All procedures involving human participants were performed in accordance to the 1964 Declaration of Helsinki and its later amendments. The animal research protocol was approved by the Animal Experiment Centre Committee of Xi'an Jiaotong University. All animal experimental procedures were performed according to the policy of Xi'an Jiaotong University Animal Experiment Centre.

A total of 20 pairs of primary osteosarcoma and matched adjacent non-cancerous paraffin-embedded specimens were obtained at the authors' institution between 2012 and 2015. The osteosarcoma diagnosis and the confirmation that the adjacent tissues were normal were performed by two independent pathologists based on the pathological changes associated with osteosarcoma and the microscopic morphology of the adjacent normal tissues. The tumour stage was assessed according to the Enneking-Musculoskeletal Tumour Staging System (18). The human osteosarcoma cell line 143B was purchased from the Type Culture Collection of the Chinese Academy of Science, (Wuhan, China) and was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Gibco) and 0.015 mg/ml 5-bromo-2′-deoxyuridine (Sigma-Aldrich, St. Louis, MO, USA) in a humidified incubator at 37°C with 5% CO₂.

miR-504 mimics or inhibitors (RiboBio, Guangzhou, China) were transfected into 143B cells with the Ribo FECT™ CP Transfection kit (all reagents from RiboBio) according to the manufacturer's instructions to achieve the overexpression or knockdown of miR-504, respectively. Using the Ribo FECT™ CP Transfection kit, a negative control mimic (normal control) (RiboBio) and siR-Ribo™ Transfection Control (Cy3; RiboBio) were transfected into 143B cells as the normal and transfection controls, respectively.

The microRNAs were extracted from the clinical samples and cell lines using the miRNAprep Pure FFPE kit (Tiangen Biotech, Beijing, China) or the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The miRcute miRNA First-Strand cDNA Synthesis kit and miRcute miRNA qPCR Detection kit (SYBR Green; Tiangen Biotech) were used for cDNA synthesis and RT-qPCR according to the manufacturer's instructions. The RT-qPCR conditions were 95°C for 5 min followed by 40 cycles of 95°C for 15 sec and 60°C for 20 sec and a final dissociation stage. The PCR was performed using the Roche LightCycler^® 96 fluorescent quantitative PCR instrument. The relative miR-504 expression level was determined using the 2^−ΔCt method with the nuclear U6 RNA as the endogenous control (19). The forward primers were designed as follows: miR-504, 5′-GACCCTGGTCTGCACTCTATCA-3′; and U6, 5′-ACGCAAATTCGTGAAGCGTTC-3′. The reverse primers were provided with the kit.

The potential targets of miR-504 were predicted by miRanda (http://www.microrna.org). The predicted miR-504 binding site at the TP53INP1 3′-UTR and corresponding mutant 3′-UTR sequence were cloned in the psiCHECK™-2 Vector (Promega, Madison, WI, USA). The psiCHECK-2-TP53INP1-WT and psiCHECK-2-TP53INP1-MT were co-transfected with miR-504 NC or miR-504 mimic into 293T cells. The relative luciferase activity was analysed using a Promega Dual-Luciferase system (Promega) after 48 h of transfection.

Four hundred cells were seeded in 60-mm dishes and cultured for nine days. Subsequently, the medium was removed. The colonies were fixed with 4% paraformaldehyde for 10 min, stained with 1% crystal violet for 15 min and then counted.

The cells were transfected and seeded in 96-well plates at a density of 1,000 cells/well. The medium was changed every three days. The cells were harvested each day from the third to the seventh day and the cell viability was detected with Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions.

The transfected cells were stained with 50 µg/ml propidium iodide (Sigma-Aldrich) and 250 µg/ml RNAase (Sigma-Aldrich). After 30 min of incubation in the dark, the cell cycle was analysed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cell apoptosis was analysed using an Annexin V-FITC/PI Apoptosis Detection kit (Wanleibio, Shenyang, China) by flow cytometry according to the manufacturer's instructions.

