A total of 24 paired samples of OS and paraneoplastic tissue were obtained from patients (10 males and 14 females; age range, 9–35 years; mean age, 17±5 years) with OS who underwent surgery at The First Hospital Affiliated with Nanchang University (Nanchang, China) between January 2010 and December 2017. None of the patients received chemotherapy or radiotherapy prior to surgical resection. Written informed consent was obtained from all patients, and the study was approved by the Ethics Committee of The First Hospital Affiliated with Nanchang University.

The human osteosarcoma cell line 143B was purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences. 143B cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml of penicillin and 100 U/ml of streptomycin. Cells were cultured at 37°C with 5% CO₂.

To construct siRNA vectors for the downregulation of VCP, reverse complement sequences as well as nonfunctional negative sequences (Invitrogen; Thermo Fisher Scientific, Inc.; Table I) were cloned into the lentivirus vector GV159 (Invitrogen; Thermo Fisher Scientific, Inc.). Fresh medium was administered to 143B cells, and they were cultured to ~80% confluence. Virus particles incorporating lentivirus vector for the downregulation of VCP (LV-down-VCP; MOI=20) and the cell transfection enhancer polybrene (6 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) were added to the appropriate cultures [LV-down-VCP and negative lentivirus vector (Neg-LV)], and incubated at 37°C, 5% CO₂ for 6–8 h. The medium was collected and replaced with fresh medium at 6 h after transfection. Transfection efficiency was evaluated using a fluorescence microscope 24 h post-transfection. 143B cells transfected with Neg-LV served as the control. A total of 6 independent experiments were performed, and subsequent experiments were started ³24 h after transfection.

Autophinib and spermidine trihydrochloride were purchased form Sigma-Aldrich (Merck KGaA). Concentrations were adjusted to 10 mM for storage according to the manufacturer's instructions. For cell treatment, autophinib and spermidine trihydrochloride were added to the cell culture medium at final concentrations of 20 and 30 nM, respectively, and incubated at 37°C with 5% CO₂ for 6 h. For cells treated with RNA interference and autophagy agonists, the drug treatment was performed 24 h after transfection.

Total protein was extracted from the OS tissues and cell lines using radioimmunoprecipitation assay lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 60 µg/ml phenylmethylsulfonyl fluoride. Protein concentrations were determined using a Bradford assay. Proteins (15 µg/lane) were separated by SDS-PAGE using a 5% concentrated gel and 15% separating gel. Following electrophoresis, the protein was transferred to a nitrocellulose membrane and the membrane was blocked with 5% skimmed milk for 30 min at room temperature. Western blot analysis was conducted using primary antibodies against VCP (cat. no. ab36047), ERK (cat. no. ab79853), NF-κβ (cat. no. ab16502), beclin-1 (cat. no. ab62557) and LC3 (cat. no. ab62721; all Abcam; dilution, 1:2,000) and GAPDH (cat. no. sc-48166, Santa Cruz Biotechnology, Inc.; dilution, 1:5,000) and horseradish peroxidase-conjugated secondary antibodies (cat. nos. sc-2004 and sc-2020, Santa Cruz Biotechnology, Inc.; dilution, 1:5,000). Membranes were incubated with primary antibodies at 4°C for ~12 h (overnight), and subsequently with secondary antibodies at room temperature for 2–3 h. Immune complexes were detected using a pro-light HRP kit (Pierce; Thermo Fisher Scientific, Inc.). The strip gray value was determined using ImageJ software (version 1.46; National Institutes of Health). A total of 6 independent experiments were performed.

Cell migration was assessed using a wound healing assay to determine the ability of cells to move into a cellular space in two-dimensions, in vitro. In brief, cells were cultured to confluence in six-well tissue culture dishes, to a density of ~5×10⁶ cells/well. The wound was created by dragging a rubber policeman (Thermo Fisher Scientific, Inc.) across the center of the plate. Cultures were rinsed with PBS and replaced with fresh medium alone or containing 10 g/l BSA (Gibco; Thermo Fisher Scientific, Inc.), and incubated at 37°C for 24 h. BSA was only used in place of FBS in the wound healing experiments. Images were captured using a light microscope at 0 and 24 h, and the migration distance was determined using ImageJ Software (National Institutes of Health).

