OS specimens (n=22) were obtained from eligible patients treated at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) between June 2014 and May 2016. Tumor and matched adjacent non-cancerous tissue samples were collected and characterized based on histopathological examination. All tissues are snap frozen in liquid nitrogen immediately following surgery and stored at −80°C until use. Prior to the use of these clinical materials for research purposes, written informed consent was obtained from all patients, and the present study was approved by the Institute Research Medical Ethics Committee of Zhengzhou University.

Total RNA from tissue samples and cultured cells was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. cDNA was synthesized using the RevertAid First Strand cDNA kit (Thermo Fisher Scientific, Inc.). RT-qPCR was performed using SYBR-Green Premix Ex Taq (Takara Bio, Inc.) to quantify miR-192 and MMP-11 expression by ABI 7500 fast apparatus, thermocycling conditions: 95°C, 5 min; 95°C, 5 min; 40 cycles, 95°C, 15 sec; 60°C, 30 sec. U6 small nuclear RNA and GADPH were used as endogenous controls. The premier sequences was has-miR-192: Forward 5′-GCGGCGGCTGACCTATGAATTG-3′; and reverse: 5′-ATCCAGTGCAGGGTCCGAGG-3′; the primer sequences of U6 were, forward: 5′-TCCGATCGTGAAGCGTTC-3′; and reverse: 5′-GTGCAGGGTCCGAGGT-3′; the primer sequences of GADPH forward: 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse: 5′-ATCCAGTGCAGGGTCCGAGG-3′; the premier sequences of MMP-11 were forward: 5′-CCTAAAGGTATGGAGCGATGT-3′ and reverse: 5′-CGATAGTCCAGGTCTCATCAT-3′. The RT-qPCR results were compared using the 2^−ΔΔCq method (23). The experiments were repeated in three times.

The human OS cell line MG63 was obtained from the Chinese Academy of Medical Sciences (Beijing, China). MG63 cells were maintained in RPMI-1640 medium that was supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO₂ humidified atmosphere. Human osteoblast hFOB1.19 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO₂ humidified atmosphere.

MG-63 cells were seeded into each well of a 6-well plate (2×10⁵ cells/well) and then transfected with either miR-192 agomir or negative control (NC), or miR-192 antagomir or NC (Shanghai GenePharma Co., Ltd., Shanghai, China) (50 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.,) when the cell confluence was ~90%. The cells were harvested for further experiments 24–48 h following transfection.

Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The transfected MG63 cells (5×10³ cells/well) were seeded into 96-well plates in a final volume of 100 µl and cultured for 24, 48, 72, 96 and 120 h. The CCK-8 solution (10 µl) was added into each well, incubated for 4 h, and the optical density at 450 nm was measured to calculate the number of viable cells. A total of five parallel wells were analyzed for each group. The apoptosis assay was conducted using caspase-3 analysis. The cells from each group were harvested 36-h post-transfection by trypsinization. The cells were then seeded into 96-well plates at a total density of 2×10⁶ cells/well in a mixed Ac-DEVD-PNA buffer and incubated at 37°C overnight. On the following day, absorbance values were measured at 415 nm.

The cells from each group were collected 36-h post-transfection and resuspended in medium at 2×10⁵ cells/ml. The upper chamber of a 24-well Transwell Permeable Support device (Corning, Corning, NY, USA) with 8-µm pores was loaded with 200 µl cell suspension, and the lower chamber was supplied with 500 µl complete medium (RPMI-1640) containing 10% FBS. The loaded device was incubated at 37°C in a humidified incubator with 5% CO₂ for 36 h. After incubation, the medium was removed from the upper chamber and the cells in this chamber were scraped off with a cotton swab. The cells that had migrated to the other side of the membrane were fixed with methanol. The cells were then stained with hematoxylin, mounted and dried at 80°C for 30 min. The number of invaded cells in three randomly selected fields was counted using an inverted microscope at a magnification of ×400. The experiments were performed in triplicate.

The cells from each group were collected 24-h post-transfection, seeded in 6-well plates (2×10³ cells/well) and allowed to adhere for a further 24 h. An artificial wound was made using a 10-µl pipette tip across the cell monolayer. The cells were then rinsed with phosphate-buffered saline (PBS) three times to clear cell debris and cells remaining in suspension, and then cultured in serum-free RPMI-1640 medium. Micrographs (taken using an inverted microscope) of the same position relative to the wound were taken at 0 and 36 h. Each assay was performed in triplicate.

Bioinformatic analysis was conducted using TargetScan (http://www.targetscan.org/vert_71/) and miRanda (http://34.236.212.39/microrna/home.do) to predict the target gene of miR-192 and to verify MMP-11 3′UTR containing the seed region of miR-192.

Regions of the human MMP-11 gene containing putative miR-192 binding sites were amplified from human genomic DNA by RT-PCR, performed as previously mentioned. These fragments were cloned into the pmirGLO vector (Promega Corporation, Madison, WI, USA) downstream of the luciferase gene to generate the recombinant vectors pGL3-MMP-11-wt and pGL3-MMP-11-mut. For luciferase reporter assays, a Dual-Luciferase Reporter Assay system (Promega Corporation) was used. MG63 cells were cultured in 96-well plates and co-transfected using 0.8 pmol Pmir-Glo with 50 nM miRNA and wild-type or mutant reporter vectors using Lipofectamine^® 2000. Luciferase values were determined using the Dual-Luciferase Reporter Assay system (Promega Corporation) 24 h post-transfection.