To determine the cell migration, 3×10⁴ cells were reseeded in the top of a Transwell chamber 48 h after transfection. The cells that remained in the upper chamber after 24 h were carefully removed using a cotton swab. The migrated cells were fixed and stained with 1% crystal violet for 20 min. The procedure for the cell invasion was similar to the migration assay, except for the following two differences: the upper Transwell chamber was precoated with 60 µl of diluted Matrigel (BD Biosciences) and incubated at 37°C overnight with 6×10⁴ transfected cells seeded in the upper chamber.

Seventy-two hours after transfection, the total cellular protein was extracted using RIPA lysis buffer and Phosphatase Inhibitor II Cocktail (50X) (Heart, Xi'an, China) according to the manufacturer's instructions. The total protein was separated and transferred onto polyvinylidene membranes (Merck Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked and incubated with primary antibodies overnight at 4°C. Following incubation with an HRP-conjugated goat anti-rabbit IgG secondary antibody (dilution, 1:5,000; Boster Biological Technology, Wuhan, China) at room temperature for 1 h, the protein signals were detected using the Immobilon™ Western Chemiluminescent HRP Substrate (Merck Millipore). The following primary antibodies were used: rabbit anti-TP53INP1 (dilution, 1:2,000; Abcam, Cambridge, MA, USA), rabbit anti-P53 (phospho S46; dilution, 1:1,000; Abcam), rabbit anti-GAPDH (dilution, 1:1000; Wanleibio), rabbit anti-P53 (dilution, 1:800; Wanleibio), rabbit anti-P73 (dilution, 1:500; Wanleibio), rabbit anti-P21 (dilution, 1:500; Wanleibio), rabbit anti-Bax (dilution, 1:500; Wanleibio), rabbit anti-caspase-3 (dilution, 1:500; Wanleibio), rabbit anti-cleaved-caspase-3 (dilution, 1:300; Wanleibio) and rabbit anti-SPARC (dilution, 1:1000; Sanying Biotechnology, Wuhan, China).

Ten five-week-old nude mice were randomly and equally divided into the miR-504 agomir and the miR-504 NC groups. Subsequently, 3×10⁶ cells were subcutaneously injected in the flanks of the nude mice. The cells had been pre-transfected with agomir-hsa-miR-504 (miR-504 agomir; GenePharma, Shanghai, China) or agomir negative control (miR-504 NC; GenePharma) based on the grouping. One week after the tumour-cell implantation, 1 nmol of miR-504 agomir or 1 nmol of agomir miR-504 NC was injected into the tumours twice a week; the tumour volume was concurrently assessed according to the formula: tumour volume = length × width²/2. All mice were sacrificed four weeks after inoculation and orthotopic tumours and lungs were harvested. After weighing the orthotopic tumours, both tumours and lungs were stained with haematoxylin and eosin (H&E).

The slides containing formalin-fixed, paraffin-embedded, 5-µm thick tissue sections were subjected to microwave antigen retrieval and then incubated with the primary antibody overnight at 4°C. Subsequently, the slides were incubated with an HRP-conjugated secondary antibody for 30 min at room temperature and the proteins were chemically visualised using a DAB kit (ZSGB-Bio, Shanghai, China).

Statistical comparisons of the results were performed using the SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) with Student's t-test, independent-samples t-test or Fisher's exact probability test, depending on the particular data type. P<0.05 was considered to indicate a statistically significant difference.

Expression of miR-504 was higher in 17 osteosarcoma specimens compared to the matched adjacent non-cancerous tissues and the remaining tumour specimens had a relatively lower expression of miR-504 based on RT-qPCR assay (Fig. 1A). As shown in Table I, miR-504 was more highly expressed in patients with distant metastasis, advanced clinical stage and larger tumours. Additionally, TP53INP1-positive specimens had a statistically lower expression of miR-504. There was no significant difference between miR-504 expression and age or sex.

