The following monoclonal mouse antibodies were used in the present study: anti-phosphatase and tensin homolg (PTEN; A2B1), sc-7074; anti-AKT1/2/3 (BDI222), sc-56878; anti-phospho (p)-AKT (11E6), sc-81433; and anti-β-actin (C4), sc-47778. All of the antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The cell counting kit-8 (CCK-8) was obtained from Beyotime Institute of Biotechnology (Nantong, China). The inhibitor, mimics and non-specific negative control oligonucleotides for miR-214 and specific small interfering (si)RNA against human PTEN (5′-UUU GUC UCU GGU CCU UAC UUC C-3′ and 5′-GGA AGT AAG GAC CAG AGA CAA A-3′) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). All other chemicals and reagents were purchased from Sigma-Aldrich China, Inc. (Shanghai, China), unless otherwise specified.

Saos-2 human OS cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified 5% CO₂ incubator at 37°C. The hFOB1.19 human osteoblastic cell line was also purchased from ATCC and was cultured in Ham’s F12/DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. A total of 15 human primary OS and matched adjacent noncancerous tissues were collected from patients attending the Affiliated Hospital of Harbin Medical University (Harbin, China). Tissue samples were frozen at −80°C until the samples were used for RNA extraction. The human OS tissues were analyzed according to a procedure approved by the Clinical Research Committees of Harbin Medical University. All procedures were approved by the Local Research Ethics Committee of Harbin Medical School (Harbin, China). The study also conformed to the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all patients.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For the detection of mature miR-214, RNA was reverse-transcribed using a specific reverse-transcription primer (Applied Biosystems, Inc., Foster City, CA, USA). The expression levels of miR-214 were quantified by qPCR using TaqMan miRNA assays (Applied Biosystems, Inc.), according to the manufacturer’s instructions, with the Rotor-Gene 6000 system (Corbett Life Science, Qiagen, Hilden, Germany). The PCR assays for human mature miR-214 and human U6 small nuclear (sn)RNA were performed as follows: 95°C for 30 sec, 40 cycles of 95°C for 30 sec, 60°C for 20 sec, and 70°C for 1 sec. The expression of U6 snRNA was used as an internal standard. The following RT primers were used: miR-214-RT, 5′-TCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACACTGCCTG-3′; and U6-RT, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGG ATACGACAAAATATGGAAC-3′. The following PCR primers were used: U6: U6-Fwd, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′; and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′; and miR-214: miR-214-Fwd, 5′-TGCGGACAGCAGGCACAGAC-3′;and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′.

miR-214 precursor sequences were released from full-length constructs with BamHI and EcoRI subcloned into the complementary sites of pcDNA3.1 (Invitrogen). Construction of the pcDNA3.1-miR-214 vector was performed as described previously (15,16). The Saos-2 cells were seeded in six-well plates at 30–50% confluency 24 h before transfection with the miR-214 mimics, or the empty parental vector as a control, using Lipofectamine 2000 reagent (Invitrogen) and stable transfectants were selected by growth in hygromycin.

At 24 h after transfection, the Saos-2 cells were digested with trypsin and replated in 96-well plates. After 6 h, the cells were labeled with Br-dU for 18 h. Subsequently, the level of Br-dU incorporation was determined using a Br-dU cell proliferation ELISA kit (Roche Diagnostics GmbH, Penzberg, Germany). At 24 and 48 h after transfection, the Saos-2 cells were incubated at 37°C in 10% CCK-8 diluted in normal culture medium until visual color conversion occurred in the culture plates. The number of viable cells was assessed by absorbance at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The apoptosis assay was performed on the Saos-2 cells 48 h after transfection with either miR-214 inhibitor or control using an Annexin V-fluorescein isothiocyanate apoptosis detection kit (KeyGen Biotech, Nanjing, China) and the cells were analyzed by fluorescence-activated cell sorting (FACS Scanner; BDi Biosciences, Franklin Lakes, NJ, USA).

For preparation of the cell extracts, the control cells and miR-214-transfected cells were washed thrice with ice-cold phosphate-buffered saline and lysed in lysis buffer, containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and PMSF 100 μg/ml, on ice for 30 min. Following centrifugation at 12,000 × g for 20 min at 4°C, the supernatants were analyzed by 10% SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane (Invitrogen). After 2 h blocking with 5% bovine serum albumin at room temperature, the membranes were incubated with anti-PTEN, anti-AKT, anti-P-AKT and anti-β-actin monoclonal antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G polyclonal secondary antibodies (Pierce, Rockford, IL, USA). Protein bands were visualized with an enhanced chemiluminescence reagent (Pierce).

Nude mice (Vital River Laboratories, Beijing, China), aged four to six weeks, were subcutaneously injected with 1×10⁶ Saos-2 cells stably expressing high levels of miR-214. The mice were manipulated and housed according to procedures approved by the Local Medical Experimental Animal Care Commission. After five weeks, the mice were euthanized by cervical dislocation and tumor growth was examined. The tumor weight was measured using an electronic balance. The tumor volume (V) was examined using a caliper and calculated using the following formula: V = (A × B²)/2; where A is the larger and B the smaller dimension of the tumor.

