A total of 26 human OS tissues and corresponding normal adjacent tissues (NATs) were obtained from patients who suffered from OS and underwent surgical resection at Central Hospital of Zibo (Zibo, China) between February 2013 and March 2017. None of the patients were treated with chemotherapy or radiotherapy before the surgery was performed. All of the tissues were immediately frozen in liquid nitrogen and stored in a super cold refrigerator at −80°C. The approval for this study was obtained from the Ethic Committee of Central Hospital of Zibo. Written informed consent was also provided by the patients with OS enrolled in this research.

Human OS cell lines (MG-63, HOS, Saos-2, and U2OS) and a normal human osteoblast hFOB1.19 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units of penicillin/ml and 100 ng of streptomycin/ml (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and maintained at 37°C in a humidified atmosphere with 5% CO₂.

miR-660 inhibitor and negative control miRNA inhibitor (NC inhibitor) were provided by GenePharma (Shanghai, China). Small interfering RNA (siRNA) against the expression of Forkhead box O1 (FOXO1 siRNA) and NC siRNA were produced by Ribobio (Guangzhou, China). Cells were plated into 6-well culture plates and transfected with miRNA inhibitor or siRNA when the cells were grown to approximately 60–70% confluence by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. At 6–8 h post-transfection, the culture medium was removed, and fresh RPMI-1640 medium containing 10% FBS was added to each well.

Total RNA was isolated from tissue specimens or culture cells by using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocol. The miR-660 expression level was detected by utilizing a One-Step SYBR^® PrimeScript™ miRNA RT-PCR kit (Takara Bio, Dalian, China) with U6 snRNA as an internal reference. To quantify the mRNA level of FOXO1, we conducted a reverse transcription with a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) and quantitative PCR with a SYBR Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.). GAPDH served as an internal control for the mRNA expression of FOXO1. Relative gene expression was analysed using the 2^−∆∆Cq method (17).

At 24 h post-transfection, the transfected cells were inoculated into 96-well plates at a density of 3,000 cells/well. In our research, four time points after inoculation (0, 24, 48 and 72 h) were chosen, and CCK-8 assays were carried out at each time point. A total of 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well. After the cells were incubated at 37°C for 2 h, the absorbance at 450 nm was determined with a SpectraMax Microplate^® Spectrophotometer (Molecular Devices LLC, Sunnyvale, CA, USA).

After 24 h of transfection, the transfected cells were collected and suspended in FBS-free RPMI-1640 medium. A total of 1×10⁵ cells were seeded on the upper surface of Matrigel-coated Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA). RPMI-1640 medium (600 µl) containing 20% FBS was added to the lower chambers. The chambers were incubated at 37°C with 5% CO₂ for 24 h. The non-invasive cells that remained on the upper surface of the chambers were removed with a cotton swab. The invasive cells on the lower surface of the chamber were fixed with 4% paraformaldehyde, stained with 0.05% crystal violet and photographed under an inverted microscope (Olympus Corporation, Tokyo, Japan). The number of invasive cells was counted in five randomly selected visual fields (magnification, 200×) from each Transwell chamber.

The putative targets of miR-660 were predicted using TargetScan (http://www.targetscan.org) and Pictar (http://www.pictar.org/).

The FOXO1 3′-UTR containing wild-type (Wt) and mutant-type (Mut) predicted miR-660 binding sequences was generated by GenePharma, cloned into the pGL3 plasmid (Promega Corporation, Madison, WI, USA), and named as pGL3-FOXO1-Wt-3′-UTR and pGL3-FOXO1-Mut-3′-UTR, respectively. Cells were seeded into 24-well plates and cotransfected using Lipofectamine 2000 with miR-660 inhibitor or NC inhibitor, and pGL3-FOXO1-Wt-3′-UTR or pGL3-FOXO1-Mut-3′-UTR. 48 h after transfection, luciferase activities were detected using the Dual-Luciferase Reporter Assay System (Promega Corporation). The firefly luciferase activity was normalized to Renilla luciferase activity.

Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Inc., Shanghai, China) supplemented with 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and 1 mg/ml aprotinin (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The concentration of total protein was quantified using the BCA Protein Assay kit (Beyotime Institute of Biotechnology, Inc.). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis before being transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Subsequent to block with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-human FOXO1 monoclonal antibody (ab52857; Abcam, Cambridge, UK) and rabbit anti-human GAPDH monoclonal antibody (ab128915; Abcam). Afterwards, the membranes were washed thrice with Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with Goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibodies (ab205718; Abcam) followed by visualization using an enhanced chemiluminescence system (EMD Millipore). GAPDH served as a normalization control.

