Fresh OS tissue specimens were collected from 26 patients who underwent surgery for OS resection at Dongfang Hospital Affiliated to Beijing University of Chinese Medicine (Beijing, China) between October 2012 and May 2014. The group of patients with OS comprised 14 males and 12 females, aged between 13 and 58 years. Without any preoperative treatment, all 26 cases were pathologically diagnosed as OS postoperatively. In addition, 22 normal bone tissue specimens were collected from the long bones of 22 healthy subjects aged 24–61 years (males, 8; females, 14). The specimens were immediately preserved in liquid nitrogen (Leshan Dongya Cryogenic Vessel Co., Ltd, Beijing, China) for subsequent analyses. All subjects provided written informed consent, and the specimen collection procedure of the present study was approved by the Medical Ethics Committee of Dongfang Hospital Affiliated to Beijing University of Chinese Medicine (Beijing, China).

The U2OS and MG63 human OS cell lines and normal osteoblast cell line (hFOB1.19) were purchased from the American Type Culture Collection (Manassas, VA, USA). hFOB1.19 normal osteoblast cell line was used as the control group when evaluating the expression levels of SCARA5 in OS cells. All OS cell lines were cultured in complete Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 1% L-glutamine, and 1% penicillin/streptomycin (both Sigma-Aldrich, St. Louis, MO, USA). The cultures were incubated at 37°C with 5% CO₂ in a humidified incubator.

OS tissues were frozen in liquid nitrogen, washed twice with phosphate-buffered saline and lysed using ice-cold radioimmunoprecipitation assay buffer and 0.1 PMSF supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Following centrifugation at 12,000 × g for 5 min, the supernatant was collected. When the cells reached 90% confluency, total RNA was extracted from the OS tissues and cells using TRIzol reagent, according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 2 µg of total RNA was reverse transcribed to first-strand cDNA using TaqMan reverse transcription reagents (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primers were used: SCARA5, sense 5'-CAGCTGGTTTCTTACCACGTAT-3' and antisense 5'-GCACAAGTTCTCCCACACTTAG-3'; and β-actin, sense 5'-CCGTGAAAAGATGACCCAGATC-3' and antisense 5'-CACAGCCTGGATGGCTACGT-3'. RT-qPCR was performed using 1 µl cDNA templates, 2 µl forward and reverse primers and 5 µl SYBR Green qPCR Master Mix. Thermal cycling was performed as follows: 95°C for 10 min, then 40 cycles of 95°C for 15 sec, 59°C for 30 sec, 72°C for 30 sec, followed by an extra extension at 72°C for 5 min. Reactions were performed using a Step One Plus Real-Time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.). For analysis, the expression levels of the target gene were normalized by the gene expression of β-actin. Based on the ΔΔCq method (13), the relative quantities of mRNA were expressed as 2^−ΔΔCq.

Proteins were extracted from the OS tissues and cells, and protein concentrations were measured using the Bradford method (14). Subsequently, 30 µg of protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (both Sigma-Aldrich). The membrane was incubated with 2% nonfat dry milk in Tris-buffered saline (TBS; Sigma-Aldrich), to block non-specific binding, at room temperature for 1 h. Subsequently, the membrane was immunoblotted with the following mouse monoclonal primary antibodies: Anti-SCARA5 (1:1,500; sc-98123), anti-phosphorylated (p)-FAK (Y397; sc-11765), anti-FAK (both 1:1,000; sc-271195), anti-p-Src (Y416; sc-101802), anti-Src (both 1:2,000; sc-24621), anti-matrix metalloproteinase 2 (MMP2; sc-13594), anti-MMP9 (sc-21733) and anti-β-actin (all 1:1,000; sc-8432; all Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C, followed by three washes in TBS with 0.05% Tween-20 (Sigma-Aldrich) for 10 min. Subsequently, the membranes were incubated with rat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:3,000; sc-2370; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The expression of the target protein was visualized using enhanced chemiluminescence (Pierce Biotechnology; Thermo Fisher Scientific, Inc.). Absorbance values of the target proteins were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

To construct the SCARA5 recombinant adenovirus vector, the cDNA encoding SCARA5 was amplified and subcloned into the adenoviral shuttle vector, pAd-CMV, and green fluorescent protein (GFP; both Invitrogen; Thermo Fisher Scientific, Inc.) was used as a non-specific control. The pAd-CMV adenoviral shuttle vector and pAdEasy-1 (Invitrogen; Thermo Fisher Scientific, Inc.) adenoviral gene expression vector were homologously recombined in the Escherichia coli strain, BJ5183 (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) at 4°C for 16 h. The newly recombined plasmid, Ad-SCARA5, was then propagated in 293 cells (Type Culture Collection of the Chinese Academy of Sciences) at 37°C for 16 h. The recombinant adenoviruses were harvested, and the titers were determined using a p24 ELISA kit (Cell Biolabs, Inc., San Diego, CA, USA) prior to use.

