We obtained 45 pairs of fresh specimens containing tumour tissues and paired adjacent tissues from patients who underwent radical resection at the Department of Orthopaedics of University-Town Hospital of Chongqing Medical University (Chongqing, China) from September 2013 to July 2017. The patients were diagnosed according to the diagnosis of bone cancer of the NCCN (National Comprehensive Cancer Network) guidelines (https://www.nccn.org/) and the pathology of the tumour tissues was evaluated by pathologist. Patients with other sarcomas including chondrosarcoma, osteochondroma and Ewing's sarcoma were excluded from this study. Patients who received prior radiotherapy or chemotherapy were also excluded. The patient clinical data including age, sex, tumour size, stage and metastasis were obtained. The age range of the patients was from 11 to 30 years and the average age was 18, the ratio of men to women was 1.812. Detailed information is listed in Table I. All tissue samples were obtained following surgical resection and stored in liquid nitrogen immediately. Written informed consent was obtained from all tissue donors, and this study was approved by the Ethics Committee of Chongqing Medical University (Chongqing, China) prior to surgery.

We purchased human OS cell lines (U2OS, Saos-2, MG-63, 143B and HOS) and normal human osteoblastic (NHOst) cell line) from the American Type Culture Collection (ATCC; Manassas, VA, USA) and Chinese Academy of Medical Science (Beijing, China). All cells were maintained in HyClone™ Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and cultured in an incubator maintained at 37°C with 5% CO₂.

We embedded OS tumour tissues into paraffin to perform the immunohistochemical analysis. First, tissue sections were dewaxed, antigen repaired with citrate solution and blocked in goat serum. Subsequently, we incubated the sections with the USP7 antibody (dilution 1:200; cat. no. ab4080; Abcam, Cambridge, UK) overnight at 4°C, following incubation with a secondary of goat anti-rabbit antibody carrying horseradish peroxidase (HRP) for 30 min at room temperature. Finally, we dyed sections with DAB dyestuff for 30 sec. This assay was performed with the immunohistochemistry kit (cat. no. SP9001; ZSGB-BIO, Beijing, China).

We used TRIzol reagent (Takara, Biotechnology Co., Ltd., Dalian, China) to extract the total RNA from OS tumour tissues or cells. Subsequently, cDNA was synthesised from the RNA samples using the PrimeScript™ RT Master Mix (Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions. The following primer sequences were used: USP7 forward, 5′-AATCATTGGTGTTCATCA-3′ and reverse, 5′-CAAGCATCTCATTCTCTT-3′; β-actin forward, 5′-TCCTTTGGAACTCTGCAGGT-3′ and reverse, 5′-GACCTACTGTGCGCCTACTT-3′. In addition, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays with the SYBR Green Master Mix kit (Takara Biotechnology Co., Ltd.). Of note, β-actin was an internal control. Furthermore, we used the 2^−ΔCq or 2^−ΔΔCq method (17) to analyse the relative USP7 expression in OS tumour tissues and cells.

We extracted the total protein of OS tumour tissues and cells using RIPA lysate (Beyotime Institute of Biotechnology, Shanghai, China). In addition, the protein concentration was tested using a BCA kit (Beyotime Institute of Biotechnology) and standardised using RIPA lysate. Overall, a 40-µg protein sample was separated using 10% SDS-PAGE gel for 2 h. These protein samples were transferred into polyvinylidene fluoride membranes, which were blocked in 5% skimmed milk for 2 h at room temperature. Subsequently, we incubated the membranes with primary antibodies against USP7 (dilution 1:1,000; cat. no. ab4080; Abcam), β-catenin (dilution 1:1,000; cat. no. ab32572; Abcam), Snail (dilution 1:1,000; cat. no. ab53519; Abcam), E-cadherin (dilution 1:1,000; cat. no. 14472; Cell Signaling Technology, Danvers, MA, USA), N-cadherin (dilution 1:1,000; cat. no. 13116; Cell Signaling Technology), vimentin (dilution 1:1,000; cat. no. 5741; Cell Signaling Technology), MMP9 (dilution 1:1,000; cat. no. 13667; Cell Signaling Technology) and β-actin (dilution 1:2,000; cat. no. 3700; Cell Signaling Technology) at 4°C overnight. The membranes were further incubated in secondary antibodies conjugated with horseradish peroxidase (HRP) (dilution 1:3,000; cat. nos. 7074 and 7076; Cell Signaling Technology) at room temperature for 2 h. Finally, we visualised images of proteins using ECL reagent (Beyotime Institute of Biotechnology). The gray value of protein expression was quantified with Quantity One software (Bio-Rad, Shanghai, China).