Two-tailed Student’s t-test was used for statistical analysis. All data are presented as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

To investigate the involvement of miR-214 in OS carcinogenesis, the expression levels of miR-214 in human OS tissues and Saos-2 human OS cells were analyzed by qPCR. The results revealed that the miR-214 expression levels were significantly increased in the human OS tissues compared with adjacent noncancerous tissues (P<0.01; Fig. 1A). Consistent with these observations, the Saos-2 human OS cells expressed significantly higher levels of miR-214 compared with the hFOB1.19 human osteoblastic cell line (P<0.05; Fig. 1B). These data indicate that miR-214 may act as a novel regulator of OS development. Thus, it was decided to further investigate the role and function of miR-214 in OS.

To examine the biological importance of miR-214 in OS tumorigenesis, Saos-2 cells stably expressing miR-214 were established using plasmids. Significantly increased miR-214 expression levels in these cells, compared with control cells, were confirmed by qPCR (P<0.05; Fig. 2A). To further investigate whether overexpression of miR-214 increases the proliferation of Saos-2 cells, the cells were examined by Br-dU incorporation and CCK-8 proliferation assays. The two cell proliferation assays revealed that cell growth was significantly increased in Saos-2 cell lines stably expressing miR-214 compared with the corresponding control (P<0.05; Fig. 2B and C). Similar results were observed in miR-214 mimic-transfected OS cells compared with either miR-control-transfected cells or untreated cells (P<0.05; Fig. 2D). The results of these in vitro assays indicate that exogenous miR-214 significantly promoted the proliferation of OS cells.

It was analyzed whether inhibition of miR-214 induces OS cell apoptosis and cell death using flow cytometric analysis to determine the number of early and late apoptotic Saos-2 cells following miR-214 inhibitor treatment (Fig. 3). As expected, few Annexin V-positive cells were detected in the miR-control-treated or untreated cells, whereas miR-214 inhibition significantly increased the percentage of apoptotic cells, compared with the control cells, as determined by Annexin V staining (P<0.05; Fig. 3). These results indicate an antiapoptotic role of miR-214 in OS cells.

Following the in vitro findings, animal experiments were conducted to further evaluate the effect of miR-214 on tumor growth in a subcutaneous xenotransplantation model with BALB/c athymic nude mice. Two groups of stably transfected cells (an Saos-2/control group, stable Saos-2 cells transfected with empty pcDNA3.1 plasmid; and a Saos-2/miR-214 group, stable Saos-2 cells transfected with pcDNA3.1-miR214 plasmid) were injected subcutaneously into the right flank of the nude mice as described. After five weeks, the mice were sacrificed and the size of the tumor nodules was examined. As expected, the mice injected with stable Saos-2 cells transfected with pcDNA3.1-miR214 exhibited a significant increase in tumor weight and tumor volume compared with the controls from three weeks onwards (P<0.05; Fig. 4A–C). These results further suggest that miR-214 may function as a tumor oncogenic miRNA in OS cells.

To analyze the underlying mechanism by which miR-214 promotes the growth of OS cells, bioinformatic analysis was performed to search for genes regulated by miR-214 using the TargetScan software program (http://www.targetscan.org). Following a preliminary screen, the miR-214 binding site located on the 3′-UTR of PTEN was found to be conserved in different species (Fig. 5A). Furthermore, a previous study reported that miR-214 suppresses PTEN expression in human ovarian tumor cells (9). Thus, PTEN was investigated as a possible target of miR-214; the effect of miR-214 on the endogenous expression levels of PTEN was examined in the Saos-2 cells. As expected, the western blot analysis demonstrated that transfection with miR-214 mimics reduced the PTEN protein expression levels in Saos-2 cells. Furthermore, miR-214 overexpression was found to increase the protein levels of P-AKT (Fig. 5B). These results suggest that PTEN is a target of miR-214 in OS cells.

PTEN has been previously demonstrated to modulate the tumor suppressor response in human ovarian tumors in vivo (9). To examine the effect of PTEN on OS cell proliferation, a specific siRNA against human or human PTEN was transfected into the Saos-2 cells. The siRNA-mediated PTEN downregulation was found to significantly increase Saos-2 cell proliferation, compared with those of the two control cell groups (P<0.05; Fig. 5C). These results suggest that the proliferation-promoting effect of PTEN knockdown was similar to that of miR-214 overexpression. Subsequently, it was determined whether restoration of PTEN reverses the oncogenic effects of miR-214. Transfection of pcDNA3.1-PTEN into Saos-2 cells stably expressing miR-214 efficiently restored PTEN expression levels and reversed the increased cell proliferation effect induced by miR-214 (Fig. 5D).

In conclusion, these results indicate that miR-214 inhibits PTEN translation, resulting in activation of the AKT signaling pathway, thus promoting OS tumor survival and growth.