The use of patient tissues was approved by the ethics committee of Qingdao West Coast Hospital, Affiliated Hospital of Qingdao University (approval no. QYFYWZLL25779A) and written informed consent was obtained from all patients. In total, 60 patients (27 males and 33 females; age range, 35–63 years old) histologically diagnosed with OS were recruited in our hospital between February 2017 and April 2019. The inclusion criteria were: i) First-time diagnosis; and ii) no prior history of radiotherapy, chemotherapy and other adjuvant therapy. The exclusion criteria were: i) Presence of other malignant tumors; and ii) treatment for OS before admission. OS tissues and adjacent normal tissues (distance from tumor margin, 2 cm) were collected from these patients prior to the administration of treatment.

The human OS cell lines (MG-63, Saos-2, U2OS and HOS) and the human osteoblast cell line hFOB1.19 were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (HyClone; Cytiva) and 1% penicillin-streptomycin, and maintained at 37°C with 5% CO₂.

U2OS and HOS cells (6×10⁵ cells/well) in the logarithmic growth phase were transfected with 20 nM pcDNA3.1 (pcDNA)-NGX6 or pcDNA-negative control (NC; both Sangon Biotech Co., Ltd.) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were randomly divided into the BLANK (untransfected cells), pcDNA-NC and pcDNA3.1-NGX6 groups. Following transfection for 48 h at 37°C, the cells were used for follow-up experiments.

To determine whether the regulatory effects of NGX6 on OS cells were associated with the Wnt/β-catenin signaling pathway, U2OS and HOS cells transfected with pcDNA-NGX6 were treated with BML284 (20 µM; Abcam), an activator of the Wnt/β-catenin signaling pathway, for 0, 24, 48, 72 or 96 h at room temperature. Cells were divided into the following groups: i) pcDNA-NC; ii) pcDNA3.1-NGX6; and iii) pcDNA3.1-NGX6 + BML284.

Total RNA was extracted from tissues or cells using TRIzol^® Plus RNA Isolation reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of total RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Total RNA (500 ng) was reverse transcribed into cDNA at 42°C for 45 min using Moloney Murine Leukaemia Virus Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was subsequently performed using SYBR^® Premix Ex Taq™ (Takara Biotechnology Co., Ltd.) on a StepOnePlus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 3 min; followed by 40 cycles at 95°C for 15 sec, annellation at 60°C for 30 sec, elongation at 72°C for 1 min, and a final extension at 72°C for 5 min. The primer sequences used for the qPCR are listed in Table I. The mRNA expression levels of target genes were quantified using the 2^−ΔΔCq method (21). GAPDH or U6 was used as the internal loading control.

Total protein was extracted from tissues or OS cell lines (U2OS and HOS) using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA Protein assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) and 50 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto a polyvinylidene fluoride membrane and blocked with 5% skimmed milk for 2 h at 25°C. The membranes were then incubated with the following primary antibodies (Abcam) overnight at 4°C: Anti-GAPDH (1:1,000; cat. no. ab9485), anti-β-catenin (1:1,000; cat. no. ab32572), anti-c-Jun (1:1,000; cat. no. ab40766) and anti-c-Myc (1:1,000; cat. no. ab32072). After washing three times with TBST (Tween-20; 0.05%) for 5 min, the membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 2 h at 25°C. Protein bands were visualized using an chemiluminescent substrate kit (Invitrogen; Thermo Fisher Scientific, Inc.) and analyzed using Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Inc.). GAPDH was used as the internal loading control.

The transfected U2OS and HOS cells (2×10⁴ cells/well) were seeded into 96-well plates. Following incubation for 24, 48, 72 or 96 h at 37°C, 20 µl MTT reagent (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added into each well. After 4 h of incubation at 37°C, 150 µl DMSO was added into each well to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Early apoptosis in transfected U2OS and HOS cells were analyzed using an Annexin V-FITC apoptosis detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, cells were centrifuged at 200 × g for 5 min at 4°C and resuspended in binding buffer. Cells were subsequently stained with 5 µl Annexin V-FITC and 5 ml propidium iodide for 15 min at 4°C in a dark room. Apoptosis was detected using a FACScan flow cytometer (version 2.0; BD Biosciences) and the data were analyzed using CellQuest software (version 5.1; BD Biosciences).

U2OS and HOS cells were plated into six-well plates containing DMEM at a density of 5×10⁵ cells/well. Upon reaching 90% confluence, the cell monolayer was scratched using a sterile 100-µl pipette tip to form wounds. The cells were then incubated in serum-free DMEM for 24 h at 37°C. Cells were observed and photographed at 0 and 24 h using an inverted light microscope (Olympus Corporation; magnification, ×200) and measured using ImageJ software (version 1.46; National Institutes of Health). The relative migration rate was calculated as follows: (original gap distance-gap distance at 24 h)/original gap distance ×100. Relative migration was normalized to the BLANK or pcDNA-NC group.

Cell invasion was assessed using Transwell chambers (Corning, Inc.) pre-coated (at 37°C for 30 min) with Matrigel^® (BD Biosciences). Briefly, transfected U2OS and HOS cells were resuspended in serum-free medium and 200 µl cell suspension (1×10⁵ cells) was placed in the upper chamber. Subsequently, 600 µl medium containing 10% FBS was plated into the lower chambers. Following 24 h of incubation at 37°C with 5% CO₂, the invasive cells in the lower chamber were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 0.5% crystal violet at 37°C for 30 min. Cells in six independent fields per well were imaged using a light microscope (Olympus Corporation; magnification, ×200), and the number of invading cells were counted.

