The present study was approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University (Nanchang, China) and written informed consent was obtained from all patients. OS tissue samples and matched adjacent non-tumor tissue samples were collected from 54 patients with OS at the Second Affiliated Hospital of Nanchang University between May 2010 and May 2013. The patients included 33 males and 21 females, with a mean age of 18.8 years (range, 10–26 years). The clinical characteristics of the patients are summarized in Table I. No patients received radiotherapy or chemotherapy prior to surgery. All tissues samples were stored at −80°C prior to use.

The normal human osteoblastic cell line hFOB 1.19 and the human OS cell lines SAOS2, U2OS, MG63 and HOS were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). hFOB 1.19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. OS cells were maintained in DMEM with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C with 5% CO₂.

Cells were cultured at 37°C with 5% CO₂ in a 6-well plate at a density of ~1×10⁶ cells/well. When a confluency of 70–80% was reached, the cells were transfected with 100 nM negative control (NC) small interfering (si)RNA (cat. no. AM4611; Thermo Fisher Scientific, Inc.), RAB31 siRNA (cat. no. AM16708; Thermo Fisher Scientific, Inc.), miR-NC (cat. no. 4464058; Thermo Fisher Scientific, Inc.), miR-26b mimic (cat. no. 4464066; Thermo Fisher Scientific, Inc.), NC inhibitor (cat. no. 4464077; Thermo Fisher Scientific, Inc.) or miR-26b inhibitor (cat. no. 4464084; Thermo Fisher Scientific, Inc.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following incubation for 48 h at 37°C, subsequent experiments were performed.

Total RNA was extracted from tissue samples and cells (1×10⁶) using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed using miScript Reverse Transcription kit (Qiagen GmbH), according to the manufacturer's protocol. qPCR was conducted using QuantiTect SYBR-Green RT-PCR kit (Qiagen GmbH) with an ABI 7500 fast real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions for PCR were as follows: 95°C for 1 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. GAPDH or U6 was used as the internal reference for mRNA and miRNA, respectively. Data were quantified using the 2^−ΔΔCq method (25). The primer sequences were as follows: RAB31 forward, 5′-GGGGTTGGGAAATCAAGCATC-3′ and reverse, 5′-GCCAATGAATGAAACCGTTCCT-3′; GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. Primers for U6 (cat. no. HmiRQP9001) and miR-16 (cat. no. HmiRQP0356) were obtained from Guangzhou FulenGen, Co., Ltd.

Tissue samples and cells (1×10⁷) were solubilized with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Total protein (50 µg) was separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk in 0.1% TBS/Tween-20 at room temperature for 1 h, followed by incubation with rabbit anti-human RAB31 (dilution, 1:100; cat. no. ab230881; Abcam), or rabbit anti-human GAPDH (dilution, 1:200; cat. no. ab181602; Abcam) primary antibodies at room temperature for 3 h. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution, 1:5,000; cat. no. ab6721; Abcam) at room temperature for 1 h. Enhanced chemiluminescent reagent (Pierce; Thermo Fisher Scientific, Inc.) was used to detect the signals on the membranes. ImageJ software (v1.46; National Institutes of Health) was used for densitometry analysis.

Cell proliferation was investigated using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. A total of 48 h after transfection, cells (3,000 cells/well) were seeded into 96-well plates and cultured for 0, 24, 48 or 72 h. The optical density value at 450 nM was measured using a microplate reader.

The transfected cells were harvested and washed twice with ice-cold PBS. Subsequently, 1×10⁶ cells were resuspended with 300 µl binding buffer. Following staining with Annexin V-fluorescein isothiocyanate and propidium iodide (BD Biosciences), according to the manufacturer's protocol, apoptotic cells were analyzed using a flow cytometer and Accuri C6 Software (version 1.0; BD Biosciences).

The transfected cells (1×10⁶ cells per well) were cultured in 6-well plates for 24 h. Subsequently, an artificial wound was created using a sterile 10 µl pipette tip in the confluent cell monolayer. Photographs were captured at 0 and 24 h using an inverted light microscope (magnification, ×40).

Matrigel (BD Biosciences) was diluted with serum-free DMEM and added to the upper chamber of a 24-well Transwell™ chamber (Corning Inc.), which was then incubated at 37°C for 1 h. Cells were diluted to a density of 2×10⁵ cells/ml with serum-free DMEM. Subsequently, 100 µl cell suspension was added to the upper chamber and 700 µl DMEM containing 10% FBS was added to the bottom chamber. Following incubation at 37°C for 24 h, the chamber was washed with PBS. The cells were then fixed with 4% paraformaldehyde at room temperature for 30 min, stained with crystal violet at room temperature for 30 min and photographs were obtained using an inverted light microscope (magnification, ×400).

TargetScan software version 7.1 was used for bioinformatics analysis (26). The wild-type (WT) or mutant type (MUT) 3′-UTR of RAB31 was inserted downstream of the luciferase reporter gene in the pMIR-REPORT vector (Thermo Fisher Scientific, Inc.), generating WT-RAB31 and MUT-RAB31. MG63 and U2OS cells were co-transfected with miR-26b mimics/miR-NC, WT-RAB31/MUT-RAB31 and pRL-SV40 (Promega Corporation) expressing Renilla luciferase. Following 48 h incubation at 37°C with 5% CO₂, the luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Promega Corporation).

