A total of 60 samples of OS tissues and adjacent non-tumor tissues were obtained from patients who underwent surgery at Tianjin Hospital between February 2011 and January 2015. Two pathologists independently evaluated the histological diagnosis and differentiation of the tissue samples, according to the World Health Organization classification system (5). All of the tissue samples collected were immediately snap-frozen in liquid nitrogen and stored at −80°C until required. The present study was approved by the Research Ethics Committee of Tianjin Hospital (Tianjin, China), and informed consent was obtained from all patients. The clinicopathological characteristics of the patients are provided in Table I; a low expression of HOTAIR was considered to be <1.2 times the normal level and a high expression of HOTAIR was considered to be >1.2 times the normal level.

The OS cell line MG-63 was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified environment at 37°C with 5% CO₂.

Specific small interfering RNA (siRNA/siR) targeting HOTAIR mRNA (siR-HOTAIR1, 3′-GAACGGGAGUACAGAGAGAUU-5′; siR-HOTAIR2, 3′-CCACAUGAACGCCCAGAGAUU-5′) and negative control with scrambled sequence (siR-NC) were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China), and transfected using Invitrogen Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 3×10⁵ MG-63 cells were seeded into 6-well plates and divided into four groups as follows: Mock group, cells without transfection; siR-NC group, cells transfected with the negative control siRNA; siR-HOTAIR1 group, cells transfected with siR-HOTAIR1; and siR-HOTAIR2 group, cells transfected with siR-HOTAIR2. After 48 h the effects of gene silencing were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. HOTAIR was silenced in order to explore the association between the expression of HOTAIR and the behavior of MG-63 cells.

The proliferation of MG-63 cells was measured using a Cell Counting Kit (CCK)-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol, after siRNA transfection for 0,12, 24 and 48 h. A total of 3×10³ MG-63 cells were seeded in 96-well plates and 10 µl CCK-8 was added and the samples were incubated for 4 h at 37°C. Subsequently, the OD value was measured at 450 nm using a Bio-Rad 3550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

MG-63 cells (3×10³) were seeded in 6-well plates. After digestion and centrifugation, the plates were fixed in 70% ethanol at 4°C overnight and then washed three times in PBS. A total of 50 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added and the plates were incubated for 30 min at room temperature in the dark. Measurement of the cell cycle was then performed using a BD FACSCalibur^™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analysis was performed using ModFit LT software (Verity Software House, Topsham, ME, USA).

Cell apoptosis was measured using an Annexin V-FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. Briefly, cells (3×10⁵) were cultured in 6-well plates, trypsinized 48 h after transfection and then washed with cold PBS. Cells were then stained by adding the Annexin V reaction mixture consisting of 10 µl Annexin V and 5 µl PI, and were incubated at room temperature for 15 min in the dark. The stained cells were measured using a BD FACSCalibur according to the manufacturer's protocol and analysis was performed using FlowJo 6 software (FlowJo, LLC, Ashland, OR USA).

Total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) from MG-63 OS cells and tissue samples. RNA Lysis Buffer (RLA) was provide by Promega Corporation (Madison, WI, USA). Complementary (c)DNA was synthesized using a cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) in accordance with the manufacturer's protocol. Then, a SYBR^® Premix Ex Taq^™ II kit (Takara Biotechnology Co., Ltd.) was used following the manufacturer's protocol to amplify the cDNA. The PCR condition s were as follows: Denaturation at 94°C for 3 min, followed by 30 cycles at 94°C for 30 sec, 55°C for 30 sec and a final elongation at 72°C for 60 sec, then 72°C for 10 min. The expression of GAPDH was used as a reference to normalize the amount of lncRNA or mRNA in each sample. The results were quantified using the 2^−ΔΔCq method (17). The PCR primers used were as follows: HOTAIR forward, 5′-GGTAGAAAAAGCAACCACGAAGC-3′ and reverse, 5′-ACATAAACCTCTGTCTGTGAGTGCC-3′; G₁/S-specific cyclin D1 (cyclin D1) forward, 5′-TCTCCAAAATGCCAGAGGCGAGGAAGTTGTTGGGGCTCCT; cyclin E forward, 5′-GTGGCTCCGACCTTTCAGTC-3′ and reverse, 5′-CACAGTCTTGTCAATCTTGGCA-3′; cyclin-dependent kinase (CDK)-4 forward, 5′-TGCCAATTGCATCGTTCACCGAG-3′ and reverse, 5′-TGCCCAACTGGTCGGCTTCA-3′; CDK2 forward, 5′-GCCTAATCTCACCCTCTCC-3′ and reverse, 5′-CCCTTTCACCCCTGTATTCC-3′; p21 forward, 5′-GTCCTGGATGTTGACTGCCTTGA-3′ and reverse, 5′-GTCCAGCAAATCCAAGCTGTCTC-3′; p27 forward, 5′-CATTAACCCACCGGAGCTGTTTAC-3′ and reverse, 5′-GGTTAGCGGAGCAGTGTCCA-3′; matrix metalloproteinase (MMP)2 forward, 5′-GTGGATGATGCCTTTGCTC-3′ and reverse, 5′-CAGGAGTCCGTCCTTACC-3′; MMP9 forward, 5′-AATCTCTTCTAGAGACTGGGAAGGAG-3′ and reverse, 5′-AGCTGATTGACTAAAGTAGCTGGA-3′; epithelial (E)-cadherin forward, 5′-CGGGAATGCAGTTGAGGATC-3′ and reverse, 5′-AGGATGGTGTAAGCGATGGC-3′; neural (N)-cadherin forward, 5′-ACAGTGGCCACCTACAAAGG-3′ and reverse, 5′-CCGAGATGGGGTTGATAATG-3′; and CD44 forward, 5′-TCCCAGACGAAGACAGTCCCTGGAT-3′ and reverse, 5′-CACTGGGGTGGAATGTGTCTTGGTC-3′. GAPDH was the control for the PCR assay, with a forward primer sequence of 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and reverse primer sequence of 5′-CATGTGGGCCATGAGGTCCACCAC-3′.

