A total of 26 paired OS and adjacent non-tumor tissues were collected from patients (sex, 15 males and 11 females; age, 60±5 years) with OS who underwent curative tumor resection between September 2010 and August 2014 at the Jinling Hospital (Nanjing, China). The tissues samples were obtained once the patients had provided written informed consent, and the process was approved by the Ethics Committee of Jinling Hospital (Nanjing, China). SAOS2, MG-63, HOS and U2OS human OS cell lines and hFOB1.19 normal osteoblast cells were purchased from the American Type Culture Collection.

All cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂. For plasmid transfection, cells were first seeded in six-well plates, and transfection was performed when 80% confluence was achieved. A total of two 8 µl (500 ng/µl) plasmids (pcDNA3.1-RUNX2, RUNX2-shRNA (5′-AAAAGCGCATTCCTCATCCCAGTATTTCGATACTGGGATGAGGAATGCGC-3′, Shanghai GenePharma Co., Ltd.) or miR-150 mimics and NC (hsa-miR-150 mimics, 5′-UCUCCCAACCCUUGUACCAGUG-3′; hsa-miR-150 mimics NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.) and 8 µl Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) were suspended in 100 µl Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and the final concentration of RUNX2 plasmids or mimics used was 1 mg/ml. Following incubation at room temperature for 30 min, the mixture was added into cell culture. After 6 h of culture, the cell medium was replaced by fresh medium. After another 24 h of culture, the cells were used to detect the rates of proliferation. The proliferation rates in the different groups of HOS and U2OS cells (blank, vector and RUNX2 overexpression groups) were detected via MTT assay. Cells were suspended in 200 µl culture medium in a 96-well plate at 1×10⁴ cells/well. At every 24 h after cell seeding for 72 h, the culture medium of the 96-well plate was replaced with DMEM plus 0.5 mg/ml MTT and incubated at 37°C for 4 h. DMEM medium was then replaced with isopycnic DMSO. Following mixing by extensive shaking, absorbance at 490 nm was detected for each well using a SpectraMax 190 (Molecular Devices in Sunnyvale) to measure the optical density value at 0, 24, 48 and 72 h.

DOX (Selleck Chemicals) was used to construct an OS cell chemotherapy resistance model, where cell viability was measured using CCK8 assay. A dose-dependent assay was performed using 0, 25, 50, 75 and 100 µM DOX treatment at 37°C for 4 h. Since cell viability did not reduce further on increasing the concentration of DOX beyond 50 µM, this concentration was chosen for subsequent experiments. OS cell lines HOS and U2OS were seeded in 96-well plates with 5,000 cells/well. Following DOX treatment, cell proliferation was assessed using a CCK-8 assay (Dojindo Molecular Technologies). Every 24 h for 72 h after seeding, 10 µl CCK-8 solution was added to the culture medium, and the cultures were incubated for 30 min at 37°C in 5% CO₂. The absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad Laboratories, Inc.).

Total RNA was extracted from the HOS and U2OS cell lines and tissues using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Reverse transcription was performed using a Takara PrimeScript™ RT reagent kit (Takara Bio Inc.) at 37°C for 1 h and 85°C for 15 sec. qPCR was performed using SYBR Green (Takara Bio Inc.). The thermocycling conditions were as follows: Initial denaturation at 90°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec) with the ABI Prism 7700 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientifc, Inc.). RUNX2 or miR-150 expression was quantified using the 2^−∆∆Cq method (21). GAPDH and U6 were used as internal controls of the mRNA or miRNA, respectively. Sequences of the primers used in the present study were as follows: miR-150 forward, 5′-TGTCGTGGAGTCGGCAATTCAGTTGAGCACTGG-3′ and reverse, 5′-ACACTCCAGCTGGGTCTCCCAACCCTTGTA-3; GAPDH forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

TargetScan bioinformatics algorithm (version 7.2; http://www.targetscan.org/vert_72/) was used to identify the potential targets of miR-150. The 293T cells (Type Culture Collection of the Chinese Academy of Sciences) were seeded in 24-well plates and cultured until they reached 60% confluence. After incubation overnight at 37°C, the cells were co-transfected pmirGLO plasmids encoding RUNX2 wild-type (WT) 3′-UTR or mutant (Mut) 3′-UTR (Shanghai GenePharma Co., Ltd.) and miR-150 mimics (Shanghai GenePharma Co., Ltd.) or the control mimics (Shanghai GenePharma Co., Ltd.) using Lipofectamine 2000. Firefly and Renilla luciferase activities were detected using Dual-Luciferase Reporter assay system (Promega Corporation) 48 h after transfection, according to the manufacturer's protocol. All transfection assays were performed in triplicate.

