A total of 18 patients (male, 10; female, 8; age ≤18 years, 13; age >18 years, 5; tumor stage I+II, 15; tumor stage III, 3) diagnosed with OS were recruited from the Second Affiliated Hospital, Chongqing Medical University (Chongqing, China). These patients were divided into two groups (metastasis, vs. no metastasis) according to radiological results. All patients provided written informed consent. The experimental protocols were approved by the Ethics Committee of the Second Affiliated Hospital, Chongqing Medical University. The OS tissues and the corresponding normal tissues (5 cm from the tumor margin) were obtained from resection and then immediately snap frozen in liquid nitrogen for storage at −80°C.

The OS-derived human HOS, Saos2, U2OS and MG-63 cell lines, and the NHOst normal osteoblast cell line were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in DMEM with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated in a humidified atmosphere containing 5% CO₂ at 37°C.

Total RNA was extracted from the tissues and cell lines using an RNeasy Plus Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Total RNA (2 µg) was purified and reverse transcribed into cDNA using a PrimeScript RT Reagent kit (Perfect Real-Time) from Takara Bio, Inc. (Otsu, Japan) according to the manufacturer's instructions. The qPCR analysis was performed on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with a SYBR Premix Ex Taq™ II kit (Takara Bio, Inc.). The reaction mix contained 2 µl cDNA, 10 µl 2X Premix Ex Taq (Probe qPCR), 0.4 µl forward/reverse primers, 0.8 µl Probe, 0.2 µl ROX Reference Dye II and 6.2 µl ddH₂O in a total volume of 20 µl. The sequences of the primers were as follows: miR-494 forward, 5′-TGACCTGAAACATACACGGGA-3′, and reverse, 5′-TATCGTTGTACTCCACTCCTTGAC-3′; U6 forward, 5′-AAAGACCTGTACGCCAACAC-3′ and reverse, 5′-GTCATACTCCTGCTTGCTGAT-3′. The PCR cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 30 sec, 62°C for 30 sec and 72°C for 30 sec. The results were calculated using the 2^−ΔΔCq method (16) and normalized to the expression of U6.

PicTar (http://pictar.mdc-berlin.de/), TargetScan (http://www.targetscan.org) and miRBase (http://www.mirbase.org/) (17–19) were used to investigate the potential binding sites of miR494.

The wild-type 3′-untranslated region (3′-UTR) of CDK6 containing the miR-494 binding site was cloned into the pGL3-control vector (Ambion; Thermo Fisher Scientific, Inc.) to construct the CDK6-3′-UTR-wild-type (WT). The CDK6 3′-UTR mutations containing mutant (mut) sequences were obtained using a QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Palo Alto, CA, USA). All plasmids were confirmed by sequencing. The miR-494 mimics were generated by Shanghai GenePharma Co., Ltd. (Shanghai, China) and the scramble sequence was purchased from Ambion; Thermo Fisher Scientific, Inc. Briefly, the MG-63 (~2–3×10⁶) and U2OS cells (~2–3×10⁶) were seeded into a 24-well plate 1 day prior to transfection, and then co-transfected with miR-494 mimics (sense, 5′UGA AAC AUA CAC GGG AAA CCU C3′ and antisense, 5′GGU UUC CCG UGU AUG UUU CAU U3′; 100 nM) or scramble (sense, 5′UUC UCC GAA CGU GUC ACG UUU 3′ and antisense, 5′ACG UAC ACG UUC GGA GAA UU3′; 50 nM), in addition to the CDK6-3′-UTR-WT or CDK6-3′-UTR-mutusing Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, the luciferase activity was determined using a Dual-Luciferase reporter assay (Promega Corporation, Madison, WI, USA), which was normalized to the activity of Renilla luciferase.

In order to evaluate the effect of the overexpression of miR-494 on cell proliferation, MTT assay and colony formation assays were performed. Briefly, 1×10³ cells (MG-63 and U2OS cells) were seeded in 96-well plates with four repeats and then transfected with the miR-494 mimics or scramble control. The cell viability was assessed 48 h following transfection using an MTT kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The results were determined at an optical density of 570 nm on a Tecan Spectra Fluor microplate reader (Tecan Group, Ltd., Männedorf, Switzerland). For the colony formation assay, ~200 transfected cells (miR-494 mimics or Scramble) were seeded in a 6-well plate with four repeats and cultured in DMEM containing 10% FBS for 2 weeks at 37°C. The medium was replaced every 3 days. The colonies were fixed with methanol, stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) and subsequently counted under a light microscope.