A lentivirus carrying a USP7-specific small-hairpin RNA (sh/USP7) and USP7 overexpression sequence were constructed at Shanghai Genechem Company (Shanghai, China). The sh/USP7 sequences were as follows: shRNA1, 5′-TGTATCTATTGACTGCCCTTT-3′; shRNA2, 5′-CGTGGTGTCAAGGTGTACTA-3′; shRNA3, 5′-CTCAGAACCCTGTGATCAA-3′. After 48 h, OS cells were transfected with the lentiviral diluent, according to the manufacturer's instructions, and selected with puromycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) after 2 weeks.

The migration and invasion abilities of the OS cells were assayed in 24-well Transwell™ Chambers (Corning Inc., Corning, NY, USA). Briefly, for the migration assay, cells were digested and adjusted to 2×10⁵ cells/ml using serum-free medium; 100 µl of this cell suspension was added to the upper chamber, and 700 µl of medium containing 10% FBS was added to the bottom chamber. After a 24-h incubation, the chamber was carefully taken out and washed with PBS buffer. Subsequently, the migrated cells were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet for 30 min and photographed using an Leica DMi1 inverted microscope (Leica Microsystems, Solms, Germany) at ×200 magnification. During optical microscope observation, three random regions were selected for the statistical analysis.

For the invasion assay, Matrigel (BD Biosciences, San Diego, CA, USA) was diluted with the serum-free medium at an appropriate ratio. Overall, 100 µl of diluent Matrigel was added to the upper chamber and placed in an incubator at 37°C until the Matrigel was completely solidified. The next steps were performed in accordance with the migration assay. After 48 h, the chamber was carefully taken out and washed with PBS buffer. Subsequently, the migrated cells were fixed with paraformaldehyde, stained with crystal violet and photographed with the Leica DMi1 inverted microscope (Leica Microsystems) at ×200 magnification. Three random regions were selected for the statistical analysis.

The total protein of OS cells was isolated using RIPA lysate (Beyotime Institute of Biotechnology). Protein A/G magnetic beads (Millipore Corporation, Billerica, MA, USA) were added and incubated at 4°C for 2 h to discard the non-specific protein in the cell lysates. The cell lysates were incubated with the primary antibodies (USP7, cat. no. ab4080; Abcam; Use at an assay dependent concentration) (β-catenin, dilution 1:200; cat. no. ab32572; Abcam) at 4°C overnight. The magnetic beads were re-added to the cell lysates and incubated at 4°C for 3 h to pull down the protein complex. Tube was placed on the magnetic stand to remove the supernatant. Then, the beads were carefully washed with PBS buffer and were added to 1X loading buffer and boiled in 100°C water for 10 min. Finally, we removed the magnetic beads using a magnetic stand to obtain the immunoprecipitated protein complex. This protein complex was further analysed using western blot assays.

All statistical analyses were performed using SPSS version 22.0 statistical software (SPSS, Chicago, IL, USA). Data are presented as mean ± SD from independent triplicate experiments. We used Student's t-test or Mann-Whitney test to analyse the difference between two independent groups. Variance (ANOVA) followed by Dunnett or Bonferroni post hoc test was used to analyse the difference of multiple comparisons. We analysed the correlation between USP7 expression and clinicopathological features with χ² statistical testing. Furthermore, Spearman correlation analysis was performed to test the correlation between USP7 and β-catenin expression. For all tests, P<0.05 was considered statistically significant.

We detected USP7 expression in 45 pairs of OS specimens using immunohistochemical assays; USP7 was highly expressed in tumour tissues compared with that noted in the paired adjacent tissues (Fig. 1A). In addition, qRT-PCR assays revealed that USP7 mRNA expression was upregulated in 30/45 (66.6%) OS tumour tissues compared with the paired adjacent tissues (Fig. 1B). Similarly, western blot assay revealed that the protein level of USP7 was markedly upregulated in the OS tumour tissues compared with the level noted in the adjacent normal tissues (Fig. 1C). We examined USP7 expression in five OS cell lines and normal human osteoblastic cells and found that USP7 mRNA and protein levels in OS cell lines (U2OS, Saos-2 and MG-63) were markedly higher than these levels in the NHOst cell line (Fig. 1D and E). However, USP7 expression was lower in the HOS cell line when compared to that noted in the NHOst cells. These findings indicated that USP7 was highly expressed in OS tissues and most OS cell lines when compared with the level in the paired adjacent tissues or the NHOst cell line.

