The U2OS and MG-63 osteosarcoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were routinely cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% FBS (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO₂ at 37°C. Rabbit anti-STAT3, phosphor-STAT3, rabbit anti-ERK1/2, phosphor-ERK1/2, rabbit anti-p38, phosphor-p38, rabbit anti-MMP-2, rabbit anti-MMP-9 and rabbit anti-Snail antibodies were all purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-E-cadherin, vimentin, N-cadherin, fibronectin and rabbit anti-MMP-7 antibodies were purchased from Abcam (Cambridge, UK). Alexa Fluor 488-conjugated goat anti-rabbit IgG was purchased from Invitrogen Life Technologies. IL-6 was purchased from PeproTech (Rocky Hill, NJ, USA). Irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). WP1066, a selective inhibitor of STAT3, was purchased from Sigma-Aldrich (St. Louis, MO, USA).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cell proliferation and viability. The U2OS and MG-63 osteosarcoma cells (5,000 cells/well) in 100 µl medium were seeded into 96-well plates. After being stimulated with irisin at different doses (0, 25, 50, 100 and 200 ng/ml) for different time-points (12, 24 and 48 h), 20 µl MTT solution (5 mg/ml) was added into each well. After incubation for 4 h, 100 µl of dimethyl sulfoxide (DMSO) was added to each well for another 15 min. Then, the absorbance values were determined using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 490 nm.

To evaluate the migration of the U2OS and MG-63 osteosarcoma cells, a scratch wound healing assay was used. Briefly, the U2OS and MG-63 cells (1×10⁶/well) were seeded in 6-well plates and cultured with DMEM supplemented with 10% FBS. When reaching confluency, each well was directly scratched with a 200 µl pipette tip. To detect the effect of irisin on the migration of U2OS and MG-63 cells, 100 ng/ml irisin was added to each well. After 24 h of incubation, the wound healing areas were photographed and the distance between the two cell edges was analyzed by ImageJ software.

The Transwell system was used to evaluate the effect of irisin on the invasion of U2OS and MG-63 osteosarcoma cells. The U2OS and MG-63 cells were cultured in Boyden chambers, with 8-µm pore filter inserts, in 24-well plates (Corning Costar, Corning, NY, USA). The pore inserts were pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA) overnight. The U2OS and MG-63 cells (1×10⁵ cells/well) were suspended in 100 µl DMEM supplemented with 1% FBS and were added to the upper chamber. DMEM supplemented with 10% FBS and irisin (100 ng/ml) were added to the lower chamber. After 24 h of incubation, the cells that had attached to the lower surface were fixed with methanol and stained with 0.1% crystal violet. Then, 5 random high-power fields (magnification, ×200) of each sample were chosen and counted to evaluate the average number of invasive cells.

Total RNA was extracted from the U2OS and MG-63 osteosarcoma cells using TRIzol reagent (Invitrogen Life Technologies) and then reverse transcribed to cDNA with the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR analysis was performed using a LightCycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences used for qPCR were as follows: E-cadherin forward, 5′-CGAGAGCTACACGTTCACGG-3′ and reverse, 5′-GGGTGTCGAGGGAAAAATAGG-3′; N-cadherin forward, 5′-TTTGATGGAGGTCTCCTAACACC-3′ and reverse, 5′-ACGTTTAACACGTTGGAAATGTG-3′; vimentin forward, 5′-TGCCGTTGAAGCTGCTAACTA-3′ and reverse, 5′-CCAGAGGGAGTGAATCCAGATTA-3′; fibronectin forward, 5′-TCTCCTGCCTGGTACAGAATATGTAGTGAG-3′ and reverse, 5′-GGTCGCAGCAACAACTTCCAGGT-3′; Snail forward, 5′-TCGGAAGCCTAACTACAGCGA-3′ and reverse, 5′-AGATGAGCATTGGCAGCGAG-3′; MMP-2 forward, 5′-TTGATGGCATCGCTCAGATC-3′ and reverse, 5′-TTGTCACGTGGCGTCACAGT-3′; MMP-9 forward, 5′-GACGCAGACATCGTCATCCA-3′ and reverse, 5′-CACAACTCGTCATCGTCGAAA-3′; MMP-7 forward, 5′-GAGTGAGCTACAGTGGGAACA-3′ and reverse, 5′-CTATGACGCGGGAGTTTAACAT-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. Melting curves were assessed to confirm the specificity of the products generated for each set of primers. Following amplification, the ΔΔCt comparative method was used to normalize the relative levels of gene expression. The experiments were performed in triplicate.

After being treated with and without IL-6 and/or irisin, the U2OS and MG-63 osteosarcoma cells were collected and lysed. Total cell protein concentrations were assessed using the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal protein from the cell lysates was loaded onto 12% SDS-PAGE. After electrophoresis, the proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA) and then blocked with 5% fat-free milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. Then, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immune complexes were detected with ECL reagents (Millipore, USA), and the blots were quantified by densitometric analysis using the Alpha Imager 2200.

After being treated with and without IL-6 and/or irisin, the U2OS osteosarcoma cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with 5% normal goat serum for 1 h. Subsequently, the U2OS cells were incubated with rabbit anti-E-cadherin and rabbit anti-vimentin overnight at 4°C. This step was followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG for 1 h at room temperature. Then the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI)/PBS (1:5,000; Sigma-Aldrich) for 5 min at room temperature. Finally, the images were captured using a Nikon Eclipse 80i fluorescence microscope.

The data are expressed as the mean ± standard deviation (SD). All of the experiments were repeated at least three times. A Student's t-test was used to compare differences between different groups. Comparisons among values of multiple groups were performed by one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at P<0.05.

