Human osteoblast cell line hFOB 1.19, and OS cell lines U2OS, Saos-2, MG-63 and MNNG/HOS were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human osteosarcoma cell lines MNNG/HOS, U2OS, Saos-2 and MG-63 were cultured in minimal essential medium, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 and DMEM, respectively, supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin and 100 IU/ml penicillin, at 37°C with 5% CO₂. The human osteoblast hFOB 1.19 cell line was maintained in DMEM/F12 medium supplemented with 10% FBS, 100 mg/ml streptomycin and 100 IU/ml penicillin, at 37°C with 5% CO₂. The cells were serum-starved for 12 h prior to each experiment.

The present study included 46 patients with OS and 46 matched normal tumor-free patients treated in the Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) between March 2008 and March 2011. The patient cohort consisted of 27 males and 19 females, with an age at diagnosis that ranged from 9 to 48 years and a mean age of 19.6 years. Inclusion criteria were a diagnosis of osteosarcoma based on the results of imaging studies and biopsies, while exclusion criteria included the presence of any other type of neoplasm, liver and kidney dysfunction, and a post-operative pathological diagnosis of osteosarcoma. Peripheral blood samples (15 ml) were obtained from the OS patients and matched normal tumor-free patients. The samples were centrifuged at 978 × g for 10 min at room temperature, serum was extracted and stored immediately in liquid nitrogen until use.

OS and matched normal non-tumor tissues (n=46) were collected from the patients with OS. All the tissues were resected at the time of surgery (including amputation and limb-sparing surgery) and immediately stored in liquid nitrogen until use. The present study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Total miRNA was obtained from all cultured cells, serum samples and tissues using RNeasy Mini and miRNeasy Mini kits (Qiagen, Inc., Valencia, CA, USA). miR-17 expression was quantified by performing RT-qPCR using TaqMan MicroRNA Assay kits (catalog no. 217004; Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) on a LightCycler 480 System II (Roche Diagnostics, Rotkreuz, Switzerland). The primer sequences were as follows: Forward, 5′-CCAGGACCAGAGGAAACCT-3′ and reverse, 5′-GCTAGCCTCTGGATTTGA-3′ for PTEN; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; miR-17 forward, 5′-TGCTTACAGTGCAGGTAG-3′ and reverse, 5′-GAACATGTCTGCGTATCTC-3′; and forward, 5′-ATGTCGTGGAGTCTACTGGC-3′ and reverse, 5′-TGACCTTGCCCACAGCCTTG-3′ for GAPDH. The PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, 95°C for 30 sec and 60°C for 30 sec, for 40 cycles. PTEN expression was detected using SYBR Premix Ex Taq II (Takara Bio, Inc., Otsu, Japan). The RT-qPCR data was evaluated using the 2⁻ΔΔ^Ct method (26) relative to GAPDH for mRNA or U6 small nuclear RNA for miRNA. Each PCR experiment was conducted in triplicate.

Data are presented as mean ± standard deviation and were analyzed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). The correlation between miR-17/PTEN expression levels and prognosis was analyzed by Kaplan-Meier survival analysis using GraphPad Prism 6 software (GraphPad Software, Inc., San Diego, CA, USA). Analysis of variance (followed by Gabriel's procedure or a two-tailed Student's t-test were used to examine the data between >2 or 2 variables, respectively, and Spearman's correlation analysis was performed to evaluate the association between miR-17 and PTEN expression levels. P<0.05 was used to indicate a statistically significant difference.

To investigate the potential significance of miR-17 in OS development and progression, the present study evaluated the expression of miR-17 in a human osteoblast cell line, and OS cell lines, serum samples and tissues using RT-qPCR. miR-17 expression levels were significantly upregulated in all four OS cell lines, U2OS, Saos-2 (P<0.05), MG-63 and MNNG/HOS (P<0.01), compared with the hFOB 1.19 human osteoblast cell line (Fig. 1A). The expression of miR-17 was also significantly increased in serum samples of patients with OS compared with normal controls subjects (P<0.05; Fig. 1B). Furthermore, the expression of miR-17 was significantly increased in OS tissues compared with matched normal tissues (P<0.05; Fig. 1C).

Previous studies demonstrated that PTEN is a target gene of miR-17 and its expression appears to be inversely correlated with miR-17 expression levels in OS tissues (27,28). In the present study, PTEN expression was significantly decreased in human OS tissues compared with in matched normal tissues (P<0.01), indicating that downregulation of PTEN may have a significant role in OS development and progression (Fig. 2A). To evaluate the association between circulating miR-17 and PTEN expression levels, Spearman's correlation analysis was performed, demonstrating that serum miR-17 expression is strongly inversely correlated with PTEN expression in OS tissue (P=0.0002; Fig. 2B).

To further determine the potential roles of miR-17 in OS development and progression, the present study analyzed the correlations between miR-17 expression in circulation and PTEN expression in OS tissues, and clinical features of patients with OS. The expression levels of miR-17 in serum and PTEN in OS tissues were analyzed in the cohort of 46 patients with OS, and divided into the following three groups: Low expression (score 1), moderate expression (score 2) and high expression (score 3). The results revealed that high expression of miR-17 in circulation was strongly associated with poor prognosis in patients with OS, while overexpression of PTEN in OS tissues was significantly correlated with good prognosis in patients with OS (P<0.05; Table I). As indicated in Table I, the expression levels of miR-17 were correlated with incidence at an early age (P>0.05).

Furthermore, Kaplan-Meier analysis demonstrated that the prognosis of patients with OS is significantly worse in those with high serum miR-17 expression compared with those exhibiting moderate and low miR-17 expression (P=0.0037; Fig. 3A). By contrast, patients with high PTEN tissue expression exhibited a significantly better prognosis than those with moderate and low PTEN expression (P=0.0187; Fig. 3B). Furthermore, patients were divided into two outcome groups: Survival group (survived for >5 years) and non-survival group (survived for <5 years). It was observed that patients in the survival group (24 patients, 52.2%) exhibited significantly lower miR-17 expression scores compared with patients in the non-survival group (22 patients, 47.8%; P=0.0026). By contrast, patients in the survival group had higher PTEN expression scores than patients in the non-survival group (P=0.0002). The mean miR-17 and PTEN expression score ranges in two groups were 2.2–2.6 (Fig. 3C) and 1.9–1.6 (Fig. 3D), respectively.