In order to investigate the effect of the overexpression of miR-494 on cell invasion and migration, the present study examined metastasis using an assay with BD Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA). A total of 4×10⁴ cells (MG-63 and U2OS cells) transfected with miR-494 mimics or scramble control were cultured in serum-free DMEM medium and subsequently placed into the upper chamber, which was Matrigel-coated for the cell invasion assay or Matrigel-free for the migration assay. Simultaneously, 10% FBS was added into the lower chambers to function as a chemoattractant. Following incubation for 48 h at 37°C, the cells on the upper surface of the membranes were removed, and the cells on the lower surface were fixed and stained (0.1% crystal violet). Images of the cells were captured and the number of cells were counted under a microscope (IX71; Olympus, Tokyo, Japan) in five randomly selected fields (magnification, ×200).

A total of 1×10⁴ MG-63 and U2OS cells were first transfected with miR-494 mimics or scramble control. After 48 h, the transfected cells were digested, collected and washed with PBS. Following washing twice with PBS, the cells were fixed with 70% ethanol at 4°C overnight. Propidium iodide (PI) staining solution (50 µg/ml; 1 mg/ml of RNase A, 0.1% Triton X-100 in PBS) was added into the cell suspension. Finally the cells were examined on a BD FACSCalibur flow cytometer (BD Biosciences). The experiments were repeated three times.

To examine the role of miR-494 in vivo, 20 female BALB/C-nu/nu mice (4–5 weeks old; weight, ~18–25 g) were used, which were purchased from the Animal Center of the Cancer Institute of Chinese Academy of Medical Science (Beijing, China). The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital, Chongqing Medical University. The mice were maintained on a standard 12:12 h light-dark cycle with free access to food and water at 18–22°C and with 50–60% humidity. The mice were randomly dived into two groups: the Lv-miR-494 group and the Lv-control group. A total of 2×10⁶ MG-63 cells transfected with miR-494 (Lv-miR-494 group) or the corresponding control (Lv-control) were subcutaneously injected into the flank region of the female nude mice. The mice were monitored every 3 days. After 5 weeks, the mice were sacrificed and the tumor tissues were measured on the basis of their width and length: (length × width²)/2.

Following transfection, the cells were washed three times with PBS and lysed with radioimmunoprecipitation assay lysis buffer to obtain the total protein. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total proteins (50 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes, as previously described (20). The membranes were incubated with blocking buffer (5% skimmed milk) for 1.5 h at room temperature and then incubated with the following primary antibodies: anti-CDK6 (1:500, ab79454; Abcam, Cambridge, MA, USA) and anti-GAPDH antibody (1:500; SAB4300645-100UG; Sigma-Aldrich, Merck KGaA) at 4°C overnight. Following incubation with the primary antibody, blots were washed with TBS-0.1% Tween and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:2,000, ab6721) or rabbit anti-mouse IgG (1:2,000, ab6709) (both from Abcam) secondary antibodies at 37°C for 2 h. Band intensity was quantified using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA). Experiments were performed in triplicate.

Statistical analysis was performed using the SPSS 13.0 software package (SPSS, Inc., Chicago, IL, USA) using one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. P≤0.05 was considered to indicate a statistically significant difference.

The present study used RT-qPCR analysis to examine the expression profiles of miR-494 in OS tumor tissues (metastatic, vs. non-metastatic), cell lines and corresponding controls. It was found that the expression levels of miR-494 in the 18 patients were significantly downregulated, compared with those in the normal tissues (P<0.05; Fig. 1A). In accordance, the expression levels of miR-494 in the HOS, Saos2, U2OS, MG-63 OS cells were significantly downregulated, compared with that in the NHOst cells (P<0.05; Fig. 1B). The association between cell metastasis and the expression of miR-494 was also investigated. The results suggested that the expression of miR-494 was decreased in the metastatic group, compared with the non-metastatic group (P<0.05; Fig. 1C). These findings indicated that the expression of miR-494, which was downregulated in OS tissues and cells, was closely linked to tumor metastasis.