RIPA lysis buffer (Beyotime Institute of Biotechnology) with protease inhibitor cocktail (Beyotime Institute of Biotechnology) was used for cell lysis. A BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to detect the protein concentration. In total, 4 µg all proteins were resolved by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were subsequently incubated with blocking buffers, consisting of 10% skimmed milk powder (dissolved in TBS supplemented with 0.1% Tween-20) for 2 h at room temperature, prior to incubation with the respective primary antibodies. The primary antibodies used in the present study were as follows: Anti-RUNX2 (1:1,000; cat. no. D1H7; Cell Signaling Technology, Inc.), anti-β-actin (1:2,000; cat. no. 20536-1-AP; Proteintech Group, Inc.), anti-caspase-8 (1:1,000; cat. no. 9746; Cell Signaling Technology, Inc.), anti-caspase-3 (1:1,000; cat. no. 9662; Cell Signaling Technology, Inc.). The membranes were incubated with the primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase conjugated-goat anti-mouse IgG anti-rabbit secondary antibodies (1:4,000; cat. no. sc-2030; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The membranes were exposed using an ECL kit (EMD Millipore) and assessed using Image Lab™ Software 2.0 (Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard error of the mean, and experiments were repeated at least three times. Significance between groups was analyzed by one-way analysis of variance followed by Student-Newman-Keuls test. Paired Student's t-test was used to analyze differences between paired samples. SPSS software (version 17.0; SPSS, Inc.) was used for the data analysis. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-150 in 26 OS tissues and non-tumor tissues were detected by RT-qPCR, and the results revealed that miR-150 was significantly decreased in OS tissues compared with non-tumor tissues (Fig. 1A). The expression levels of miR-150 were also significantly decreased in the four human OS cell lines (HOS, SAOS2, MG-63 and U2OS) compared with the normal osteoblast cells (hFOB1.19) (Fig. 1B), indicating that miR-150 exhibits low expression levels in OS.

In order to investigate the biological role of miR-150 in OS tumorigenesis, miR-150 was overexpressed in OS cell lines HOS and U2OS in the present study (Fig. 2A and B). Cell proliferation was assessed in order to determine the effects of miR-150 on OS cell growth using CCK-8. The data revealed that overexpression of miR-150 significantly inhibited the proliferation of HOS and U2OS cells (Fig. 2C and D). In order to investigate the role of miR-150 in OS cell chemotherapy resistance, an OS cell chemotherapy model was constructed using DOX. Cell viability was assessed in order to determine the effect of DOX treatment on OS cell viability. A dose-dependency assay was performed using 0, 25, 50, 75 and 100 µM DOX treatment. The results revealed that the cell viability of both HOS (Fig. 2E) and U2OS (Fig. 2F) was significantly decreased with DOX treatment in a dose-dependent manner. No further reductions in cell viability was observed when the concentration of DOX exceeded 50 µM. Furthermore, miR-150 overexpression significantly decreased the cell viability of HOS and U2OS in the DOX treatment group, compared with the NC mimic + DOX group (Fig. 2G and H). Thus, these data suggested that miR-150 inhibited OS cell proliferation and sensitized OS cells to DOX.

In order to assess the downstream target and underlying molecular mechanism of miR-150 in OS, a TargetScan bioinformatics algorithm was used to identify the potential target gene of miR-150. The results revealed that RUNX2 was a potential target of miR-150 based on a putative 3′-UTR sequence (Fig. 3A). A luciferase assay was performed in order to investigate the association between miR-150 and RUNX2. Luciferase reporter constructs containing the WT or Mut 3′-UTR of the RUNX2 were constructed. The results revealed that miR-150 significantly decreased the luciferase activity of the WT RUNX2 3′-UTR but not the Mut RUNX2 3′-UTR (Fig. 3B). Overexpression of miR-150 significantly downregulated the expression of RUNX2 both in HOS and U2OS cells (Fig. 3C and D). The results indicated that RUNX2 was a target gene of miR-150 in OS.

The aforementioned results indicated that miR-150 inhibited OS cell growth by targeting RUNX2. Therefore, the roles of RUNX2 in OS cell proliferation and chemotherapy resistance were further detected in the present study. The data revealed that RUNX2 expression was significantly decreased by transfection with miR-150 mimic in both HOS (Fig. 4A and B) and U2OS (Fig. 4E and F) cells, compared with the NC mimic group. Treatment of the miR-NC group with DOX also significantly reduced the expression of RUNX2 (Fig. 4B and F), suggesting that sensitization of OS to DOX chemotherapy is associated with RUNX2 expression. RUNX2 was then overexpressed using the pcDNA3.1 vector to rescue RUNX expression. Proliferation assays revealed that the suppressive effect of miR-150 on HOS (Fig. 4C) and U2OS (Fig. 4G) cells was comparable to that of shRUNX2. As presented in Fig. 4D and H, overexpression of RUNX2 reversed the effects of miR-150 on the DOX sensitization of HOS and U2OS cells, respectively. Furthermore, following RUNX2 knockdown (Fig. 4I and J), sensitization of HOS and U2OS cells to DOX was also significantly increased (Fig. 4K and L). Taken together, the results indicated that RUNX2 was an important functional target of miR-150 involved in the proliferation and chemotherapy sensitization of OS cells.

Expression levels of RUNX2 were decreased with increased concentrations of DOX treatment in HOS and U2OS cells in a dose-dependent manner (Fig. 5A and B). The levels of apoptosis proteins, including cleaved caspase-3 and cleaved caspase-8 were tested. The results revealed that miR-150 significantly increased the expression of cleaved caspase-3 and cleaved caspase-8 in both HOS and U2OS cells, while RUNX2 significantly decreased the expression levels of cleaved caspase-3 and cleaved caspase-8 (Fig. 5C-J). Therefore, the data from the present study demonstrated the molecular mechanism underlying the miR-150-RUNX2 axis in OS resistance to DOX.