Animal experiments were approved by the ethics committee of Qingdao West Coast Hospital (approval no. QYFYWZLL25779) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (version 1996) (20). Male specific pathogen free mice (BALB/c; age, 4 weeks; weight, 20–25 g; n=30) were purchased from the Medical College of Shanghai Jiaotong University (Shanghai, China). The animals were housed in a sterile environment at a controlled temperature of 20°C with 40% relative humidity, 12-h light/dark cycles, and free access to food and water. A volume of 100 µl U2OS cells (0.1×10⁸ cells/ml) at the logarithmic growth phase from the different groups (pcDNA-NGX6, pcDNA-NC, pcDNA-NGX6 + BML284) were resuspended in PBS and subcutaneously injected into the posterior limb of mice (n=10/group) to create a subcutaneous tumor-bearing model. The tumor volume was measured with a Vernier caliper every week following injection. At the end of the 4th week, mice were anesthetized with 50 mg/kg pentobarbital sodium and sacrificed by cervical dislocation. The tumors were removed and weighed. The experiment lasted for 4 weeks and no mice died during this period.

Statistical analysis was performed using SPSS 22.0 software (IBM Corp.). All experiments were performed in triplicate and data are presented as the mean ± SD. A paired Student's t-test was used to determine the significant differences between two groups (Fig. 1A), while a one-way ANOVA test followed by Tukey's post hoc test was used to determine the statistical differences between >2 groups. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis revealed that NGX6 expression levels were significantly downregulated in the OS tissues compared with the normal tissues from patients (P<0.001; Fig. 1A). Consistently, NGX6 expression levels were also significantly downregulated in OS cell lines (MG-63, Saos-2, U2OS and HOS) compared with the human osteoblastic cell line hFOB1.19, but especially in U2OS and HOS cells (P<0.01; Fig. 1B). Therefore, U2OS and HOS cells were selected for subsequent experiments. These findings suggested that NGX6 expression levels may be downregulated in OS tissues and cell lines.

To determine the effect of NGX6 on OS progression, NGX6 was overexpressed by the transfection of pcDNA-NGX6 into U2OS and HOS cells. RT-qPCR illustrated that the NGX6 expression levels were significantly upregulated in the pcDNA3-NGX6 groups compared with the BLANK groups in both cell lines (P<0.01; Fig. 2A). The results of the MTT assay revealed that the optical density (OD)450 values of the pcDNA-NGX6 groups were significantly decreased at 48 h in both cell lines compared with the BLANK groups (P<0.05; Fig. 2B). The levels of apoptosis of U2OS and HOS cells in the pcDNA-NGX6 groups were significantly increased compared with the BLANK groups (**P<0.01; Fig. 2C). Conversely, the transfection of cells with pcDNA-NC did not influence the viability or apoptotic rate of U2OS and HOS cells (Fig. 2B and C). These results indicated that the overexpression of NGX6 may impede the viability and promote the apoptosis of U2OS and HOS cells.

The relative migration and invasion rates were significantly decreased in the pcDNA-NGX6 groups compared with the BLANK groups in both cell lines (P<0.01; Fig. 3A and B). The transfection of cells with pcDNA-NC did not influence the migration and invasion rates of U2OS and HOS cells. Taken together, these data indicated that the overexpression of NGX6 may repress the migration and invasion of U2OS and HOS cells.

To determine the potential mechanism of NGX6 in OS, transfected U2OS and HOS cells were treated with BML284 (Wnt/β-catenin signaling pathway activator). In both cell lines, the expression levels of β-catenin, c-Jun and c-Myc were all significantly downregulated in the pcDNA-NGX6 groups compared with the pcDNA-NC groups (P<0.01; Fig. 4). Conversely, BML284 treatment significantly reversed the inhibitory effects of pcDNA-NGX6 on the expression levels of β-catenin, c-Jun and c-Myc in U2OS and HOS cells (^##P<0.01; Fig. 4). These results indicated that the transfection of pcDNA-NGX6 may block the Wnt/β-catenin signaling pathway, while the intervention with BML284 may reverse the inhibitory effect of pcDNA-NGX6 on the Wnt/β-catenin signaling pathway.

To explore the possible effect of the Wnt/β-catenin signaling pathway on the occurrence and development of OS in vitro, U2OS cells were treated with an activator of the Wnt/β-catenin signaling pathway (BML284). The results indicated that treatment with BML284 reversed the inhibitory effects of pcDNA-NGX6 on the viability, migration and invasion, and the promoting effect on the apoptosis of U2OS cells (P<0.05, P<0.01; Fig. 5A-D). Collectively, the results implied that treatment BML284 could reverse the effect of NGX6 overexpression on the progression of OS in vitro.

To verify the inhibitory effect of pcDNA-NGX6 on the tumorigenesis of OS, xenograft tumor model mice were established. A significant decrease was observed in both the tumor volume (P<0.05; Fig. 6A) and tumor weight (P<0.01; Fig. 6B) in the pcDNA-NGX6 group compared with the pcDNA-NC group. However, the addition of BML284 treatment partially rescued the inhibitory effect of pcDNA-NGX6 on the tumor growth and weight of mice (P<0.05, P<0.01). In addition, the expression levels of β-catenin, c-Jun and c-Myc in the tumor tissues were significantly downregulated in the pcDNA-NGX6 group compared with the pcDNA-NC group (P<0.01; Fig. 6C), while the addition of BML284 partially rescued this inhibitory effect of pcDNA-NGX6 on the expression levels of β-catenin, c-Jun and c-Myc in the tumor tissues of mice (P<0.05; Fig. 6C). The results indicated that treatment with BML284 reversed the suppressive effect of NGX6 overexpression on the growth of xenograft tumors in vivo.