Human OS cell lines, U2-OS, SAOS-2, MG-63 and SOSP-9607, and the human osteoblast cell line h-FOB, were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 100 U/ml penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cell lines were cultured in a humidified incubator at 37°C in an atmosphere containing 5% CO₂.

A total of 8 pairs of human OS tissues and tumor-adjacent normal tissues (TATs) were obtained from 8 patients (4 females and 4 males; age range, 31–55) with OS at the Department of Orthopedics, Guangzhou First People's Hospital (Guangzhou, China) from August 2014 to October 2015. The present study was approved by the Ethics Committee of Guangzhou First People's Hospital (Guangzhou, China). OS diagnosis was confirmed pathologically by 2 pathologists independently. Informed consent was obtained from all patients for tissue collection during surgery. Tissues were immediately frozen in liquid nitrogen, and stored at −80°C until use.

miR-544 mimics (HmiR0234), miR-544 inhibitor (miR-544-in, HmiR-AN0623), and the negative control miRNAs (CmiR0001 and CmiR-AN0001) were purchased from GeneCopoeia, Inc., (Rockville, MD, USA), and each miRNA (30 nM) was transfected into OS cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The time interval between transfection and subsequent experimentation was 48 h.

AXIN2-siRNA (5′-GCAGAGGGACAGGAATCAT-3′) and the negative control siRNA (5′-GCAGGGACAAGGTAGACAT-3′) were purchased from Qiagen, Inc., (Valencia, CA, USA). Transfection with 50 nM siRNA was performed using Lipofectamine^® 2000 reagent, according to the manufacturer's protocol. The time interval between transfection and subsequent experimentation was 48 h.

Total RNA, including miRNA, was extracted from OS cells and clinical tissues using TRIzol^® (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. miRNA was converted to cDNA using a TaqMan^® miRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression levels of miR-544 (HmiRQP0623, GeneCopoeia^™, Guangzhou, China) were quantified using primers within a miRNA-specific TaqMan^® miRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc), according to the manufacturer's protocol. PCR was performed to detect the expression of Cyclin D1 (CCND1, HQP016204, GeneCopoeia^™, Guangzhou, China) and c-Myc (HQP011597, GeneCopoeia^™, Guangzhou, China using the ABI 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) with SYBR Green Mix Taq kit (Takara Bio, Inc., Otsu, Shiga, Japan). The thermocycling conditions were as follows: At 95°C for 30 sec, followed by 40 cycles of amplification at 95°C for 5 sec, at 59°C for 30 sec and at 72°C for 30 sec. U6 (HmiRQP9001, GeneCopoeia^™, Guangzhou, China) and GAPDH (HQP064347, GeneCopoeia^™, Guangzhou, China) served as internal controls for the miRNA and mRNA assays, respectively. Expression was quantified using the 2^−ΔΔCq method (11).

Transfected SOSP-9607 cells were seeded into 96-well plates, in medium containing 10% FBS, at a density of 1×10³ cells/well. The cells were stained with 20 µg MTT dye (0.5 mg/ml; Sigma-Aldrich; Merck KGaA). The formazan crystals formed were dissolved in 150 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) at 1, 2, 3, 4 or 5 days, and the absorbance was recorded at 490 nm using a spectro-photometric plate reader.

SOSP-9607 cells were seeded into 6-well plates (1×10³ cells/well) and incubated for 10 days in medium containing 10% FBS. The colonies were stained with 0.5% crystal violet at room temperature for 15 min following fixation in 4% paraformaldehyde for 5 min at room temperature. The number of colonies, each defined as a group of >50 cells, was counted per plate.

SOSP-9607 cells were trypsinized, and 2×10³ cells were suspended in complete medium (Dulbecco's modified Eagle's medium supplemented with 10% FBS and 100 U/ml penicillin/streptomycin) containing 0.3% agar (Sigma-Aldrich; Merck KGaA), and applied onto a layer of 1% agar in complete medium in 6-well plates. Cells were incubated for 2 weeks at 37°C prior to subjection to a colony formation assay, as aforementioned, and cell colonies were imaged at magnification ×100. Only cell colonies >0.1 mm in diameter were counted.

Based on the miR sequences, target genes were predicted using TargetScan (version 3.1; http://www.targetscan.org/mamm_31/). AXIN2 was amplified from SOSP-9607 cell cDNA and was sub-cloned into a firefly luciferase reporter pGL3 plasmid (cat. no., GUR100013-P-2; Guangzhou RiboBio Co., Ltd., Guangzhou, China). Cells were seeded in triplicate in 24-well plates (5×10⁴ cells/well) and cultured for 24 h. pGL3-AXIN2-3L3-luciferase reporter pGL3 plasmids were co-transfected into the cells with the pRL-TK Renilla plasmids (Promega Corporation, Madison, WI, USA) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After 48 h of transfection, the luciferase activities of the transformed cells were assayed using a Dual-Luciferase Reporter assay system (Promega Corporation), according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