To explore the effect of irisin on the proliferation of osteosarcoma cells, an MTT assay was applied as previously described in Materials and methods. As shown in Fig. 1A, various doses (0, 25, 50, 100 and 200 ng/ml) of irisin were added into the cultured U2OS and MG-63 osteosarcoma cells. After 24 h of incubation, irisin significantly suppressed the viability of U2OS and MG-63 osteosarcoma cells at a concentration of 200 ng/ml. After a 48-h incubation, irisin significantly inhibited the proliferation of U2OS and MG-63 osteosarcoma cells at a dose above 25 ng/ml (50, 100 and 200 ng/ml). Therefore, irisin inhibited the proliferation of U2OS and MG-63 osteosarcoma cells in a dose- and time-dependent manner. Based on these results, we selected irisin at a concentration of 100 ng/ml for 24 h and conducted the following migration and invasion experiments, so as to eliminate the effect of irisin on U2OS and MG-63 osteosarcoma cell proliferation.

The wound healing assay was applied to detect the effect of irisin on the migration of osteosarcoma cells. The U2OS and MG-63 osteosarcoma cells were treated with 100 ng/ml irisin for 24 h. As shown in Fig. 1B, the migration of U2OS and MG-63 cells was suppressed by 100 ng/ml irisin. The results of the wound healing assay revealed that the relative scratch width was significantly decreased when treated with irisin. To further explore the effect of irisin on osteosarcoma cell invasion, a Transwell assay was used. The U2OS and MG-63 cells were treated with 100 ng/ml irisin for 24 h. As shown in Fig. 1C, the invasion of U2OS and MG-63 cells was inhibited by 100 ng/ml irisin. The results of the Transwell assay revealed that irisin may inhibit the invasive ability of osteosarcoma cells.

Evidence that EMT plays an important role in tumor invasion and metastasis have been well demonstrated (42,43). In addition the role of IL-6 in the induction of EMT has been established (44). To further explore the effect of irisin on osteosarcoma cell EMT, the expression of EMT markers (E-cadherin, N-cadherin, vimentin, fibronectin, MMP-2, MMP-7 and MMP-9) when treated with IL-6 (100 ng/ml) and irisin (0, 50 and 100 ng/ml) was detected using qPCR and western blot analysis. As illustrated in Fig. 2A and B, IL-6 inhibited E-cadherin protein expression and irisin restored the effect of IL-6 in a dose-dependent manner. The expression of N-cadherin, vimentin, fibronectin, MMP-2, MMP-7 and MMP-9 was upregulated when treated with IL-6 and irisin suppressed the effect in a dose-dependent manner. We also used immunofluorescence staining to evaluate the effect of irisin on the expression of EMT markers. After being treated with IL-6 (100 ng/ml) and irisin (100 ng/ml) for 24 h, the U2OS cells were stained with E-cadherin and vimentin and were analyzed by fluorescence microscopy. As shown in Fig. 2C, IL-6 downregulated E-cadherin expression and upregulated vimentin expression, while irisin effectively reversed the effect of IL-6. Collectively, these results indicated that irisin treatment prevented IL-6-induced EMT. Furthermore they revealed that the suppressive effect of irisin on osteosarcoma cell invasion and migration may be related to EMT.

The MAPK and STAT3 signaling pathways play a crucial role in IL-6-induced cancer cell EMT (44–46). To further explore the potential mechanism of irisin on osteosarcoma cell EMT, the STAT3, ERK1/2 and p38 signaling pathways were detected. As shown in Fig. 3A, STAT3, ERK1/2 and p38 phosphorylation was upregulated while being treated with IL-6 (100 ng/ml). STAT3 phosphorylation was suppressed when treated with irisin (100 ng/ml). The suppression was statistically significant, as quantified by densitometry (Fig. 3B). However, irisin did not change the phosphorylation of ERK1/2 and p38. These results revealed that the STAT3 signaling pathway may be involved in irisin-mediated osteosarcoma cell EMT.

To further demonstrate whether STAT3 is involved in irisin-regulated osteosarcoma cell EMT, we selected WP1066 (a STAT3 inhibitor) to block STAT3 phosphorylation (Fig. 4A). As shown in Fig. 4B and C, at the mRNA and protein level, the suppression of STAT3 phosphorylation with WP1066 (10 µM) enhanced the expression of E-cadherin and further downregulated the expression of some EMT markers (N-cadherin, fibronectin, MMP-2 and MMP-7) which were induced by irisin. These results revealed that irisin-mediated osteosarcoma cell EMT may be STAT3-dependent. Blockade of STAT3 activation could further enhance irisin-mediated osteosarcoma cell EMT.

Evidence has established the expression and activation of Snail transcriptional factor during EMT in E-cadherin suppression and cancer progression (47). In the present study we further explored the effects of irisin on the IL-6-induced Snail expression and the relationship between STAT3 and Snail. Firstly, U2OS and MG-63 osteosarcoma cells were treated with IL-6 (100 ng/ml) and irisin (0, 50 and 100 ng/ml) for 24 h and the expression of Snail was detected using qPCR and western blot analysis. As shown in Fig. 5A and B, IL-6 induced Snail mRNA and protein expression and irisin inhibited the effect in a dose-dependent manner. Then, to further evaluate whether STAT3 participated in irisin-regulated Snail expression, U2OS and MG-63 osteosarcoma cells were also pretreated with WP1066 for 30 min before IL-6 and irisin were added. As displayed in Fig. 5C and D, inhibition of STAT3 phosphorylation with WP1066 further suppressed the mRNA and protein expression of Snail which was induced by irisin. These results indicated the crucial role of STAT3 in regulating Snail expression and irisin inhibition of the expression of Snail via the STAT3 pathway. Collectively, these results demonstrated that the suppression of metastasis by irisin was mediated by the inhibition of EMT via the STAT3/Snail signaling pathway in osteosarcoma cells.