The human osteosarcoma cell line U2OS was obtained from the China Center for Type Culture Collection (Wuhan, China). U2OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% (v/v) antibiotics (10⁵ U/ml penicillin, 10⁵ µg/ml streptomycin; GE Healthcare Life Sciences, Logan, UT, USA). Cells were propagated in a humidified environment at 37°C with 5% CO₂ and 100% humidity. Cell viability was determined using trypan blue staining (Thermo Fisher Scientific, Inc.). Medium was replaced every three days.

Small interfering RNA (siRNA) gene expression knockdown studies were performed according to the manufacturer's protocol. Each 27mer RNAi duplex was transfected into cells using Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. siRNA was synthesized (Guangzhou RiboBio Co., Ltd., Guangzhou, China) using the following sequences: Snail, 5′-CCACAGAAAUGGCCAUGGGAAGGCCUC-3′; and negative control, 5′-UCACAAGGGAGAGAAAGAGAGGAAGGA-3′.

The cells were seeded into 96-well culture plates and cultured at 37°C for 24 h to attach. Subsequently, various doses (0, 2, 4, 6, 8, 10, 12, 14 or 16 µmol/l) of cisplatin (Sellack Chemicals, Houston, TX, USA) were used to treat cells as indicated and cultured at 37°C for 24 h. The cells in each well containing 100 µl medium were incubated with 10 µl cell counting kit-8 reagent (Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 2 h. The optical density of each well was subsequently measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

Total RNA was isolated by the RNeasy Plus Mini kit (Qiagen China Co., Ltd, Shanghai, China). The concentration and purity of RNA was determined by an ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Reverse transcription was performed using the TaqMan Reverse Transcription Reagents (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was subsequently performed using an ABI 7900 HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in the presence of SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The gene-specific primers used are listed in Table I. Target sequences were amplified at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. β-actin was used as an endogenous normalization control. All assays were performed in triplicate. The fold change in mRNA expression was determined according to the method of 2^ΔΔCq (16).

The cells were seeded on square coverslips in six-well plates for 24 h to allow them to attach. Subsequently, the cells were fixed, permeated and blocked using the Immunol Fluorence Staining kit (Beyotime Institute of Biotechnology). The cells were then incubated with anti-E-cadherin antibody (diluted at 1:100; 701134; Thermo Fisher Scientific, Inc.), anti-N-cadherin antibody (diluted at 1:200; PA5-19486; Thermo Fisher Scientific, Inc.) and anti-vimentin antibody (diluted at 1:200; PA5-27231; Thermo Fisher Scientific, Inc.) overnight at 4°C. Secondary antibody (diluted at 1:200; ab150077; Abcam, Cambridge, MA, USA) was applied for 1 h at room temperature. The cells were counterstained with DAPI and washed with PBS following each step of the staining procedure. Coverslips were mounted using Anti-fade Fluorescence Mounting Medium (Beyotime Institute of Biotechnology). The long and short axes of cells were measured using the Zeiss LSM Image Examiner software (version 4.2.0.121; Carl Zeiss AG, Oberkochen, Germany), and the long/short axis ratio was determined by counting 100 cells per experiment.

Cell lysates were extracted using radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). Protein concentrations were determined using the bicinchoninic acid method (Sigma-Aldrich; EMD Millipore). Cell lysates containing 40 µg protein were loaded and separated on 10% SDS-PAGE gels and subsequently transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked in Tris Buffered Saline with 5% (w/v) skimmed milk and 0.05% Tween 20 (Thermo Fisher Scientific, Inc.) for 1 h at 37°C. Primary antibodies were incubated overnight at 4°C. The primary antibodies and mouse monoclonal anti-β-actin were purchased from Abcam (anti-Snail antibody; ab180714; diluted at 1:1,000; anti-Slug antibody; ab27568; diluted at 1:1,000; anti-N-cadherin antibody; PA5-19486; diluted at 1:1,000; anti-β-actin antibody; ab8226; diluted at 1:2,000) The pH2AX antibody (MBS837487; diluted at 1:2,000) was purchased from MyBioSource, Inc. (San Diego, CA, USA). The membranes were washed and incubated with secondary antibody (ab6721; Abcam) at 1:5,000 dilution for 2 h at room temperature. The membranes were washed again and developed using enhanced chemiluminescence substrate (Sigma-Aldrich; EMD Millipore). Quantitative analysis was performed using QuantiOne imaging software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A total of 5×10⁵ cells were seeded into 6-well plates and cultured overnight at 37°C to attach. When adherent cells reached ~90% confluence, a scratch was made using a 200-µl pipette tip. The cells were washed three times and further incubated at 37°C for 24 h. The migration was observed and recorded under a phase contrast microscope (Nikon Eclipse TE2000-U; Nikon Corporation, Tokyo, Japan).

Matrigel-coated Transwell invasion assay plates (Corning Inc., Corning, NY, USA) were used for this assay. Cells were placed in the upper chamber (1×10⁵ cells/well) in DMEM medium with 0.1% FBS. The lower chambers were filled with DMEM medium with 10% FBS. Following culturing for 24 h at 37°C, the inserts were removed and the inner side was wiped with cotton swabs. The filters were stained with Harris's hematoxylin solution (Sigma-Aldrich; EMD Millipore) for 20 min and peeled off following washing three times. The migrated cells were counted by a light microscope (Nikon Eclipse TE2000-U).

