chamber of Transwell inserts (Corning), and HCC cells in serumfree medium were inoculated into the upper chamber. Cells were cultured for 24 Hours, and those in the lower chamber were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and imaged under an inverted microscope (Nikon, DS-Ri2).

Half-life and inhibitor analysis
Cells were treated with 10 mM cycloheximide (CHX; Selleck, S7418) to block protein synthesis, and proteins were extracted at various time points and protein levels assessed by Western blotting for half-life determination.
Hep3B and Huh7 cells were treated with dimethyl sulfoxide (DMSO) or 10 mM TCID (Selleck, S7140) for 24 h, and UCHL3 and Vimentin expression was determined by Western blotting. Western blotting was used to evaluate polyubiquitinated Vimentin following co-immunoprecipitation of Vimentin.

Statistical analysis
Means ± standard deviations for at least three replicates are presented. Statistical analyses were performed by GraphPad Prism 9 (San Diego, CA, USA), and differences were assessed by Student's ttest. A value of p < 0.05 was considered statistically significant.

Results
UCHL3 was upregulated in HCC and associated with poor prognosis UCHL3 expression was analyzed by the UALCAN (35) platform and found to be upregulated in HCC by comparison with normal hepatic tissue ( Figure 1A). Analysis via the GEPIA platform indicated that high UCHL3 expression was associated with shorter overall survival among HCC patients ( Figure 1B). Kaplan-Meier analysis also demonstrated that high UCHL3-expressing HCC patients had reduced survival ( Figure 1C). UCHL3 was also highly expressed in the HCC cell lines, namely, Hep3B, Huh7, and HepG2, by comparison with the normal hepatocyte cell line, MIHA ( Figure 1D). In summary, it is suggested that UCHL3 may contribute to liver tumorigenesis and may be a useful prognostic biomarker in HCC.

UCHL3 overexpression promoted migration and stem-like properties of HCC cells
Huh7 and Hep3B cells stably overexpressing UCHL3 were generated (Figures 2A, B). UCHL3 overexpression promoted cell migration during wound healing ( Figure 2C) and Transwell assays ( Figure 2D). UCHL3 Overexpressing cells formed larger spheroids, indicating increased stem-like properties, compared with control cells ( Figure 2E). Overall, UCHL3 may contribute to malignant progression by promoting migration and stem-like properties of HCC cells.

UCHL3 knockdown inhibited migration and reduced stem-like properties of HCC cells
Stable knockdown of UCHL3 was established in HCC cell lines ( Figures 3A, B). UCHL3 knockdown inhibited cell migration and stem-like properties, as revealed by wound healing and Transwell assays ( Figures 3C, D) and spheroid formation assays ( Figure 3E). Loss of UCHL3 thus impaired migration and stem-like properties of HCC cells.

Identification of Vimentin as a new substrate of UCHL3
The above results demonstrate the contribution of UCHL3 to migration and stem-like properties of HCC cells, but downstream targets of this de-ubiquitinase remain unknown. Coimmunoprecipitation experiments with UCHL3 antibody followed by mass spectrometry analysis (IP-MS) were performed to identify potential substrates of UCHL3. UCHL3-binding protein complex contained peptide fragments of Vimentin, indicating a Vimentin-UCHL3 interaction ( Figure 4A). Co-immunoprecipitation experiments in HCC cells confirmed the interaction of UCHL3 with Vimentin ( Figure 4B). Immunofluorescence results show the colocalization of UCHL3 and Vimentin in cells ( Figure 4C). The findings suggest that Vimentin is a novel protein-binding partner of UCHL3.