The invasiveness of OS cells was assessed using the BD BioCoat™ BD Matrigel™ Invasion Chamber (BD Bioscience) according to the manufacturer's protocol. Cells (2×10⁵) were resuspended in serum-free DMEM and added to the upper chambers; the medium in the lower chambers contained 5% FBS as a chemo-attractant. At 24 h, cells that had migrated through the Matrigel-coated membrane were stained with Diff-Quik (Sysmex Corporation, Kobe) at room temperature in Diff-Quik A for 10–20 sec, Diff-Quik B for 5–10 sec and Diff-Quik C for 5–10 sec, and images were captured under a light microscope (magnification, ×200).

Osteosarcoma 143B cells (2×10⁶ cells) were cultured in suspension for 7 days and subsequently cultured for 6 h. MTT solution (5 mg/ml; 500 µl/well) was added to a 24-well plate, incubated for 4 h at 37°C, 1 ml DMSO was added to each well, and the plate was agitated for 10 min to completely dissolve the crystals. The absorbance at 490 nm was measured using a microplate spectrophotometer.

All statistical analyses were conducted using SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Bivariate correlation analysis (Spearman's Rho) was performed to evaluate the association between VCP expression and autophagy in OS tissues. One-way analysis of variance followed by a Fisher's Least Significant Difference test was used to analyze multiple samples. P<0.05 was considered to indicate a statistically significant difference.

To investigate the association between VCP expression and autophagy in OS, VCP and autophagy-associated proteins beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3)-I/II were analyzed in 24 tissue samples from patients with OS, using western blot analysis. The association between VCP and autophagy-associated proteins was evaluated by bivariate correlation (Spearman's Rho). The results revealed that VCP, beclin-1 and LC3-I/II expression was greater in OS tissues compared with paracancerous tissues (Fig. 1). There was a positive correlation between increased expression levels of VCP and autophagy (Spearman's Rho=0.658; data not shown). Therefore, increased expression levels of VCP may contribute to cell autophagy in OS.

To determine if VCP was able to induce autophagy and anoikis resistance, VCP expression was inhibited in 143B cells by RNA interference. After 7 days in suspension culture, LC3-II/I expression levels and cell survival were analyzed (Fig. 2). The results illustrated that VCP inhibition lead to a significant decrease in LC3-II/I expression (Fig. 2A) and cell survival (Fig. 2B). Further studies performed with an autophagy inhibitor autophinib and autophagy stimulator spermidine trihydrochloride revealed that autophagy inhibitors and stimulator could cause relative changes in the levels of autophagy-related proteins in cells (Fig. 2A), and inhibiting autophagy decreased cell survival; conversely, autophagy stimulation promoted cell survival and counteracted the changes caused by VCP inhibition (Fig. 2B). Collectively, these results indicate that VCP induced autophagy to enhance cell survival and possibly anoikis resistance.

To identify whether VCP expression effects autophagy, and may therefore alter the malignant phenotype of OS cells, lentivirus vectors and autophagy-inhibitory and stimulating agents were used to treat OS cells. Wound-healing and Matrigel-invasion assays were used to evaluate the malignant phenotype of OS cells. The results demonstrated that after 24 h, the migration rate and number of invaded cells were reduced in cells where VCP was downregulated, compared with those transfected with Neg-LV. By contrast, there was no marked difference in the migration rate and invasive ability of cells treated with autophagy inhibitor or stimulator, compared with that of Neg-LV cells (Fig. 3). Collectively, the results revealed that VCP can induce cell autophagy and promote OS cell invasiveness or migration, but altering autophagy levels in vitro did not affect cell invasiveness or migration.

To investigate the mechanism by which VCP induced autophagy, 143B cells were treated with LV-down-VCP vector, and the expression levels of VCP, ERK, NF-κβ and autophagy-associated protein beclin-1 were determined using western blotting. The results illustrated that VCP inhibition led to a marked decrease in the expression levels of ERK, NF-κβ and beclin-1 (Fig. 4).