MG-63 cells were transfected with siR-HOTAIR1 or siR-NC for 48 h, and the proteins were extracted using a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). The Bradford assay was used to determine the protein concentration. A total of 50 µg proteins were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk at 4°C overnight and then incubated at 4°C overnight with rabbit or mouse antibodies specific for the following human proteins: CD44 (1:1,500, sc-65265), CDK4 (1:1,500, sc-23896), MMP2 (1:1,500, sc-13594) and MMP9 (1:1,500, sc-21733) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), cyclin D1 (1:1,500, #2926), cyclin E (1:1,500, #2925), p27 (1:1,500, #3686) (all from Cell Signaling Technology, Inc., Danvers, MA, USA). Antibodies against phosphoinositide 3-kinase (PI3K, 1:1,500, #4255), RAC α serine/threonine-specific protein kinase (AKT, 1:1,500, #4060), mammalian target of rapamycin (mTOR, 1:1,500, #2983), phosphorylated (p)-PI3K (tyr458, 1:1,000, #4228S), p-AKT (ser473, 1:1,000, #4060) and p-mTOR (ser2448, 1:1,000, #5536) were purchased from Cell Signaling Technology, Inc. Mouse anti-human GAPDH antibodies (1:3,000, G8795), which were used as a control, were purchased from Sigma-Aldrich (Merck KGaA). The next day the membranes were probed with secondary anti-rabbit IgG HRP-linked antibody (1:5,000; #7074) or anti-mouse IgG HRP-linked antibody (1:5,000; #4408) (both from Cell Signaling Technology, Inc.). After washing three times in TBS-Tween 20, the membranes were incubated with the corresponding secondary antibodies for 1 h and then detected by an enhanced Pierce chemiluminescence system (ECL; Thermo Fisher Scientific, Inc.).

Cell migration and invasion were assayed using a Transwell invasion assay (invasion Transwell chambers; EMD Millipore, Billerica, MA, USA) with and without Matrigel^™ (BD Biosciences). The cells were seeded at a density of 1×10⁵ cells on the upper chamber with 200 µl serum-free DMEM. Following transfection for 48 h, 600 µl DMEM supplemented with 20% FBS, which served as a chemoattractant, was added to the lower chamber. After 48 h incubation at 37°C, the upper side of the membrane was wiped with cotton wool to remove noninvasive cells; the membranes were then fixed with 4% methanol at 4°C and stained with 0.1% crystal violet at room temperature. Five visual fields with a magnification ×200 were randomly selected from each membrane and the cell numbers were counted using a light microscope.

All data are expressed as the mean ± standard deviation (n=3). The significance of differences between two groups was estimated using a Student's t-test. The significance of differences between multiple groups was determines using one-way analysis of variance followed by a post hoc Tukey's range test. SPSS software (version 21.0; IBM Corp., Armonk, NY, USA) was used to perform the statistical analysis. All tests performed were two sided. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis determined that the expression of HOTAIR was significantly increased in clinical OS specimens compared with adjacent normal tissues (P<0.001; Fig. 1). Clinicopathological analysis demonstrated that when the 60 tissue pairs were tested, the expression of HOTAIR demonstrated no difference between age, gender or tumor size; however, it was significantly associated with tumor grade (P=0.035) and distant metastasis (P=0.007) (Table I). These results indicate that HOTAIR serves an oncogenic role in OS.