RIPA buffer was prepared for protein isolation, and the cell lysis solution was centrifuged at 13,000 × g for 5 min at 4°C, and then the supernatants were collected. Protein concentration was determined using the BCA assay. Proteins were loaded for SDS-PAGE on 10% polyacrylamide gels followed by transfer to polyvinylidene difluoride membranes. The membrane was blocked for 0.5 h at room temperature in 5% skimmed milk, followed by incubation with primary antibodies (1:300; Ruiqi Biotechnology, Shanghai, China) at 4°C overnight. After washing three times with TBST (5 min each), the membranes were incubated with a 1:2,500 dilution of goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) secondary antibody for 1 h at room temperature and washed again. Immunoblotting was performed using a chemiluminescence detection kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The signals from target proteins were normalized to those of GAPDH. The software used for densitometric analysis was Image J (version 1.8.0_112; National Institutes of Health, Bethesda, MD, USA).

All analyses were performed using SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. The differences were evaluated using a paired Student's t-test or one-way analysis of variance as appropriate. A value of P<0.05 was used to indicate a statistically significant difference.

The levels of miR-192 and MMP-11 expression were detected by RT-qPCR in human OS and matched adjacent non-tumor tissues as well as a normal human osteoblastic cell line (hFOB1.19) and an OS cell line (MG63) in order to investigate the role of miR-192 in the development of human OS. These results showed that miR-192 expression was significantly downregulated in OS tissues compared with matched adjacent non-tumor tissues (P<0.05; Fig. 1A). By contrast, the levels of MMP-11 mRNA were higher in OS tissues compared with matched adjacent non-tumor tissue (P<0.05; Fig. 1A). Similarly, the level of miR-192 expression in MG63 cells was lower compared with hFOB1.19 cells. Conversely, MMP-11 mRNA was upregulated in MG63 cells compared with hFOB1.19 cells (P<0.05; Fig. 1B). Furthermore, western blotting showed that MMP-11 protein levels were also markedly upregulated in OS tissues compared with matched adjacent non-tumor tissues, and in MG63 cells compared with hFOB1.19 cells (Fig. 1C). These data revealed that there is an inverse correlation between the expression of miR192 and MMP-11 in OS tissues and matched adjacent non-tumor tissues (Fig. 1D).

To investigate the functional roles of miR-192, miR-192 agomir or NC constructs were transfected into MG63 cells and miR-192 expression was detected after 36 h. Subsequently, CCK-8 and caspase-3 assays were conducted to evaluate the changes in proliferation and apoptosis, respectively. RT-qPCR analysis revealed that miR-192 expression was increased by transfection of miR-192 agomir compared with the control group (P<0.01; Fig. 2A). The CCK-8 assay showed that transfection of miR-192 agomir was able to significantly suppress the proliferation of MG63 cells at five consecutive time points compared with the NC and control groups (Fig. 2B). Moreover, the caspase-3 assay indicated that the upregulation of miR-192 in MG-63 cells was able to promote apoptosis compared with the NC and control groups (P<0.05; Fig. 2C).

Next, the NC or miR-192 antagomir was transfected into MG63 cells. Following incubation for 36 h, miR-192 expression, cell proliferation and apoptosis were detected. miR192 expression was sharply decreased by miR-192 antagomir transfection compared with the control (P<0.01; Fig. 3A). The miR-192 antagomir was also able to markedly promote proliferation (Fig. 3B) and significantly block apoptosis in MG63 cells (P<0.05; Fig. 3C). These data indicated that miR-192 had a strong effect on the proliferation and apoptosis of OS cells.

Next, the effects of miR-192 on the migration and invasion of MG63 cells were investigated. In wound healing assays, the upregulation of miR-192 was able to suppress the migration of MG63 cells to a significant extent, as observed by the degree of wound closure after 36 h under a microscope (Fig. 4A). Similarly, Transwell assays indicated that the overexpression of miR-192 led to a significantly decreased invasive ability of MG63 cells compared with the NC and control groups (Fig. 4B). Then, miR-192 expression was decreased by transfection of miR-192 antagomir, and wound healing and Transwell assays were performed. After 36 h, the downregulation of miR-192 was able to promote the migration and invasion of MG63 cells, which was on the contrary to the miR-192 overexpression results (Fig. 4A and B). Moreover, statistical analyses demonstrated that the overexpression of miR-192 was able to suppress the migration and invasion of MG63 cells. By contrast, the inhibition of miR-192 enhanced migration and invasion (P<0.05; Fig. 4C and D).

To gain further insight into the molecular mechanism through which miR-192 modulates the behavior of OS cells, target genes of miR-192 were searched using two commonly used target prediction programs, TargetScan (http://www.targetscan.org/vert_71/) and miRanda (http://34.236.212.39/microrna/home.do). The results showed that MMP-11 was a potential miR-192 target gene (Fig. 5A). To confirm that MMP-11 is a direct miR-192 target, dual-luciferase reporter plasmids containing the 3′-UTR of MMP-11 with either a wild-type or mutant putative miR-192 binding site were constructed. Following the overexpression of miR-192 in MG63 cells, the relative luciferase activity of the construct containing the wild-type 3′-UTR of MMP-11 was significantly downregulated compared with the mutant MMP-11 3′-UTR (Fig. 5B). In subsequent western blot assays, MMP-11 protein levels were decreased in MG63 cells when miR-192 expression was upregulated via transfection of agomir (Fig. 5C-E). These results suggested that miR-192 suppressed MMP-11 expression by directly targeting its 3′-UTR.