TP53INP1 was detected in nine osteosarcoma specimens (45%) using immunohistochemical staining, which was tan-coloured and located in the nucleus. Seventeen adjacent non-cancerous specimens (85%) exhibited TP53INP1 expression (Fig. 1B). Furthermore, the detectable expression of TP53INP1 was infrequent in patients with advanced clinical stage according to Fisher's exact probability test (Table I).

We searched for potential targets of miR-504 using the miRanda databases and found a potential miR-504-binding site in the 3′UTR of the TP53INP1 mRNA. To validate the direct binding between miR-504 and the predicted seed sequence, the predicted potential binding sequence and paired mutated sequence were inserted into the psiCHECK™-2 vector (Fig. 1C). Luciferase activity was significantly decreased after co-transfection of psiCHECK-2-TP53INP1-WT and miR-504 mimic, whereas miR-504 had no effect on luciferase activity when co-transfected with psiCHECK-2-TP53INP1-MT (Fig. 1D). The feasibility of the transfection was verified using Cy3-labelled normal control siRNA and RT-qPCR in 143B cells (Fig. 1E and F). Furthermore, the results of western blot analysis confirmed that the expression of TP53INP1 was downregulated in the 143B cells after the miR-504 mimic transfection (Fig. 1G).

The CCK-8 assay was performed to investigate the effects of miR-504 on the proliferation of 143B cells. Proliferation was significantly enhanced in 143B cells transfected with miR-504 mimic and decreased with miR-504 inhibition compared with the normal control and blank groups (Fig. 2A). Consistently, the cells transfected with the miR-504 mimic had more colonies in the colony formation assay, whereas fewer colonies survived in the normal control and blank groups. The lowest number of colonies was found in the miR-504 inhibitor group (Fig. 2B).

Based on a cell cycle assay, the G1 subpopulation proportion of cells was decreased in the miR-504 mimic-transfected cells compared with other groups and the proportion of cells in the G2 subpopulation was increased. In contrast, G1 arrest was obvious in the cells transfected with the miR-504 inhibitor, which blocked the G1 to S-phase transition, compared with the blank and normal control groups (Fig. 2C). The transfection of miR-504 mimic mitigated apoptosis in the 143B cells compared with the normal control and blank groups and the cells transfected with the miR-504 inhibitor had the highest proportion of apoptosis (Fig. 2D).

To investigate the effects of miR-504 on migration and invasion in 143B cells, a Transwell assay was performed. Cells in the miR-504 mimic group exhibited a strengthened migration and invasion capacity compared with the normal control and blank groups, while minimal numbers of cells traversed the polycarbonate membrane of the Transwell chamber in the miR-504 inhibitor group for both the migration and invasion assays (Fig. 2E and F).

Finally, we evaluated TP53INP1, phosphorylated P53, P73, P21, Bax, cleaved-caspase-3 and SPARC expression and found that TP53INP1, P73, P21, Bax and cleaved-caspase-3 expression was decreased 72 h after the miR-504 mimic transfection, but upregulated in the miR-504 inhibitor group. SPARC is correlated with pancreatic cancer cell migration and was upregulated in 143B cells after the miR-504 mimic transfection compared with the normal control and blank groups. However, phosphorylated P53 was undetectable in all four groups (Fig. 3).

The tumour growth was faster in the miR-504 agomir group than in the miR-504 NC group (Fig. 4A). The tumours were harvested and weighed at the fourth week and the tumour weights ascertained faster growth in the miR-504 agomir group (1.29 vs. 0.95 g) (Fig. 4B). The microscopical morphology of the tumour was also visualised using H&E staining (Fig. 4C).

All lungs were harvested and stained with H&E to detect distal metastases. Only one mouse developed pulmonary metastasis in the miR-504 NC group and three mice developed pulmonary metastasis in the miR-504 agomir group. Mice in the latter group developed larger metastatic tumours (Fig. 4D).

Although all tumours had detectable TP53INP1 expression, the expression level was lower in the miR-504 agomir-treated tumours (Fig. 4E).