For the in vivo assay, male NOD/SCID mice (n=14; 6-week-old; 18–23 g; SPF) were purchased from and maintained maintained (humidity, 50–60%; temperature, 18–22°C; light cycle, 10–14 h a day) at the Wuhan University Center for Animal Experiment (Wuhan, China). The care and use of animals was reviewed and approved by the Institutional Animal Care and Use Committee (approval number, 2011006). A total of 5×10⁶ cells were subcutaneously injected into 2 mice, and the xenografts were obtained following two weeks of growth, and the 2 mice were sacrificed by CO₂. The tumor xenografts were divided into small pieces of ~5 mm³ and transplanted subcutaneously. The mice were divided into a cisplatin treated group (peritoneal injection of 5 mg/kg cisplatin once a week; n=6) and the control group (receiving the same amount of saline once a week; n=6). Following 4 weeks of rearing, the mice were sacrificed by CO₂. Tumor samples and lung tissues were obtained and usde for subsequent immunohistochemistry experiments.

Tissues were fixed in 10% neutral-buffered formalin, processed and embedded in paraffin. Tissue sections were deparaffinized and rehydrated in an ethanol series. Sections were blocked for nonspecific binding with 1% normal serum (Thermo Fisher Scientific, Inc.) and incubated with the primary anti-Snail (ab53519; diluted at 1:500; Abcam) and anti-N cadherin (PA5-19486; diluted at 1:300; Thermo Fisher Scientific, Inc.) antibodies overnight at 4°C. Subsequently, immunostaining was developed using 3,3′-diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA, USA) followed by hematoxylin counterstaining (Sigma-Aldrich; EMD Millipore). Immunostaining was visualized using a fluorescence microscope (Eclipse 80i Fluorescence Microscope; Nikon Corporation).

Each sample was analyzed in triplicate, and experiments were repeated at least two times. The mean, standard error and P-values base on the two-sample two-tailed t-test were calculated with Excel 2013 software (Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference.

Cells were treated with 5 µM of cisplatin for 24 h and maintained in normal conditions for 5 days to recover from the chemotherapeutic stress. The surviving cells were observed to exhibit increased resistance to cisplatin (Fig. 1A). The DNA damage marker pH2AX was investigated by western blotting to observe the effect of cisplatin on the cells, and the drug was confirmed to be effective (Fig. 1B). The present study observed cell morphology changes in these cisplatin-resistant cells, in which they appeared to possess a more spindle-like shape. Although there were no differences between the two groups in terms of E-cadherin and vimentin expression, N-cadherin expression was observed to be significantly increased in the cisplatin treated group compared with the control cells (Fig. 1C). EMT is frequently accompanied by an alteration from a rounded to spindle shape in terms of cell morphology (17). Therefore, the present study assessed the long/short axis ratio in various cells. The cells treated with cisplatin were observed to have an average ratio of 3.591±0.119, which was increased compared with the cells of the control group (average ratio, 2.232±0.041; P=0.001; Fig. 1D). In addition, the expression of epithelial and mesenchymal markers was examined by western blot analysis. The epithelial markers, E-cadherin and cytokeratin, were not detectable in either the cisplatin of the control group. Mesenchymal-marker N-cadherin was highly expressed in the cisplatin group (Fig. 1E). EMT-inducing TFs, including Snail/Slug and Zeb1/2, suppress epithelial marker expression and induce the expression of mesenchymal markers, facilitating qPCR analysis that led to the observation that these four EMT-TFs were significantly upregulated (Snail, P<0.001; Slug, P<0.001; Zeb1, P=0.0011; Zeb2, P<0.001) in the cisplatin treated cells (Fig. 2A), among which the expression of the Snail gene exhibited the most marked increase with levels of relative mRNA expression of 35.44±2.35. Furthermore, western blotting confirmed the upregulated expression of Snail and Slug in the cisplatin treated cells (Fig. 2B). In addition, the in vivo xenograft assay confirmed that the expression of N-cadherin and Snail was increased following cisplatin exposure (Fig. 2C).

Subsequently, the present study investigated the migratory and invasive capacity following cisplatin treatment. The cisplatin treated cells exhibited significantly increased cell migration compared with the control group (P=0.001; Fig. 3A). The invasive potential through the Matrigel of the cisplatin treated group was also enhanced, with an average fold increase of 1.31±0.05 (P=0.002; Fig. 3B). To investigate the in vivo metastatic capacity, the present study examined pulmonary lesions in both groups. It was observed that cisplatin exposure promoted pulmonary metastasis and induced more severe lung destruction, although the primary tumor was inhibited (Fig. 3C).

The present study applied RNAi techniques to knockdown the expression of Snail. The efficiency of RNAi was confirmed by qPCR and western blotting (blank vs. mock group, P=0.88; blank vs. siRNA group, P<0.001; Fig. 4A and B). The IC₅₀ for cisplatin was decreased when Snail was inhibited, which indicated that the sensitivity of osteosarcoma cells to cisplatin was enhanced (Fig. 4C). When the Snail knockdown cells were exposed to cisplatin, they exhibited a less spindle-like shape, with a decreased long/short axis ratio compared with mock cells (P<0.001; Fig. 4D). Furthermore, Snail inhibition blocked cisplatin-induced cell migration (P<0.001) and invasion (P<0.001) in vitro (Fig. 4E and F).