Total protein was extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China), and protein concentrations were determined with BCA Protein Assay Kit (Beyotime, Institute of Biotechnology), accoding to the manufacturer's protocol. A total of 50 µg protein extracts were separated via 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk in TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween-20) for 0.5 h at room temperature, and then incubated overnight with anti-AXIN2 (catalog no., 2151), anti-CCND1 (catalog no., 2978) and anti-c-Myc (catalog no., 13987) antibodies (all at a dilution of 1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA) at 4°C overnight. α-tubulin (catalog no., 2144; 1:5,000; Sigma-Aldrich; Merck KGaA) was used as the loading control. The blots were then incubated for 2 h with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin secondary antibody (cat no. P0023D; 1:5,000; Beyotime Institute of Biotechnology) at 37°C. Signals were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) as a substrate, and images were analyzed using an automated chemiluminescence system (LAS500; GE Healthcare, USA) according to protocol of the manufacturer.

All data are presented as the mean ± standard deviation, and all experiments were repeated independently at least 3 times. Statistical analyses, specifically a one-way analysis of variance (ANOVA) or the Student's t test, were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of miR-544 in OS development, miR-544 expression was examined in OS cells and clinical tissue samples. RT-qPCR analysis indicated that miR-544 expression was significantly increased in the OS cell lines, U2-OS, SAO-2, MG-63 and SOSP-9607, compared with in the human osteoblast h-FOB cells (Fig. 1A). Furthermore, miR-544 expression was markedly upregulated in the OS tissues, as compared with in the matched TATs (Fig. 1B). This suggests that miR-544 is upregulated in OS and that it may serve a role in promoting OS development.

To explore the effect of miR-544 on OS cell proliferation, SOSP-9607 cells were transfected with miR-544 mimics, a miR-544-in or the respective controls, and the expression of miR-544 in the stably transfected SOSP-9607 cell line was confirmed by RT-qPCR (Fig. 2A). MTT and colony formation assays revealed that the overexpression of miR-544 increased the proliferation of SOSP-9607 cells, as compared with those cells transfected with negative control miRNA (Fig. 2B and C). Overexpression of miR-544 in SOSP-9607 cells also significantly enhanced their anchorage-independent growth ability (P<0.05; Fig. 2D). By contrast, SOSP-9607 cells were transfected with miR-544-in or the respective control, and the expression of miR-544 in the stably transfected SOSP-9607 cell line was confirmed by RT-qPCR (Fig. 3A). MiR544-in-transfected SOSP-9607 cells exhibited decreased proliferation rates, colony formation ability, and anchorage-independent cell growth ability, compared with in the negative control cells (Fig. 3B-D). Collectively, these data indicate that miR-544 acted as a tumor promoter to endorse OS cell proliferation.

According to bioinformatical predictions, AXIN2 is a putative target gene of miR-544. To verify this prediction, WT AXIN2 3′UTRs were generated (Fig. 4A). To determine whether miR-544 affected AXIN2 expression, AXIN2 expression was analyzed by western blotting. Overexpression of miR-544 inhibited the protein expression of AXIN2, while SOSP-9607 cells transfected with miR-544-in exhibited enhanced protein expression of AXIN2 (Fig. 4B). To investigate whether AXIN2 could be regulated by miR-544, AXIN2 3′-UTR was co-transfected into SOSP-9607 cells with miR-544 mimics, miR-544-in or miR-544-mut, followed by the measurement of luciferase activity. As demonstrated in Fig. 4C, luciferase activity was markedly reduced in cells that were co-transfected with the WT AXIN2 3′-UTR and miR-544. By contrast, miR-544-in transfection increased luciferase activity in cells transfected with WT AXIN2 3′-UTR. miR-544-mut transfection did not alter the luciferase activity of cells transfected with AXIN2 3′-UTR. These findings indicate that miR-544 downregulated the protein expression of AXIN2 via direct binding to the seed sequences in its 3′-UTR.

It has been reported that the Wnt signaling pathway serves an essential role in the cancer cell cycle and proliferation (12,13). C-Myc, CCND1, and AXIN2 are well-known target genes of the Wnt signaling pathway (14–17). The expression of these Wnt/β-catenin signaling pathway downstream genes was determined in miR-544-transfected cells. Using RT-qPCR and western blotting, it was demonstrated that the mRNA and protein expression levels of CCND1 and c-Myc were upregulated in miR-544-transfected SOSP-9607 cells, while transfection with miR-544-in resulted in the opposite effect (Fig. 4D and E). This suggests that miR-544 modulated downstream genes of the Wnt signaling pathway (AXIN2, CCND1 and c-Myc).

The effects of AXIN2 downregulation on proliferation were examined in OS cells transfected with miR-544-in. siRNA-mediated knockdown of AXIN2 was performed in miR-544-in-transfected SOSP-9607 cells, and confirmed through western blotting (Fig. 5A). Results from the colony formation and anchorage-independent growth assays indicated that the suppression of AXIN2 expression by AXIN2-siRNA reversed the effects of miR-544-in in SOSP-9607 cells (Fig. 5B and C). Taken together, these results demonstrated that the downregulation of AXIN2 counteracts the cell proliferation arrest induced by miR-544-in.