A total of 36 pairs of OS tissues and adjacent normal bone tissues were obtained from the Department of Orthopedics, Shengjing Hospital of China Medical University. Tissue samples were rapidly frozen in liquid nitrogen and stored at −80°C following surgery. Written informed consent was obtained from all patients. The study was approved by the Ethics Committee of Shengjing Hospital of China Medical University.

A normal osteoblast cell line (Nhost) and four different OS cell lines (KHOS, LM7, U2OS and MG-63) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNA was extracted from tissues and cells using TRIzol^® reagent (Takara Biotechnology, Co., Ltd., Dalian, China), following the manufacturer's protocol. RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China). miR-491-5p expression was detected using a SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.). Relative mRNA expression was normalized to that of β-actin and U6 small nuclear RNA. The RT-qPCR assays were performed using an Applied Biosystems 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primer sequences were as follows: miR-491-5p forward, 5′-GGAGTGGGGAACCCTTCC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; PKM2 forward, 5′-CTGTGGACTTGCCTGCTGTG-3′ and reverse, 5′-TGCCTTGCGGATGAATGACG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; β-actin forward, 5′-AGAAAATCTGGCACCACACC-3′ and reverse, 5′-TAGCACAGCCTGGATAGCAA-3′.

Cell proliferation was measured using a Cell Counting Kit-8 (CCK8; Dojindo Molecular Technologies, Kumamoto, Japan). U2OS or MG-63 cells (2×10³ cells/well) were seeded in 96-well plates. Cells were transfected with 100 nM miR-NC (cat. no., QPG-04191), miR-491-5p mimics (cat. no., QPG-04192) or miR-491-5p inhibitors (cat. no., QPG-04193) (sequence unavailable) purchased by Shanghai Genepharma Co., Ltd. (Shanghai, China) using the Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols at a final concentration of 100 nM. Cells were incubated for 1, 2, 3, 4 and 5 days at 37°C. Then, 10 µl CCK-8 solution was added to each well for 2 h at 37°C. Cell viability was measured using a microplate reader and the absorbance at 450 nm was recorded.

U2OS or MG-63 cells were transfected with 100 nM miR-NC, miR-491-5p mimics or miR-491-inhibitors, as aforementioned. Cells were incubated for 14 days, after which the cells were fixed in 100% methanol for 20 min at room temperature, stained with 0.1% crystal violet for 20 min at room temperature, and counted in 5 random fields of view using a light microscope (magnification, 200×).

Total protein was extracted from cells following transfection with miR-NC, miR-491-5p mimics or miR-491-5p inhibitors. The protein concentration was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's protocol. Equal amounts of protein (40 µg) were separated via 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then incubated with specific antibodies against PKM2 (dilution, 1:500; cat. no., sc-65178,) and GAPDH (dilution, 1:1,000; cat. no., sc-25778; both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. This was followed by incubation at room temperature with horseradish peroxidase-conjugated anti-mouse IgG (dilution, 1:1,000; cat. no., 7074; Cell Signaling Technology, Inc., Danvers, MA, USA). The protein bands were assessed using an Enhanced Chemiluminescence Western Blotting kit (Pierce; Thermo Fisher Scientific, Inc.). Protein expression was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Protein expression was normalized to that of GAPDH.

The putative targets of miR-491-5p were predicted by the online software Targetscan (http://www.targetscan.org/vert_71/) and miRanda (http://www.microRNA.org).

The wild-type 3′-UTR region of the PKM2 mRNA (3′UTR-PKM2-WT) containing a putative miR-491-5p-binding site and the corresponding mutant type 3′-UTR region of the PKM2 mRNA (5′-CCCCAC-3′ to 5′-GGGGUG-3′) (3′UTR-PKM2-MUT) were generated by Guangzhou RiboBio Co., Ltd., (Guangzhou, China). U2OS cells were seeded into a 96-well plate (1×10⁴ cells/well) and co-transfected with 3′UTR-PKM2-WT/MUT plasmids (Promega Corporation, Madison, WI, USA), along with miR-491-5p or the miR-negative control (NC), using Lipofectamine 2000^® (Invitrogen; Thermo Fisher Scientific, Inc.). Relative luciferase activity was measured at 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase was used to normalize the luciferase activity.