For in vitro transfection, the OS cells (3×10³) were seeded in 96-well plates. The cells grown to 30–50% confluence and were transfected with Ad-SCARA5 at final concentrations of 50 nM, using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

To analyze cell proliferation, an MTT assay was performed. The transiently transfected cells were seeded in a 96-well plate at a cell density of 2×10³ and were cultured at 24 h intervals for 4 days at room temperature. Subsequently, the initial culture medium was replaced with fresh medium containing MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and incubated for an additional 4 h at room temperature. The formazan was dissolved in dimethylsulfoxide (150 µl/well; Sigma-Aldrich) for 10 min. The absorbance was measured at 570 nm using a Spectra Max 190 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). All experiments were independently repeated at least three times.

For the soft agar colony formation assay, 2×10⁴ cells were plated in 24-well plates and grown on a plate containing 1% base agar (Sigma-Aldrich) and 0.5% top agar for 21 days at room temperature, until colonies formed. The colonies were stained with 1% crystal violet for 30 sec following fixation with 10% formaldehyde (both Sigma-Aldrich) for 5 min. The numbers of colonies were counted under a dissecting microscope (BIX-103G, Luxi Chemical Group Co., Ltd., Beijing, China). All experiments were independently repeated at least three times.

OS cells (1×10⁴ cells/well) in 200 µl serum-free DMEM were added to the upper chamber of a Transwell (Invitrogen; Thermo Fisher Scientific, Inc.) with an 8 µm microporous filter (Sigma-Aldrich), following which 500 µl DMEM containing 10% FBS was added to the lower chamber. Following 24 h incubation at 37°C, the cells on the lower surface of the filter were fixed in methanol, stained with Giemsa (Sigma-Aldrich) and examined under a Leica DM5000B microscope (Leica Microsystems GmbH, Wetzlar, Germany). The average numbers of migrated cells from five randomly-selected optical fields and triplicate filters were determined.

For in vitro invasion assays, the cells were suspended in a volume of 50 µl serum-free medium, which was then added to the upper chamber of a chemotaxis chamber (Neuro Probe, Inc., Gaithersburg, MD, USA). Complete medium was added to the lower chamber. A polycarbonate membrane (Sigma-Aldrich) was placed between the two chambers, and culture medium supplemented with 20 µl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was applied. After 24 h at room temperature, the non-invading cell remaining on the upper surface were removed using cotton-tipped swabs, following which the filters were fixed in methanol for 3 min and stained with 0.05% crystal violet in phosphate-buffered saline for 15 min. The cells on the underside of the filters were visualized and counted under a Leica DM5000B microscope (Leica Microsystems GmbH). Each sample was assayed in triplicate.

All experiments were performed independently, at least three times. Differences between groups were analyzed via Student's t-test using SPSS 13.0 software (IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation from three independent experiments performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

The present study first determined the mRNA and protein levels of SCARA5 in human OS tissues. As shown in Fig. 1, the mRNA (Fig. 1A) and protein (Fig. 1B) expression levels of SCARA5 in the OS tissues were significantly lower, compared to those in the normal bone tissues (P<0.05). The expression levels of SCARA5 in human OS cells (MG-63 and U2OS) were also analyzed. Consistent with the observation from the tissue samples, the expression levels of SCARA5 were significantly decreased in the two cell lines, compared with those in the normal human primary osteoblasts (Fig. 1C and D). These results indicated that SCARA5 may function as a tumor suppressor in OS.

To examine the biological significance of SCARA5 in OS tumorigenesis, the present study generated SCARA5-overexpressing MG-63 and U2OS OS cell lines. The transfection efficiency was confirmed using RT-qPCR and western blot analyses. Following SCARA5 transfection, the mRNA and protein levels of SCARA5 were significantly increased in the MG-63 (Fig. 2A) and U2OS (Fig. 2B) cells, respectively. The present study also examined the effect of SCARA5 overexpression on cell proliferation and colony formation. As shown in Fig. 3, transfection of the cells with Ad-SCARA5 significantly inhibited the growth of the MG-63 cells (Fig. 3A). It also significantly suppressed colony formation in the MG-63 cells (Fig. 3C). Similarly, the overexpression of SCARA5 suppressed growth (Fig. 3B) and colony formation (Fig. 3C) in the U2OS cells.

The present study used Transwell assays to assess the effects of the expression of SCARA5 on cell migration and invasion. The results showed that cell migration was significantly inhibited by SCARA5 overexpression in the MG-63 (Fig. 4A, left) and U2OS cells (Fig. 4B, left), respectively. In addition, compared with the cells transfected with the empty vector, SCARA5 overexpression significantly inhibited the invasion of the MG-63 (Fig. 4A, right) and U2OS cells (Fig. 4B, right).

The FAK signaling pathway is important in cancer cell growth and invasion (15). To examine the molecular mechanisms by which SCARA5 contributes to these malignant features, the present study examined the effect of SCARA5 on the levels of tyrosine phosphorylation at specific sites of certain molecules involved in the FAK signaling pathway. The results of the Western blot analysis revealed that the overexpression of SCARA5 significantly inhibited the phosphorylation of the FAK residue (Tyr-397; Fig. 5A and B) and Src residue (Tyr-416; Fig. 5A and C) in the MG63 cells.

The present study also assessed the expression levels of MMP-2 and MMP-9, which are crucial downstream molecules in the FAK signaling pathway (16). As shown in Fig. 6, the overexpression of SCARA5 significantly inhibited the expression levels of MMP-2 (Fig. 6A and B) and MMP-9 (Fig. 6A and C) in the MG63 cells, compared with the cells in the Ad-GFP group.