A total of 68 pairs of fresh and frozen osteosarcoma tumor tissues and the adjacent normal tissues were obtained from patients with histologically verified osteosarcoma who underwent surgical resection between 2004 and 2009 at Changzheng Hospital (Shanghai, China). All specimens were collected after obtaining written informed consent according to a protocol approved by the Ethics Committee of the Second Military Medical University (Shanghai, China). The study was performed according to the principles of the Declaration of Helsinki.

Total RNA was extracted from 25 mg tissue or 5×10⁵ cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). A total of 500 ng total RNA from each sample was reverse transcribed using PowerScript reverse transcriptase (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. qPCR was performed using a FastStart SYBR Green Master kit (Roche Diagnostics GmbH, Mannheim, Germany) on an ABI Prism 7900HT Sequence Detection system (Applied Biosystems, Inc., Foster City, CA, USA). The following gene-specific primers were used for the qPCR: Forward, 5′-CGCCCTTCTACGGCAACTA-3′ and reverse, 5′-CCCTCGCTGGCATCTTTCT-3′ for IRX2; and forward, 5′-TTAGTTGCGTTACACCCTTTC-3′ and reverse, 5′-GCTGTCACCTTCACCGTTC-3′ for β-actin. The mRNA expression levels of IRX2 were analyzed using SDS software, version 2.3 (Applied Biosystems, Inc.) and normalized to those of β-actin mRNA, which was used as an internal control. The relative mRNA levels are presented as ΔCt. Three independent experiments were completed and each reaction was performed in triplicate. All data are presented as the mean ± standard deviation (SD).

The hFOB 1.19 human normal osteoblastic cell line (ATCC, Manassas, VA, USA) was maintained in Dulbecco’s modified Eagle’s medium/F-12 (1:1) with 10% fetal bovine serum (FBS), 2.5 mM L-glutamine (without phenol red) and 0.3 mg/ml G418. The U2OS and SaOS2 human osteosarcoma cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and were cultured in RPMI-1640 medium, which was supplemented with 10% FBS, 1% L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere containing 5% CO₂ at 37°C. All cell lines were authenticated by Cancer Research UK (London, UK) in July 2010 using short tandem repeat profiling.

Specific IRX2 short hairpin RNA (shRNA) was designed to silence the expression of IRX2 (sense, 5′-CCGGCTACACAAACTACGGGAACTTCTCGAGAAGTTCCCGTAGTTTGTGTAGTTTTTG-3′ and antisense, 5′-AATTCAAAAACTACACAAACTACGGGAACTTCTC GAGAAGTTCCCGTAGTTTGTGTAG-3′) and was cloned into the EcoRI and AgeI sites of the pLKO.1-TRC cloning vector plasmid (Addgene, Cambridge, MA, USA). The firefly luciferase target sequence CTTACGCTGAGTACTTCGA replaced the IRX2 target sequence as a control. The construction of the IRX2-shRNA was confirmed by DNA sequencing.

Lentiviral particles containing pLKO.1-anti-LUC shRNA (SCR) or pLKO.1-anti-IRX2 shRNA (shIRX2) were produced using FuGENE HD Transfection reagent (Roche Diagnostics GmbH) according to the manufacturer’s instructions. For infection, the U2OS and SaOS2 cells were grown in 25-cm² flasks and transduced at 40–50% confluence with the lentiviral particles, in the presence of polybrene (8 μg/ml). After infection (24 h), the stable cells were selected in puromycin (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 5 μg/ml for 48 h.

Cells in the logarithmic growth phase (3×10³ cells/well) were transferred to each well of a 96-well plate. The cell proliferation was evaluated by cell counting kit-8 reagent (Dojindo, Kumamoto, Japan), at the time points of 0, 3, 6, 9 and 12 days after seeding and the cells were incubated in the reagent at 37°C for 2 h. The absorbance was read at 450 nm with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). This assay was performed three times in triplicate.

A total of 500 cells from each group were suspended in 2 ml culture medium and seeded in six-well plates for 14 days. The colonies were fixed and stained with 1% crystal violet for 20 min and then washed three times. The number of colonies with >50 cells in each well was counted as one colony. The experiment was repeated three times in triplicate.

Transwell systems were coated with Matrigel (diluted 1:4) on the upper surface of the polycarbonic membrane (Costar; 8.0-μm pore size; Corning Incorporated, Corning, NY, USA). Cells from each group were resuspended in serum-free RPMI-1640 media at a concentration of 3×10⁵ cells/ml. An aliquot of 100 μl cell suspension was added to the upper chamber and 600 μl containing 10% FBS and RPMI-1640 was added to the lower chamber. Following incubation at 37°C in a 5% CO₂ incubator for 24 h, the cells that had penetrated through the membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The invaded cells were washed and the upper surface of the membranes was wiped with a cotton-tipped applicator to remove the non-migratory cells. The invaded cells were counted in five non-overlapping fields and photographed, and the average numbers of cells of at least five fields from each well were assayed. Three independent assays were performed.