As primary cells from human OS tissues are difficult to culture, MG-63 and U2OS cells were selected for the subsequent experiments, due to their routine maintenance in cell culture and ubiquitous use in investigations of OS (21,22). In addition, MG-63 and U2OS cells are OS-derived human cell lines with differing proliferation potential, providing the opportunity to examine different types of the disease: MG-63 cells exhibit a low level of proliferation, whereas U2OS cells exhibit more malignant proliferation potential (23). In the present study, miR-494 mimics were transfected into MG-63 and U2OS cells, MTT and cell colony formation assays were used to investigate the effect of the promotion of miR-494 on cell proliferation in vitro (48 h post-transfection). The results of the RT-qPCR analysis showed that the expression of miR-494 was significantly elevated in the cells transfected with miR-494 mimics (P<0.05; Fig. 2A). The results of the MTT assay suggested that the ectopic expression of miR-494 resulted in cell growth inhibition, compared with the scramble control group in MG-63 and U2OS cells (P<0.05; Fig. 2B and C). The results of the cell colony formation assays confirmed that the cells transfected with miR-494 mimics exhibited decreased cell numbers (P<0.05; Fig. 2D and E), which was in accordance with the MTT assays. To further determine the underlying regulatory effects of miR-494 in vivo, female nude mice were subcutaneously injected with miR-494 lentivirus (Lv-miR-494) and Lv-control, respectively. The data indicated that injection of miR-494 significantly suppressed tumor volume and weight, compared with those in the Lv-control group, and this effect was time-dependent. (P<0.05, P<0.01 and P<0.001; Fig. 2F and G). Taken together, these findings demonstrated that the ectopic expression of miR-494 caused marked cell growth inhibition in vitro and in vivo.

As the overexpression of miR-494 inhibited proliferation in vitro and in vivo, the present study performed invasion and migration assays to evaluate the effect of the restoration of miR-494 on cell metastasis. As shown in Fig. 3A and B, the numbers of MG-63 and U2OS cells in the miR-494 group were decreased, compared with the number in the scramble group (P<0.05), which indicated that cell invasion in the miR-494 group was repressed relative to the control. In addition, the numbers of migrated MG-63 and U2OS cells in the miR-494 group were decreased, compared with the number in the scramble group, which was consistent with the results of the cell invasion assay (P<0.05; Fig. 3C and D). The effect of miR-494 on cell cycle progression was then investigated in MG-63 and U2OS cells. The results showed that the ectopic expression of miR-494 in the MG-63 cells and U2OS cells significantly increased in the number of G1-phase cells and reduced the number of S-phase cells (P<0.05; Fig. 3E-H). Taken together, these findings suggested that miR-494 inhibited cell metastasis and induced cell cycle arrest of the OS cells.

As miR-494 has been reported to be involved in tumor progression via base-pairing to the 3′-UTR of its target genes, bioinformatics analysis was performed using PicTar, TargetScan and miRBase to predict the potential target of miR-494. The results revealed an miR-494 binding site in the CDK6 3′-UTR (Fig. 4A). In order to validate whether the 3′-UTR of CDK6 is a functional target of miR-494, a luciferase reporter assay was performed. The results showed that the promoted expression of miR-494 suppressed the luciferase activity of the CDK6 3′-UTR-WT construct, but not the CDK6 3′-UTR-mut construct in OS cells (P<0.05; Fig. 4B). In addition, RT-qPCR and western blot analyses were performed to evaluate the effect of promoted miR-494 on the expression of CDK6. The elevated expression of miR-494 resulted in reductions in the mRNA and protein expression of CDK6 in the MG-63 and U2OS cells (P<0.05; Fig. 4C and D). These data indicated that miR-494 directly targeted CDK6 and negatively regulated the expression of CDK6.

As it was found that miR-494 directly targeted CDK6 and regulated the expression of CDK6, the present study investigated the expression pattern of CDK6 in the OS tissue and cells. The results of the RT-qPCR analysis indicated that CDK6 mRNA was upregulated in the 18 OS tissue samples (P<0.05; Fig. 5A) and OS cells (P<0.05; Fig. 5B), compared with the corresponding controls, respectively. In addition, the expression of CDK6 was examined in tissues with differing features of metastasis. It was observed that the tissues in the metastasis group exhibited increased levels of CDK6, compared with those in the no metastasis group (P<0.05; Fig. 5C).