Statistical analysis was performed using Statistical Package for Social Sciences version 19.0 (IBM Corp., Armonk, NY, USA). Data were shown as mean ± standard deviation and analysed with Student's t-test or one-way ANOVA plus multiple comparisons combined with Tukey's post hoc test. The relationship between miR-660 and FOXO1 mRNA in OS tissues was examined through Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the miR-660 expression level in OS, RT-qPCR was performed to measure the miR-660 expression in 26 pairs of OS tissues and the corresponding NATs. The results showed that the miR-660 expression level was significantly higher in the OS tissues than that in the NATs (P<0.05; Fig. 1A). We next sought to examine whether miR-660 upregulation also occurred in OS cell lines. miR-660 was overexpressed in all of the four human OS cell lines, namely, MG-63, HOS, Saos-2 and U2OS, compared with that in the normal human osteoblast hFOB1.19 (P<0.05; Fig. 1B). These results suggested that miR-660 is upregulated in OS tissues and cell lines.

To illustrate the biological roles of miR-660 in the development of OS, miR-660 inhibitor was introduced to MG-63 and Saos-2 cells to decrease the endogenous miR-660 level. The transfection efficiency was evaluated through RT-qPCR, and the results demonstrated that miR-660 was markedly downregulated in miR-660 inhibitor-transfected MG-63 and Saos-2 cells (P<0.05; Fig. 2A). The transfected MG-63 and Saos-2 cells were tested to examine their proliferation rate through the CCK-8 assay. The CCK-8 assay revealed that miR-660 downregulation reduced the proliferation of MG-63 and Saos-2 cells compared with that in the NC inhibitor groups (P<0.05; Fig. 2B). Transwell invasion assay was performed to determine the effect of miR-660 inhibition on OS cell invasion. In Fig. 2C, the invasion abilities of the MG-63 and Saos-2 cells transfected with miR-660 inhibitor were significantly lower than those of the cells transfected with NC inhibitor (P<0.05; Fig. 2C). These results suggested that miR-660 may play an oncogenic role in OS progression.

Bioinformatics analysis was performed to predict the potential targets of miR-660 and to explore the mechanisms underlying the oncogenic roles of miR-660 in OS. A putative binding site for miR-660 was observed in the 3′-UTR of FOXO1 (Fig. 3A). FOXO1 was selected for further experimental confirmation because FOXO1 played tumour suppressive roles in the onset and progression of OS (18–20). Luciferase reporter assay was subsequently carried out in the MG-63 and Saos-2 cells to confirm this prediction. miR-660 inhibition led to a significant increase in the luciferase activities of pGL3-FOXO1-Wt-3′-UTR in the MG-63 and Saos-2 cells (P<0.05), but no significant change was occurred in the pGL3-FOXO1-Mut-3′-UTR group (Fig. 3B). RT-qPCR and Western blot analysis confirmed that the mRNA (P<0.05; Fig. 3C) and protein (P<0.05; Fig. 3D) expression levels of FOXO1 were upregulated in the MG-63 and Saos-2 cells transfected with miR-660 inhibitor. These results demonstrated that FOXO1 is a direct target of miR-660 in OS.

Considering that FOXO1 is identified as a direct target of miR-660, we next sought to further examine the relationship between miR-660 and FOXO1 in OS. Firstly, RT-qPCR was conducted to detect the mRNA level of FOXO1 in 26 pairs of OS tissues and NATs. The mRNA expression level of FOXO1 was weakly expressed in the OS tissues in comparison with that in the NATs (Fig. 4A, P<0.05). Secondly, the protein expression of FOXO1 was determined in several pairs of OS tissues and NATs. In Fig. 4B, the FOXO1 protein was downregulated in the OS tissues relative to that in the NATs. An inverse association between miR-660 and FOXO1 mRNA was also validated in the OS tissues (r=−0.5476, P=0.0038; Fig. 4C).

On the basis of our findings described above, we hypothesised that FOXO1 mediated the oncogenic roles of miR-660 in OS cells. A series of rescue experiments was conducted to test this hypothesis. Firstly, we transfected MG-63 and Saos-2 cells with NC siRNA or FOXO1 siRNA and detected the protein level of FOXO1. As shown in Fig. 5A, FOXO1 expression was efficiently knocked down in MG-63 and Saos-2 cells after transfection with FOXO1 siRNA (P<0.05). Next, MG-63 and Saos-2 cells were transfected with miR-660 inhibitor and FOXO1 siRNA or NC siRNA. Western blot analysis indicated that the FOXO1 protein expression was restored by FOXO1 siRNA co-transfection in MG-63 and Saos-2 cells (P<0.05; Fig. 5B and C). Furthermore, CCK-8 and Transwell invasion assays revealed that FOXO1 knockdown could abrogate the effects of miR-660 inhibition on the proliferation (P<0.05; Fig. 5D) and invasion (P<0.05; Fig. 5E and F) of MG-63 and Saos-2 cells. Thus, our data evidently demonstrated that miR-660 probably performs an oncogenic function in OS at least partially by regulating FOXO1 expression.