Cells from each group were lysed in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing Complete Protease Inhibitor Cocktail tablets (one tablet=10 ml cocktail; Roche Diagnostics GmbH). The protein expression levels of the cells were quantified using a bicinchoninic acid assay (Sangon, Shanghai, China). Equal quantities of protein were separated on SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA), which was blocked using 6% not-fat milk in Tris-buffered saline with 1% Tween-20. The membrane was incubated overnight at 4°C with primary antibodies (anti-IRX2 antibody, catalog code: ab72975; anti-MMP2 antibody, catalog code: 10373-2-AP; anti-AKT antibody, catalog code: #9272; anti-p-AKT antibody, catalog code: #9271; anti-MMP9 antibody, catalog code: #3852; and anti-actin antibody, catalog code: CW0097). Following incubation, the membrane was washed and incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000; Kangcheng, Shanghai, China). The immunoblot images were all captured with an ImageQuant™ LAS-4000 (Fujifilm) and the chemiluminescence was directly analyzed and quantified with ImageQuant TL software (GE Healthcare, Little Chalfont, UK).

Data are presented as the mean ± SD Statistical analyses were performed using two-tailed Student’s t-test. Kaplan-Meier survival functions and log-rank test were used to assess the disease-free survival and overall survival times based on the mean expression levels of IRX2. All statistical analyses were two-sided and performed with SPSS software, version 15 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the functions of IRX2 in human osteosarcoma development, the expression levels of IRX2 in the hFOB 1.19 human normal osteoblastic cell line were initially analyzed in comparison with those in the U2OS and SaOS2 human osteosarcoma cell lines by western blot analysis and qPCR. The protein and mRNA expression levels of IRX2 were significantly increased in the U2OS and SaOS2 osteosarcoma cells compared with those in the hFOB 1.19 normal osteoblastic cells (Fig. 1A and B).

Furthermore, the expression levels of IRX2 in the 68 pairs of human primary osteosarcoma tumor and adjacent normal tissue samples were assayed. As expected, the IRX2 expression levels were significantly increased in the tumor tissues compared with those in the paired adjacent non-cancerous tissues (Fig. 1C). These results indicated that the expression levels of IRX2 are increased in osteosarcoma cells and tissues, and IRX2 may be involved in human osteosarcoma development.

Subsequently, it was determined whether the upregulation of IRX2 expression levels was correlated with the metastasis of osteosarcoma. The analysis of the mRNA expression levels of IRX2 identified that they were markedly increased in the metastatic tissues compared with those in the non-metastatic tissues (Fig. 2A). Furthermore, Kaplan-Meier analysis using the log-rank test was performed and the results revealed that the patients with low IRX2 expression levels in their osteosarcoma tumors had a longer median survival time of 58.0 months, compared with those with high IRX2 expression levels whose median survival time was 37.0 months (Fig. 2B). These findings suggested that the IRX2 expression levels increased with the aggressiveness of the tumors.

In order to study whether altered expression levels of IRX2 affect the functions of cells, a lentivirus-based RNAi delivery system with shRNAs against IRX2 was used in U2OS and SaOS2 cells (with high expression levels of endogenous IRX2) that are highly aggressive and exhibit a high metastatic potential. Efficient inhibition of IRX2 expression was confirmed by qPCR (Fig. 3A) and western blot analyses (Fig. 3B). In order to determine the effect of IRX2 on cell proliferation, an MTT assay was performed. The results showed that knockdown of the IRX2 expression levels had a significant inhibitory effect on the viability of the U2OS and SaOS2 cells compared with that of the control cells (Fig. 3C). Subsequently, the action of IRX2 on cell invasion was analyzed. The results demonstrated that IRX2 knockdown significantly reduced the invasiveness of the U2OS and SaOS2 cells compared with that of the control group (Fig. 3D). These results showed that silencing IRX2 expression suppressed osteosarcoma cell proliferation, invasion and migration, suggesting that IRX2 is involved in bone tumor cell growth and invasiveness.

Further experiments were conducted to reveal the underlying molecular mechanisms of the effect of IRX2 on osteosarcoma. The PI3K/Akt signaling pathway controls several cellular response functions, including proliferation, invasion, survival and metabolism. To explore whether PI3K/Akt signaling mediates IRX2-induced cell proliferation and invasion, this study investigated whether silencing IRX2 may reduce the expression levels of p-AKT. As shown in Fig. 4, the expression levels of p-AKT were markedly reduced following the knockdown of the expression levels of IRX2. Previous studies have shown that the upregulation of the expression levels of matrix metalloproteinases (MMPs) is a key step for promoting cell invasion (16). In the present study, the expression levels of MMP2 and MMP9 were determined by western blot analysis, and the results showed that the expression levels of MMP9 were markedly suppressed and that the expression levels of MMP2 were not clearly altered (Fig. 4). These data showed that the depletion of IRX2 in the U2OS and SaOS2 cell lines resulted in reduced levels of AKT activation and MMP9 protein.