FGFR1 cDNA was amplified from the human genome by polymerase chain reaction (PCR) and the amplified fragments were digested with HindIII and XhoI (Takara Biotechnology Co., Ltd., Dalian, China) and were inserted into the HindIII and XhoI sites of the pcDNA3.1-Flag vector (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. Sequences were verified using DNA electrophoresis and sequencing. Dulbecco's modified Eagle's medium (DMEM; Corning Incorporated, New York, NY, USA), dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) were all purchased from commercial sources, as described. The anti-FGFR1 and actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Primary antibodies against CDK1 and CDK2 were obtained from Abcam (Cambridge, MA, USA).

The MG63 human osteosarcoma cell line was obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM with 10% FBS at 37°C in a 5% CO₂ humidity-controlled incubator. The medium was refreshed every 2 days. The Flag-FGFR1 plasmid, which was constructed using the pcDNA3.1-basic vector, was transfected into the MG63 cells (1×10⁶ cells/6 cm dish) prior to cell confluence reaching 80%. Lipofectamine 2000 (Invitrogen Life Technologies) was used as the transfection regent. Transfection efficiency was verified using a fluorescence microscope (DMI3000 B; Leica Microsystems GmbH, Wetzlar, Germany) and subsequent western blotting.

A total of 65 patients who underwent surgery at The Sixth People's Hospital Affiliated to Shanghai Jiaotong University (Shanghai, China) between 2012 and 2014 were chosen subsequent to histological verification of osteosarcoma. None of the 65 patients received preoperative chemotherapy or radiotherapy. The examined population consisted of 65 patients (41 men and 24 women) with a median age of diagnosis of 51 years old (range, 33–75 years). The tissue, which was separate from the edge of tumor foci and was histologically judged to be free from adenocarcinoma cells, was used as a normal tissue control. All tissue samples were fixed in 4% formalin immediately subsequent to removal and were embedded in paraffin for immunohistochemical staining. The study was approved by the Ethics Committee of Shanghai Jiaotong University, and all patients provided informed consent.

IHC analysis was performed using osteosarcoma tissues and paired normal bone tissues. Paraffin-embedded tissues were first cut into 4 µm-thick sections, followed by deparaffinization and rehydration with xylene and ethanol. The sections were then subjected to antigen retrieval in 0.1 M citrate acid buffer (Sangon Biotech Co., Ltd., Shanghai, China). Following washing in phosphate-buffered saline (PBS) three times, the sections were incubated with primary antibodies overnight at 4°C. Following incubation, the sections were incubated with horseradish-peroxidase-labeled secondary antibody for 1 h at room temperature. Following washing in PBS three times, the sections were visualized using diaminobenzidine and images were captured (DM13000 B; Leica Microsystems GmbH).

Total RNA were isolated from samples from 65 patients using TRIzol reagent (Sigma-Aldrich). The cDNA (500 ng) was then prepared using an AMV Reverse Transcriptase synthesis kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. qPCR was performed using a QuantiTect SYBR Green PCR kit (Qiagen, Shanghai, China). The qPCR cycle conditions were as follows: 95°C for 30 sec 38 cycles of 95°C for 5 sec and 60°C for 34 sec. The amplification specificity was evaluated by melting curve analysis. The relative mRNA levels were determined using the 2^−ΔCT (CT; cycle threshold) formula, where ΔCT = CT (target gene) − CT (actin). All assays were performed in triplicate. β-actin was selected as the internal control. The following primers were used: Forward 5′-CTAAGATCCCGTCCATCGCC-3′ and reverse 5′-GGAGCCCAGCACTTTGATCT-3′.

Following culture for 24 h, the MG63 cells were lysed using radioimmunoprecipitation assay buffer (Sigma-Aldrich), containing 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM Tris-HCl (pH 7.5) and 2 mM EDTA), on ice for 20 min. Following centrifugation at 4°C for 20 min (12,000 × g), the supernatant was collected. Protein concentration was determined using a Bicinchoninic Acid Assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal quantities of protein extract (40 µg) were loaded into each lane of a 12% SDS-PAGE gel (Sangon Biotech Co., Ltd.) for separation, and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following antigen blocking in Tris-buffered saline with 0.1% Tween 20, supplemented with 5% skim milk for 1 h, the membranes were incubated with the following primary antibodies overnight at 4°C: Monoclonal rabbit anti-EGFR1 (1:1,000; cat. no. ab32152; Epitomics, Inc., Burlingame, CA, USA) and monoclonal rabbit anti-CDK1 (1:1,000; cat. no. 5524; Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, the membranes were incubated with the goat anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology, Inc.) secondary antibodies for 1 h at room temperature and were processed for electrochemiluminescent detection using an ECL system (Pierce Biotechnology).

