Osteosarcoma cell lines (Saos-2 and U2OS) were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Gibco^® RPMI-1640 (Thermo Fisher scientific, Inc., Beijing, China) supplemented with 10% Gibco^® fetal bovine serum (Thermo Fisher Scientific, Inc.). Tumor tissues and adjacent noncancerous normal tissues were collected from routine therapeutic surgery at the Department of Orthopaedics, Pudong New Area Zhoupu Hospital. A total of 25 samples (male; median age, 57 years; range, 48–65 years) were obtained with informed consent from patients with osteosarcoma, and the procedures were approved by the Institutional Review Board of the hospital. Subjects were excluded if they had other diseases, including biliary obstructive diseases, and acute or chronic virus hepatitis. The individuals with an alcohol consumption of ≥120 g/week for men at the time of the study or in the prior 6 months were also excluded from the study.

miRNA from tissue samples and cell lines was harvested using the Ambion^® mirVana miRNA Isolation kit (Thermo Fisher Scientific, Inc., Grand Island, NY, USA), following the manufacturer’s instructions. All RNA samples were examined as to their concentration and purity. RNA purity was measured using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Based on the absorbance ratio at 260/280 nm (mean±standard deviation = 1.86±0.03), all RNA samples were pure and protein free. Expression of mature miRNAs was assessed with the Applied Biosystems^® TaqMan^® microRNA assay (Thermo Fisher Scientific, Inc.) with probes specific to the human hsa-miR25 (5′-aggcggagacuugggcaauug-3′) and the small nuclear U6 snRNA, used as an internal control (5′-cauugaccauggacauacgacug-3′). The quantitative (q)PCR reaction was performed using a TaqMan Universal PCR Master mix on an Applied Biosystems^® 7900HT Real-Time PCR system (all from Thermo Fisher Scientific, Inc.). Briefly, PCR conditions included an initial holding period at 95°C for 5 sec and 60°C for 30 sec for 45 cycles. Relative quantitation analysis of the gene expression data was conducted according to the 2^−ΔΔCt method.

To construct the miR-25 expression plasmid, the precursor sequence of the human miR-25 was cloned into Ambion^® pSilencer™, while the negative control (NC) plasmid contained a scrambled sequence (both from Thermo Fisher Scientific, Inc.). For transfection, Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) was employed following the manufacturer’s instructions.

To assess cell proliferation, an enzyme-linked immunosorbent assay (ELISA) was used, based on the incorporation of BrdU during DNA synthesis, (BrdU kit; Beyotime Institute of Biotechnology, Shanghai, China), following the manufacturer’s protocols. All experiments were performed in triplicate. The absorbance of the samples at 450 nm (A₄₅₀) was measured on a SpectraMax 190 ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

Cells were harvested and lysed with ice-cold lysis buffer containing 50 mM Tris-HCl, pH 6.8, 100 mM 2-Mercaptoethanol, 2% w/v sodium dodecyl sulfate (SDS) and 10% glycerol. Following centrifugation at 4°C, proteins in the supernatants were quantified by a BCA quantification kit (Beyotime Institute of Biotechnology), separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Anti-p27 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) was used at 1:2,000 overnight at 4°C, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology, Inc.) was used at 1:5,000 overnight at 4°C. The membranes were then incubated with the HRP-linked secondary antibodies (Cell Signaling Technology, Inc.). The signals were detected by SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Inc., Rockford, IL, USA) according to manufacturer’s instructions. The images were visualized by a LAS-4000 Luminescent Image analyzer (Fujifilm, Tokyo, Japan). The p27 protein level was quantified using Quantity One software (Bio-Rad, Hercules, CA, USA) and was normalized to that of GAPDH.

Total cDNA from Saos-2 cells was synthesized from total RNA using random hexamers with the Superscript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The reactions were incubated in a thermal cycler for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and then held at 4°C. The cDNA was used to amplify the 3′ untranslated region (3′ UTR) of p27 by PCR. Mutations were introduced in potential miR-25-binding sites (5′-GCUAUUACGAAUACAUCCGUUAAC-3′ was changed into 5′-GCUAUUACGAAUACAUCCGAAUUC-3′) using the QuickChange^® Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). The pRL-SV40 vector (Promega Corp., Madison, WI, USA) carrying the Renilla luciferase gene was used as an internal control of transfection efficiency. Luciferase values were determined using the Dual-Luciferase^® Reporter Assay system (Promega Corp.).

Male BALB/c nude mice, 4 weeks-old, were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). Saos-2 cells (2×10⁵) were subcutaneously injected into the skin under the legs of the mice. The mice were observed for 5 weeks for tumor formation. Following sacrifice, the tumors were recovered and the wet weights of each tumor were measured.

Data were expressed as the mean ± SEM from at least three independent repetitions of the experiment. Differences between groups were analyzed using Student’s t-tests or one way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.

First, in order to examine whether the miR-25 is differentially expressed in human osteosarcoma compared to noncancerous tissues, its expression level was determined using reverse transcription (RT)-qPCR in 25 pairs of human osteosarcoma tissues and pair-matched adjacent noncancerous tissues. The results demonstrated that the expression level of miR-25 was significantly increased in osteosarcoma tissues compared to the adjacent noncancerous tissues (Fig. 1).

In order to assess the effects of miR-25 on osteosarcoma cell growth, Saos-2 and U2OS cells were transfected with a plasmid bearing the miR-25 precursor or the NC plasmid, and cell growth was subsequently examined. Transfection with the miR-25 precursor-bearing plasmid increased miR-25 expression compared to the NC transfection (Fig. 2A and B), and significantly increased the cell number and proliferation in both cell lines (Fig. 2C–F).

To study the role of miR-25 overexpression in tumorigenesis in vivo, we generated Saos-2 cells stably overexpressing miR-25 and injected the cells into a xenograft mouse model. miR-25 overexpression markedly promoted tumorigenesis in comparison with the NC, as evidenced by tumor weights and sizes (Fig. 3A and B).

Since miR-25 overexpression promoted cell proliferation and tumor growth, we examined the underlying mechanisms. Through a stringent bioinformatics approach (miRWalk software; http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/), we identified the gene p27, encoding a cell-cycle inhibitor, as a candidate target of miR-25, since p27 bears a potential miR-25-binding site (Fig. 4A). The 3′ UTR of p27 was cloned into a reporter luciferase system. When the reporter construct contained the p37 3′ UTR, overexpression of miR-25 led to a reduction in luciferase activity (Fig. 4B). By contrast, mutation of the conserved miR-25-binding motif abrogated the reduced luciferase expression (Fig. 4B). In addition, overexpression of miR-25 in osteosarcoma cells led to a reduction in the protein level of p27 (Fig. 4C and D). In addition, the p27 protein level was reduced in miR-25-overexpressing tumors and human osteosarcoma tissues (Fig. 4E and F), further supporting that p27 may be a target of miR-25 in osteosarcoma cells.

In order to confirm the functional connection between miR-25 and p27, Saos-2 cells were transfected with p27 expression plasmids following transfection with miR-25 (Fig. 5A). As shown in Fig. 5B and C, the re-introduction of p27 reversed miR-25-induced cell proliferation, indicating that the interaction between miR-25 and p27 is involved in this process. Taken together, our results suggest that the gene p27 is an important target of miR-25 in osteosarcoma cells.