To determine whether HOTAIR affects tumorigenesis, MG-63 cell lines were transfected with siR-HOTAIR1/2 or siR-NC. RT-qPCR results revealed that, after being transfected with siR-HOTAIR1/2, HOTAIR mRNA expression in the MG-63 cells decreased significantly when compared to the mock group (P<0.01; Fig. 2A). A CCK-8 assay demonstrated that when HOTAIR was knocked down, the survival rate of MG-63 cells was significantly inhibited (P<0.05; Fig. 2B). This data supports the hypothesis that HOTAIR serves a role in OS cell proliferation.

Flow cytometry analysis was performed to determine whether the reduced expression of HOTAIR contributed to cell cycle arrest or apoptosis. It was revealed that MG-63 cells of siR-HOTAIR1 group and siR-HOTAIR2 group had a significant increase in the proportion of cells in the G₁ phase compared with the mock group (P<0.05; Fig. 3A). This suggests that the cells have been prevented from moving into the next phase of the cell cycle and have therefore been prevented from proliferating. It was also revealed that the reduction in HOTAIR expression did not significantly influence the apoptosis of MG-63 cells (Fig. 3B). This data suggests that the inhibitory effect HOTAIR silencing has on the proliferation of MG-63 cells is through cell cycle arrest and not apoptosis.

To assess the effects of HOTAIR on cell migration and invasion, a Transwell migration assay was performed. MG-63 cells transfected with siR-HOTAIR1 or siR-HOTAIR2 exhibited a significant reduction in migration compared to the mock group (P<0.01; Fig. 4A and B). A Boyden chamber coated with Matrigel was used to determine the effect of siR-HOTAIR on cell invasion after incubation for 48 h. The results revealed that HOTAIR silencing significantly decreased the invasion of MG-63 cells (P<0.01; Fig. 4A and C). The suppressive effects of siR-HOTAIR explain the association observed between high HOTAIR expression and distant metastasis in patients with OS. This data indicates that siR-HOTAIR can inhibit a migratory and invasive phenotype in OS cells.

To investigate the evidence that siR-HOTAIR serves a role in G₁/S arrest, RT-qPCR (Fig. 5A) and western blot analysis (Fig. 5B) were used to evaluate the effect of HOTAIR silencing on the expression of CDK2, CDK4, cyclin D1 and cyclin E, all of which are associated with progression from the G₁ to S phase in the cell cycle. The CDK inhibitor p27 was also studied. The results demonstrated that HOTAIR silencing significantly reduced the mRNA expression of CDK2, CDK4 and cyclin E compared to the controls (P<0.01), while cyclin D1 exhibited little variation. Conversely, the mRNA expression of p27 was significantly increased compared to the control (P<0.05). Similar results were observed for protein expression. These results suggest that HOTAIR mediates OS cell proliferation by affecting cyclin E, CDK2, CDK4 and p27 expression.

To explore the molecular mechanisms underlying the effect of HOTAIR on the invasion and metastasis of OS cells, potential targets involved in tumor invasion and metastasis were investigated. The results revealed a marked decrease in CD44, MMP2, MMP9 and N-cadherin expression, and a notable increase in E-cadherin expression at the protein level (Fig. 6A). At the mRNA level a similar trend was observed, which was significant in the siR-HOTAIR groups compared to the mock group (P<0.01; Fig. 6B). This data suggests an association between HOTAIR and MMP2, MMP9, CD44, E-cadherin and N-cadherin expression, as HOTAIR treatment resulted in increased expression levels of MMP2, MMP9, CD44 and N-cadherin, but decreased expression levels of E-cadherin, which indicates that these factors may serve an important role in the HOTAIR-mediated promotion of OS cell invasion and metastasis.

The AKT/mTOR signaling pathway serves a key role in regulating cell growth, survival and metabolism. To further explore the molecular mechanisms by which HOTAIR affects OS cell proliferation and metastasis, the effects of HOTAIR silencing on the AKT/mTOR signaling pathway were investigated. In the present study the expression of three key proteins, p-mTOR (ser2448), p-PI3K (tyr458) and p-AKT (ser473), was detected. HOTAIR silencing reduced the expression of p-mTOR, p-PI3K and p-AKT, while no notable changes were observed in the unphosphorylated proteins (Fig. 7). These results suggest that HOTAIR promotes OS cell growth through activation of the AKT/mTOR signaling pathway.