All data were assessed using SPSS v.20.0 software (IBM Corp., Armonk, NY, USA) and are presented as the means ± standard deviation from ≥3 independent experiments. Differences between two groups were compared using a two-tailed paired Student's t-test; one-way analysis of variance (ANOVA) was used for comparisons between multiple groups. The Student Newman-Keuls test was used as a post-hoc test following ANOVA. The association between PKM2 expression and miR-491-5p in OS tissues was determined by Spearman's correlation analyses. P<0.05 was considered to indicate a statistically significant difference.

Firstly, the relative expression of miR-491-5p in 36 paired of OS tissues and adjacent normal tissue samples was analyzed via RT-qPCR. The results demonstrated that miR-491-5p expression was significantly decreased in OS tissues compared with in the adjacent normal tissues (Fig. 1A; P<0.01). Next, relative miR-491-5p expression levels in Nhost cells and 4 different OS cell lines (KHOS, LM7, U2OS, and MG-63) were examined. miR-491-5p expression was significantly decreased in the 4 OS cell lines compared with in the Nhost cells (Fig. 1B). Therefore, these results indicated that miR-491-5p expression is significantly downregulated in OS tissues and cells.

To investigate whether miR-491-5p expression affects the biological function of OS cells, according to the expression of miR-491-5p in the 4 OS cells, gain- and loss-of-function assays were performed using miR-491-5p mimics and miR-491-5p inhibitors, respectively, in U2OS and MG-63 cells. CCK8 assays indicated that cells transfected with miR-491-5p mimics had significantly inhibited proliferation abilities compared with the control; however, the miR-491-5p inhibitor promoted cell proliferation abilities, compared with the control groups in U2OS and MG-63 cells (Fig. 2A and B; P<0.05). Concurrently, cell colony formation assays demonstrated that miR-491-5p mimics reduced the number of colonies formed compared with the control, while the miR-491-5p inhibitor exhibited the opposite effect, in U2OS and MG-63 cells (Fig. 2C and D; P<0.05). These results indicated that miR-491-5p inhibited proliferation in OS cells.

Using miRanda and TargetScan target prediction software, PKM2 was identified to be a potential target of miR-491-5p (Fig. 3A). Furthermore, the luciferase reporter vector containing the putative 3′-UTR PKM2 target site and the mutant site for miR-491-5p was constructed (Fig. 3A). To demonstrate that PKM2 was a target of miR-491-5p, U2OS cells were co-transfected with 3′-UTR PKM2-WT or 3′-UTR PKM2-MUT and miR-491-5p mimic or miR-NC. Luciferase activity analysis results indicated that the miR-491-5p mimic significantly suppressed the luciferase activity of the 3′-UTR PKM2-WT reporter vector, but that of 3′-UTR PKM2-MUT was not altered (Fig. 3B). Furthermore, it was demonstrated that PKM mRNA expression levels were significantly reduced with miR-491-5p mimics, but increased with miR491-5p inhibitors, compared with the miR-NC in U2OS and MG-63 cells (Fig. 3C and D). Consistently, the protein expression levels of PKM2 were significantly reduced in the miR-491-5p mimic group, but increased in the miR491-5p inhibitor group, compared with the miR-NC group in U2OS and MG-63 cells (Fig. 4A-D). Therefore, these results indicated that PKM2 was a direct target of miR-491-5p.

Furthermore, the expression of PKM2 was analyzed and identified that PKM2 expression was increased in OS tissues and adjacent normal tissue samples using the RT-qPCR assay (Fig. 5A). Increased PKM2 expression was negatively associated with miR-491-5p in OS tissues as determined by Spearman's correlation analyses (r=−0.433, Fig. 5B). To investigate whether PKM2 mediated the proliferation-inhibiting effects of miR-491-5p in OS, CCK8 assays were used and demonstrated that the miR-491-5p mimic suppressed cell proliferation compared with the control group, but was reversed by co-transfection with pcDNA-PKM2 plasmid in U2OS and MG-63 cells (Fig. 5C and D). These results indicated that PKM2 overexpression reversed the proliferation-inhibiting effects of miR-491-5p in OS cells.