This study was approved by the Ethics Committee of The Second Affiliated Hospital of Mudanjiang University (approval no. 200934; Mudanjiang, China). A total of 104 patients diagnosed with OS at The Second Affiliated Hospital of Mudanjiang University were recruited between October 2010 and November 2014. The inclusion criteria for patients with OS were as follows: i) Patients were diagnosed with OS as a primary malignancy; ii) none of the patients had received any anti-tumor therapy, radiotherapy or chemotherapy; iii) all patients signed written informed consent and participated in a 5-year follow-up survey; and iv) the clinicopathological characteristics of patients with OS was provided. Patients with a history of other cancers were excluded from this study. Paired OS tissues and adjacent normal tissues were collected during resection surgery and confirmed by at least two pathologists. Tissues were immediately frozen in liquid nitrogen and stored at -80˚C prior to following experiments.

The OS cell lines MG63, U2OS, KHOS-240S and SAOS-2, and the human osteoblast cell line hFOB 1.19 were purchased from the American Type Culture Collection. All cells were cultured in DMEM medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and placed at 37˚C in a humidified incubator containing 5% CO₂.

In order to regulate the expression of miR-588 in OS cells, miR-588 mimic (50 nM; 5'-UUG GCCACAAUGGGUUAGAAC-3'), miR-588 inhibitor (50 nM; 5'-GUUCUAACCCAUUGUGGCCAA-3'), mimic negative control (mimic NC; 50 nM; 5'-UUCUCCGAACGUGUCACG UU-3') and inhibitor negative control (inhibitor NC; 50 nM, 5'-UUGCUAGUGCGACACUACUUCTT-3') (all from Shanghai GenePharma Co., Ltd.) were transfected into OS cells using Lipofectamine 2000™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection mix was prepared at room temperature and cells were incubated with the transfection complexes at 37˚C for 4 h. Transfected cells were subjected to subsequent experiments after 48 h of transfection. Non-transfected cells were used as the mock group.

Total RNA from tissues and cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following reverse transcription to cDNA using the TaqMan MicroRNA Reverse Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.), RT-qPCR was used to determine the expression of miR-588 with the SYBR Green I Master Mix kit (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences of the primers were as follows: miR-588, forward 5'-GGGUUG GCCACAAUGGGU-3', reverse 5'-GTCGTATCCAGTGCG TGT-3'; and U6, forward 5'-CTCGCTTCGGCAGCACA-3' and reverse 5'-AACGCTTCACGAATTTGCGT-3'. The thermocycling conditions were as follows: 95˚C for 30 sec, followed by 40 cycles of annealing at 95˚C for 10 sec and extension at 60˚C for 30 sec. The relative expression levels of miR-588 were normalized to endogenous control U6 and were expressed as 2^-ΔΔCq (23).

OS cell proliferation was evaluated with the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay. Briefly, 5x10³ cells were seeded per well into a 96-well plate for 0, 24, 48 and 72 h. A total of 10 µl CCK-8 was added to each well and cells were incubated for 4 h at 37˚C with 5% CO₂. The absorbance was read at 450 nm using a microplate reader (Beckman Coulter, Inc.). Experiments were performed in triplicate.

Transwell assays were used to detect the migratory and invasive abilities of OS cells. Briefly, 1x10⁴ cells were seeded into the upper chamber of a 24-well Transwell chamber (8-µm pore size; Corning, Inc.) and cultured with serum-free medium. The bottom chamber was filled with medium supplemented with 10% FBS used as a chemoattractant. For the invasion assay, the upper chamber was pre-coated with Matrigel solution (BD Biosciences) at 37˚C for 1 h prior to cell seeding. Following 48 h incubation at 37˚C, the number of cells that have migrated to the bottom chamber were stained with 1% crystal violet at room temperature for 30 min and counted using a light microscope (magnification, x200). Experiments were performed in triplicate.

Data were expressed as the means ± standard deviation obtained from at least three independent experiments and were analyzed by SPSS 20.0 software (IBM Corp.) and GraphPad Prism 7.0 software (GraphPad Software, Inc.). The difference between groups was evaluated by paired Student's t-test or one-way ANOVA followed by Tukey's post hoc test. The association between miR-588 expression and the clinicopathological characteristics of patients with OS was analyzed using χ² test. The overall survival of patients with OS according to their miR-588 expression level was estimated by Kaplan-Meier analysis and log-rank test. The prognostic value of miR-588 in OS was further evaluated by Cox regression analysis. P<0.05 was considered to indicate a statistically significant difference.

The results from RT-qPCR demonstrated that miR-588 expression was significantly decreased in OS tumor tissues compared with adjacent normal tissues (P<0.001; Fig. 1A). Similarly, miR-588 expression was significantly downregulated in OS cells (MG63, KHOS-240S, SAOS-2, and U2OS) compared with hFOB1.19 cells (P<0.001; Fig. 1B). miR-588 downregulation in OS tissues and cells suggested that it may have a role in the development of OS.

According to the mean expression level of miR-588 in OS tumor tissues (0.384), 104 patients with OS were divided into a low miR-588 expression group (n=63) and a high miR-588 expression group (n=41). The results demonstrated that miR-588 expression was associated with MSTS staging (P=0.033) of patients with OS (Table I). No significant association was found between miR-588 expression and the other clinicopathological characteristics of patients (age, sex, tumor size and location; P<0.05; Table I).

The Kaplan-Meier curve of patients with OS demonstrated that patients with high miR-588 expression levels had a higher 5-year survival rate than patients with low miR-588 expression levels (log-rank P=0.027; Fig. 2). The prognostic value of miR-588 was further assessed by Cox regression analysis. The results showed that miR-588 expression [hazard ratio (HR) =2.533; 95% confidence interval (CI) =1.187-5.407; P=0.016] and MSTS staging (HR=2.517; 95% CI=1.088-5.824; P=0.031) were independent predictors for the prognosis of patients with OS (Table II).

Cell transfections with miR-588 mimic or miR-588 inhibitor were performed to regulate the expression of miR-588 in MG63 and U2OS cells, which presented the lowest miR-588 expression (Fig. 1B). Compared with the Mock and NC groups, miR-588 expression in MG63 and U2OS cells transfected with miR-588 mimic was significantly increased, whereas its expression was significantly decreased in miR-588 inhibitor transfected cells (P<0.001; Fig. 3A). Furthermore, the effect of transfections with miR-588 mimic and miR-588 inhibitor on OS cell proliferation was determined with the CCK-8 assay. The results demonstrated that miR-588 overexpression significantly inhibited the proliferation of MG63 and U2OS cells, whereas miR-588 knockdown significantly stimulated the proliferation of OS cells (P<0.01 and P<0.05; Fig. 3B).

The migratory and invasive abilities of OS transfected cells were evaluated by Transwell assays. The results demonstrated that the number of migrated and invasive cells in the miR-588 mimic group was significantly decreased compared with that in the Mock and NC groups. Conversely, miR-588 knockdown significantly increased the number of migrated and invasive MG63 and U2OS cells (P<0.001; Fig. 4A and B).