All data are presented as the mean ± standard deviation from a minimum of three independent experiments. Statistical analysis was performed using SPSS 20.0 (IBM Corp.). Student's t-test was used for comparisons between two groups. Multiple comparisons were performed using one-way analysis of variance followed by Tukey's test. The patients with OS were divided into a high RAB31 expression group and low RAB31 expression group using the median expression level of RAB31 (1.91) as the cut-off, and χ² test was used to perform statistical analysis in Table I. Correlations between the mRNA expression levels of miR-26b and RAB3 were analyzed using Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

First, the mRNA and protein expression levels of RAB31 were examined in tissue samples from OS patients. RT-qPCR and western blot analysis demonstrated that the expression levels of RAB31 were significantly higher in OS tissues compared with adjacent non-tumor tissues (Fig. 1A and B). The patients with OS were then divided into a high RAB31 expression group and low RAB31 expression group using the median expression level of RAB31 (1.91) as the cut-off. As presented in Table I, high expression of RAB31 was identified to be significantly associated with lung metastasis and advanced clinical stage. Furthermore, the patients with high RAB31 expression exhibited a significantly shorter survival time compared with those with low RAB31 expression (Fig. 1C), which suggested that RAB31 upregulation was associated with a poor prognosis for patients with OS. In addition, the protein expression levels of RAB31 were markedly higher in the OS cell lines SAOS2, U2OS, MG63 and HOS compared with the normal human osteoblastic cell line hFOB 1.19 (Fig. 1D). MG63 and U2OS cells exhibited the highest expression levels of RAB31 protein (Fig. 1D); these cell lines were therefore selected for subsequent experiments.

To inhibit the expression of RAB31, MG63 and U2OS cells were transfected with RAB31 siRNA. As presented in Fig. 2A and B, the mRNA and protein expression levels of RAB31 were significantly lower in MG63 and U2OS cells transfected with RAB31 siRNA compared with the NC siRNA group. A CCK-8 assay was then performed to examine cell proliferation. As presented in Fig. 2C and D, knockdown of RAB31 significantly inhibited OS cell proliferation. The cell cycle distribution and cell apoptosis were then examined by flow cytometry. The data from this experiment indicated that silencing of RAB31 was significantly associated with cell cycle arrest at the G1 stage and significantly promoted OS cell apoptosis (Fig. 2E-H). The aforementioned results suggested that RAB31 may serve a promoting role in OS growth.

To study the potential role of RAB31 in OS metastasis, a wound healing assay and Transwell assay were performed to examine cell migration and invasion, respectively. As presented in Fig. 3, silencing of RAB31 expression significantly inhibited the migration and invasion of MG63 and U2OS cells compared with the NC siRNA group. In summary, RAB31 silencing inhibited OS cell proliferation, cell cycle progression, migration and invasion, and induced cell apoptosis.

TargetScan software data predicted that RAB31 is a putative target gene of miR-26b (Fig. 4A). To confirm the association between RAB31 and miR-26b, wild-type (WT) and mutant (MT) RAB31 luciferase reporter plasmids were generated (Fig. 3B) and a luciferase reporter gene assay was performed. The data demonstrated that transfection with miR-26b mimic significantly inhibited the luciferase activity in MG63 and U2OS cells transfected with WT RAB31 3′UTR reporter plasmid; however, no effect was observed on the luciferase activity in cells transfected with the MT RAB31 3′UTR reporter plasmid (Fig. 4C and D). This indicates that RAB31 is a target gene of miR-26b in OS cells.

The effects of miR-26b on RAB31 expression were then investigated in OS cells. MG63 and U2OS cells were transfected with miR-26b mimic or miR-NC. Following transfection, miR-26b expression was significantly upregulated in the miR-26b mimic group compared with the miR-NC group (Fig. 5A). Further experiments revealed that the mRNA and protein expression levels of RAB31 were significantly decreased following miR-26b overexpression (Fig. 5B and C). To further confirm these findings, MG63 and U2OS cells were transfected with miR-26b inhibitor or NC inhibitor. RT-qPCR analysis revealed that miR-26b was significantly downregulated in the miR-26b inhibitor-transfected group compared with the NC inhibitor-transfected group (Fig. 5D). As presented in Fig. 5E and F, the mRNA and protein expression levels of RAB31 were significantly increased in the miR-26b inhibitor-transfected group compared with the NC inhibitor-transfected group. These data indicated that RAB31 was negatively regulated by miR-26b in OS cells.

RT-qPCR analysis identified that miR-26b levels were significantly lower in OS tissues compared with matched adjacent non-tumor tissues (Fig. 6A). Additionally, an inverse correlation was identified between the miR-26b and RAB31 expression levels in OS tissues (Fig. 6B), which suggested that the increased expression levels of RAB31 in OS tissues may be due to downregulation of miR-26b. Furthermore, miR-26b levels were demonstrated to be decreased in OS cell lines compared with hFOB 1.19 cells (Fig. 6C).