UCHL3 stabilized Vimentin protein via de-ubiquitination
We performed immunohistochemical staining for UCHL3 and Vimentin in HCC tissues. The results showed a positive correlation between the expression of UCHL3 and Vimentin in HCC ( Figure 4D). Next, cells in which UCHL3 was either overexpressed or knocked down were used to investigate its regulatory impact on Vimentin. UCHL3 promoted Vimentin protein expression, and UCHL3 knockdown inhibited Vimentin protein expression ( Figure 4E). UCHL3 overexpression enhanced the stability of Vimentin protein, while UCHL3 inhibition suppressed its stability ( Figure 4F). Consistent with the role of UCHL3 as a DUB, UCHL3 overexpression decreased the ubiquitination level of endogenous Vimentin ( Figure 4G), and Vimentin ubiquitination was increased when UCHL3 was knocked down ( Figure 4H). In conclusion, the role of UCHL3 as a DUB allowed stabilization of Vimentin.

Inhibition of UCHL3 impaired HCC cell migration
TCID is an UCHL3 inhibitor (22) and was found to reduce Vimentin expression without any change in UCHL3 ( Figure 5A). TCID treatment also significantly increased Vimentin ubiquitination in Hep3B and Huh7 cells (Figures 5B, C). In a similar manner to UCHL3 knockdown, TCID treatment also suppressed HCC cell migration ( Figures 5D, E). In summary, inhibition of UCHL3 by TCID impaired HCC cell migration.

UCHL3 facilitated HCC cell migration through regulating Vimentin
Functional rescue experiments were performed to demonstrate actions of UCHL3 on Vimentin. Knockdown of Vimentin inhibited HCC cell migration and suppressed UCHL3-induced HCC cell migration, as illustrated by wound healing and Transwell assays ( Figures 6A-C). Vimentin overexpression promoted HCC cell migration and attenuated the inhibition of cell migration due to UCHL3 knockdown (Figures 6D-F). In conclusion, the findings suggest that UCHL3 promotes HCC cell migration through stabilizing Vimentin. Together, the abovementioned results show that the DUB activity of UCHL3 stabilizes the expression of Vimentin and promotes the migration of HCC. And TCID can inhibit the DUB activity of UCHL3 (Figure 7).

Discussion
Worldwide HCC incidence is on the increase and is estimated to rank third for cancer-related deaths by 2030 (36). Indeed, liver cancers, along with pancreatic cancer, generally have low survival rates (37). Ubiquitin dysregulation, due to imbalances between ubiquitinase and de-ubiquitinase activities, is thought to affect tumor development (38)(39)(40), but UCH de-ubiquitinases have been rarely studied in HCC, by contrast with USP enzymes (41). Only UCH37 has been investigated in liver cancer cells (42), but other family members may also have an impact. The current study correlated abnormally high UCHL3 expression with the occurrence and malignant development of HCC. UCHL3 promoted HCC cell migration and stem-like properties.
Cell migration and stem-like properties contribute to the aggressiveness of tumors and poor patient prognosis (43). Many proteins involved in HCC cell migration are regulated by ubiquitination, such as b-catenin (44), snail (45,46), and ZEB1 (47). The EMT marker, Vimentin, may also influence tumor progression. Indeed, the ubiquitination of Vimentin influenced by symmetric dimethylarginine (sDMA) is thought to be necessary for the roles of   (19,20,22) and demonstrated its contribution to carcinogenesis (14). UCHL3 promoted pancreatic cancer cell proliferation and migration by deubiquitinating FOXM1 (20). The Aryl hydrocarbon receptor (AhR) protein was stabilized by UCHL3 de-ubiquitination in NSCLC and promoted stem-cell-like properties (22). In addition, UCHL3 induced tumorigenesis in ovarian cancer by deubiquitinating and stabilizing TRAF2 (19). UCHL3 promoted migration of the liver cancer cells of the current study by deubiquitinating Vimentin. We acknowledge some limitations to the current in vitro study, and in vivo experiments are needed to confirm the findings.
In conclusion, UCHL3 was found to be highly expressed in HCC and indicated a poor prognosis. The DUB activity of UCHL3 stabilized Vimentin and promoted HCC malignant progression. The present findings give new insights into HCC pathogenesis, indicating that the UCHL3/Vimentin axis may provide an innovative therapeutic target.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The studies involving human participants were reviewed and approved by Medical Ethics Committee of Zhejiang Provincial People's Hospital. The patients/participants provided their written informed consent to participate in this study.