To investigate the possible pathways contributing to the onset of FGFR1-mediated osteosarcoma, the present study performed microarray analysis in osteosarcoma samples. The focus of this investigation was predominantly on cell cycle changes, and bone remodeling changes were selected as a control. Data from the scanned arrays were extracted using Genome Studios software (v 1.6; Illumina, San Diego, CA, USA). Data processing and statistical analysis were performed using MATLAB 2011a (MathWorks, Natick, MA, USA), which was also used for multidimensional scaling analysis of the detected probes. For gene analysis, the microarray data were analyzed using the filtering criteria of adjusted P<0.05 and >15% change. To reveal genes with significant changes in expression, heatmap analysis was performed for visualization using MATLAB.

A Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to measure cell viability following FGFR1 transfection. The MG63 cells (10³) were seeded into a 96-well plate and incubated for 48 h at 37°C in 5% CO₂. At 48 h post-transfection, 10 µl CCK-8 solution was added to each well and the cells were incubated for a further 4 h at 37°C, following which the absorbance at 450 nm was measured using a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific). Each experiment was repeated in triplicate, and observation of the cells was continued for 4 days to determine changes.

Data are representative of at least three independent replicate experiments performed in triplicate, and are expressed as the mean ± standard deviation. Student's t-test was used to analyze the difference between two groups. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed with SPSS statistical software, version 20.0 (IBM SPSS, Armonk, NY, USA).

To determine the expression level of FGFR1 in osteosarcoma tissues, the present study performed IHC analysis of osteosarcoma tissues and paired normal bone tissues using the FGFR1 antibody. The results demonstrated that the protein level of FGFR1 in the osteosarcoma tissues was notably higher than that in the paired normal bone tissues (Fig. 1A). The staining of FGFR1 was predominantly located in the plasma (Fig. 1A). In addition, the mRNA level of FGFR1 was determined in the two tissue groups. Consistently, FGFR1 exhibited a relative mRNA level of ~4.5 in the normal bone, whereas the relative mRNA level reached almost 7.5 in the osteosarcoma tissues (Fig. 1B). The elevated protein and mRNA levels of FGFR1 in osteosarcoma suggested that FGFR1 may be involved in osteosarcomagenesis.

To investigate the possible pathways, which contributed to the effects of FGFR in osteosarcoma, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/kegg/pathway.html) was accessed (Fig. 2). Notably, cell cycle was significantly affected in osteosarcoma (Fig. 2A(a); P=0.003). Bone remodeling is a ubiquitous process in bone diseases, therefore, the bone remodeling process was also assessed as a control. As expected, bone remodeling was not significantly affected (Fig. 2A(b); P=0.369). As shown in Fig. 2C and D, the cell cycle was significantly affected in osteosarcoma, with the upregulation of multiple cell cycle-associated genes and the downregulation of other genes downegulated. Furthermore, the present study performed microarray analysis of osteosar-coma using MATLAB, and to identify the genes and pathways that are involved in disease development, heatmap analysis was performed. The heatmap analysis demonstrated that multiple genes associated with the cell cycle were elevated, whereas certain other genes were decreased in osteosarcoma (Fig. 2B). Taken together, the data from the KEGG and microarray analyses suggested that the cell cycle is significantly affected by FGFR1 in osteosarcoma.

To further investigate the downstream molecules regulated by FGFR1, the MG63 cells were transfected with the Flag-FGFR1 expression plasmid. Transfection efficiency was visualized using a fluorescence microscope (Fig. 3A). Subsequently, total RNA was extracted and RT-qPCR analysis was performed to examine the expression of cell cycle molecules, including CDK1 and CDK2. Notably, the mRNA expression of CDK1 increased significantly in response to FGFR1 overexpression, whereas no significant change was observed in the mRNA level of CDK2 (Fig. 3B). Consistently, western blotting also confirmed that the protein level of CDK1 was upregulated following MG63 cell transfection with the pcDNA3-Flag-FGFR1 plasmid (Fig. 3C). These data suggested that FGFR1 targeted CDK1 to regulate the cell cycle in osteosarcoma.

To investigate the role of FGFR1 in osteosarcoma development, the MG63 cells were transfected with the pcDNA3-Flag-FGFR1 plasmid and a cell viability assay was performed. The absorbance at 450 nm, which represents cell numbers was continuously determined for 5 days. The results demonstrated that the number of cells in the FGFR1-overexpressed group were higher than in the control group from day 2. Subsequently, the cell numbers increased markedly, with the FGFR1-overexpressed MG63 cells increasing ~7-fold by day 4. Notably, the number of FGFR1-overexpressed MG63 cells increased by almost 1.7-fold, compared with the control group on day 4 (Fig. 4). These data lead to the conclusion that FGFR1 overexpression promoted MG63 cell growth.