A total of 64 pairs of OS tissues and their matched adjacent normal bone were obtained from patients who underwent surgery at The Xuanwu Hospital of Capital Medical University (Beijing, China) from March 2008 to October 2009. All patients' operation treatments were performed by the same senior doctors according to the patients' Enneking staging. None of the patients had received radiotherapy or chemotherapy before surgery. This study was approved by the Research Ethics Committee of the Capital Medical University with the permit no. 2014A083. Written informed consent was acquired from all the patients. Tissue specimens were immediately frozen in liquid nitrogen after surgery and stored at −80°C until use.

OS cell lines (KHOS-240S, SaOS2, MG-63, SOSP-9607 and U2OS), and osteoblastic cell line (hFOB1.19) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium or Ham's F12/Dulbecco's modified Eagle's medium (both GE Healthcare, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml of penicillin and 100 mg/ml of streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

Total RNA was extracted from the tissue samples and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The first-strand cDNA was reverse-transcribed from 500 ng of total RNA using a PrimeScript^™ II 1st Strand cDNA Synthesis kit (Takara, Dalian, China). qRT-PCR was performed using the FastStart Universal SYBR-Green Master Mixes (Roche Diagnostics Corp., Indianapolis, IN, USA) on a 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The following primers were used to detect the expression of XIST and GAPDH: XIST sense, 5′-CTCTCCATTGGGTTCAC-3′ and reverse, 5′-GCGGCAGGTCTTAAGAGATGAG-3′; GAPDH sense, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′. The expression of XIST was normalized to GAPDH.

The small interfering RNA (siRNA) specifically targeting XIST was designed and commercially constructed by GenePharma (Shanghai, China). The scrambled nucleotide was used as the negative control (si-NC). The sequences were as follows: siRNA1 sense, 5′-GUAUCCUAUUUGCACGCUAdTdT-3′; siRNA2 sense, 5′-GCCCUUCUCUUCGAACUGUdTdT-3′; negative control siRNA sense, 5′-UUCUCCGAACGUGUCACGUdTdT-3′. Cells were transfected separately with the siRNAs and si-NC using FuGENE 6 transfection reagent (Roche Diagnostics) to knockdown the XIST. The interfering efficiency was confirmed by qRT-PCR after transfection for 48 h.

Cell proliferation assay was used to examine the effect of XIST knockdown on viability of OS cells. In brief, 2×10³ cells transfected with MG-63 and U2OS were seeded into 96-well plates. At 24, 48, 72, and 96 h, 10 µl Cell Counting kit-8 (CCK-8) solution (Dojindo, Tokyo, Japan) was added into each well and incubated for 2 h at 37°C. The absorbance was measured on a microplate reader at 450 nm by Biotek Elx800 ELISA (BioTek, Winooski, VT, USA).

For cell apoptosis analysis, cells were harvested and stained with FITC-Annexin V and propidium iodide (PI; BD Biosciences, San Jose, CA, USA). The fluorescence of stained cells was then examined using flow cytometry (BD Biosciences) according to the manufacturer's protocol. For cell cycle analysis, cells were harvested, washed with cold PBS, and fixed in 70% ethanol at 4°C overnight. The cells were then stained with PI in the dark for 15 min at room temperature. The proportion of cells in the G0/G1, S, and G2/M phases were determined by flow cytometry.

Cell migration ability was assessed using a 24-well plate with 8-µm pore size chamber inserts (Corning Inc., New York, NY, USA). Cell invasion ability was assessed using a 24-well plate with 8-µm pore size chamber inserts coated with Matrigel (BD Biosciences). 5×10⁴ cells were suspended in 150 µl of serum-free medium and were placed in the upper chamber. Six hundred µl of medium containing 10% FBS was added to the lower chamber as the chemoattractant. Following incubation for 36 h at 37°C, the non-invading cells were gently removed with a cotton swab. Invasive cells located on the lower side of the chamber were fixed in 95% ethanol and stained with 0.5% crystal violet. The numbers of cells in five randomly selected fields were counted, and the cells were imaged through a CKX41 inverted microscope (Olympus Corp., Tokyo, Japan).

Total protein from cells was extracted by lysing cells in RIPA buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing protease and phosphatase inhibitors (Roche Diagnostics). Equivalent protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked using blocking buffer and then incubated with primary antibody overnight at 4°C. After the membranes were incubated with secondary antibody, the signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). Antibodies against Vimentin, c-Myc, E-cadherin, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

All the experiments were repeated in triplicate, and data was expressed as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 18.0 software package (SPSS Inc, Chicago, IL, USA). One-way or two-way analyses of variance was performed with Dunnett's post hoc test to determine statistical differences between >2 groups. The Chi-squared test was used to evaluate the association between the XIST expression and the clinicopathological features. The Kaplan-Meier method and log-rank test were used for the survival analysis. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the expression of XIST, we measured the expression of XIST in OS cell lines and our results indicated that the expression level of XIST was relatively high in four OS cell lines, including KHOS-240S, SaOS2, MG-63 and U2OS, compared to the osteoblastic cell line hFOB1.19 (Fig. 1A). Furthermore, we determined the levels of XIST expression in 64 pairs of OS tissues and adjacent non-tumor tissues. The results suggested that XIST was significantly increased in the cancerous tissues compared with the adjacent normal samples (Fig. 1B). Our results suggested that significant upregulation of XIST was frequently observed in the majority of the OS tumors and cell lines.

To assess the clinical significance, OS tissues were divided into two groups including the high XIST expression group (n = 32) (higher than median value) and the low XIST expression group (n = 32) (lower than median value) according to the median value of XIST expression level of all samples. As shown in Table I, the high XIST expression group was positively associated with Enneking stage and metastasis. However, there was no significant correlation between XIST expression and other clinicopathological features such as sex, age, tumor location, tumor size and pathologic fracture. In addition, Kaplan-Meier survival curves showed that patients in the high XIST expression group had a worse overall survival rate than those in the low XIST expression group (Fig. 2).

Since XIST was significantly upregulated in OS tissues, we investigated the biological function of XIST silencing in OS cells. XIST was downregulated by transfecting the siRNAs against XIST into the MG-63 and U2OS cell lines, which harbored the highest expression level of XIST (Fig. 3A). Knockdown of XIST markedly impaired the growth of MG-63 and U2OS cells compared to the NC-transfected cells (Fig. 3B). The siRNA#1 against XIST had more efficient inhibition and was chosen for the following experiments. Furthermore, compared to si-NC, transfection with XISTsiRNA#1 resulted in a considerable increase of the apoptotic percentage in MG-63 and U2OS cells (Fig. 3C). Additionally, XIST silencing exhibited a significant decrease in the percentage of cells in S phase and an increase in cells in the G2/M phase compared with si-NC in both MG-63 and U2OS cells (Fig. 3D). These data suggested that XIST promoted cell proliferation by mediating cell apoptosis and cell cycle in vitro.

To investigate the potential role of XIST in OS metastasis, the present study detected the effect of XIST on the migration and invasion capacity of OS cells. We found that XIST knockdown dramatically decreased their migration and invasion capabilities in both MG-63 (Fig. 4A) and U2OS (Fig. 4B) cells. We then sought to explore whether there was some interaction between proliferation- and metastasis-related markers and XIST expression. Interestingly, XIST knockdown upregulated E-cadherin and repressed Vimentin and c-Myc (Fig. 4C), which are clearly involved in OS cell proliferation and invasion. These results indicated that XIST functioned as an oncogene and promoted the invasion of OS cells.