Since USP7 was revealed to be upregulated in OS tumour tissues, we assessed the clinical association of USP7 expression. USP7 expression in OS tumour tissues, detected beforehand by qRT-PCR, revealed that USP7 expression in patients with advanced stages, including stages II–IV, was higher than that in patients with early stage OS (Fig. 1F). In addition, patients with metastasis exhibited high USP7 expression, compared with those without metastasis (Fig. 1G). Furthermore, χ² test revealed that USP7 expression was positively correlated with TNM stage and metastasis in patients with OS. However, we observed no marked correlation between its expression and other clinicopathological features, including patient age, sex and tumour size (Table I).

In this study, U2OS cells, which presented high USP7 expression, were stably transfected with a lentivirus carrying the USP7 shRNA target sequence (sh/USP7) or negative control sequence (NC/shRNA). We extracted the total RNA and protein of cells to assess the efficiency of USP7 knockdown. qRT-PCR results indicated that USP7 mRNA expression in U2OS cells transfected with the sh/USP7 target sequence was significantly less compared with that in cells transfected with the control sequence (Fig. 2A). As anticipated, the protein level of USP7 in sh/USP7-transfected cells was decreased compared to that in NC/shRNA cells (Fig. 2B).

Wound-healing assay that was performed to assess the effect of USP7 knockdown on OS cell migration revealed that the wound-healing ability of the U2OS cells transfected with sh/USP7 was substantially weaker than that of the control cells (Fig. 2C). In addition, Transwell migration assays further suggested that the migratory ability of the sh/USP7-transfected U2OS cells was markedly inhibited compared with that in the control cells (Fig. 2D and F). These findings suggest that USP7 knockdown impaired the migratory ability of OS cells. Furthermore, the invasion assays revealed that the U2OS cell invasive capability was markedly blocked after USP7 knockdown (Fig. 2E and F).

We selected HOS cells, which exhibited low USP7 expression, to further identify the regulatory role of USP7 on OS cell migration and invasion; these cells were stably transfected with a lentivirus carrying the USP7 overexpression sequence (USP7). qRT-PCR revealed that USP7 mRNA expression in the HOS cells transfected with the USP7 overexpression sequence was significantly higher than that in the negative control cells (NC/USP7; Fig. 3A). Moreover, the protein level of USP7 in HOS cells transfected with the USP7 overexpression sequence was increased compared with that in the control cells (Fig. 3B).

Wound-healing assay revealed that the healing ability of HOS cells transfected with the USP7 overexpression sequence was considerably better than that of the control cells (Fig. 3C). Likewise, Transwell migration assay revealed that the migratory ability of the USP7 overexpression sequence-transfected cells was substantially enhanced compared with that in the control cells (Fig. 3D and F). As expected, the invasion assay presented that USP7 overexpression significantly promoted OS cell invasive capability (Fig. 3E and F). These findings revealed that USP7 plays a crucial role in contributing to OS cell migration and invasion.

USP7 was able to promote OS cell migration and invasion; however, the mechanism for this regulatory process is unclear. Currently, it is widely recognised that epithelial-mesenchymal transition (EMT) is the key process for promoting tumour cell invasion and metastasis (18). Several studies have revealed that EMT is involved in OS malignancy process (19,20). Thus, the effect of USP7 on EMT was evaluated. The results revealed that the epithelial marker (E-cadherin) was significantly increased and mesenchymal markers (N-cadherin, vimentin and MMP9) were significantly decreased in the U2OS cells after USP7 knockdown (Fig. 4A). In contrast, the epithelial marker was significantly decreased and the mesenchymal markers were significantly increased in the HOS cells after USP7 overexpression (Fig. 4B). Therefore, USP7 may induce EMT of OS cells.

A previous study demonstrated that when the Wnt/β-catenin signalling pathway is activated, β-catenin accumulates abnormally in the cytoplasm and transfers into the nucleus; subsequently, the increment of β-catenin can recruit the transcription factor Snail1, which is a key upstream regulating factor of EMT (21). Surprisingly, we observed that USP7 knockdown markedly decreased the expression of β-catenin and Snail1 in the U2OS cells (Fig. 4C) and USP7 overexpression increased their expression in the HOS cells (Fig. 4D). Overall, these findings revealed that USP7 could activate the Wnt/β-catenin signalling pathway to induce EMT of OS cells.

USP7 can regulate various oncoproteins by directly binding with the targets and increasing their stability. In the present study, significant efforts were made to determine the correlation between USP7 and β-catenin in OS cells. Thus, CO-IP assays were performed using the total endogenous protein of U2OS cells. Remarkably, the results revealed that USP7 and β-catenin interact directly (Fig. 5A and B). The re-examination of USP7 and β-catenin expression in OS tumour tissues demonstrated that β-catenin exhibited strong positive staining in tumour tissues that presented high USP7 expression (Fig. 5C), and the correlation analysis revealed that their expression levels were markedly positively correlated (Fig. 5D).