The human VCR-resistant osteosarcoma cell line MG63/VCR and its parental cell line MG63 were obtained from the Scientific Research Center, China-Japan Union Hospital of Jilin University (Jilin, China) (11). The cells were cultured in high-glucose Dulbecco's modified Eagle's medium (H-DMEM; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) in a humidified atmosphere with 5% CO₂ at 37°C; three biological replicates were conducted using the MG63 and MG63/VCR cells.

The cells were suspended at a density of 8×10³ cells per well and plated into 96-well plates in 100 µl H-DMEM supplemented with 10% FBS. Following incubation at 37°C for 24 h, the cells were treated with VCR (New Hualian Pharmaceutical Co., Shanghai, China) at the following final concentrations: 2,000, 1,000, 500, 250, 125, and 62.5 ng/ml; drug-free medium was used as the control. MG63 cells were treated with the following concentrations: 64, 16, 4, 1, 0.25 and 0.0625 ng/ml. The half-maximal inhibitory concentration (IC₅₀) of VCR in MG63/VCR and MG63 cells was reported to be 453.4 and 0.952 ng/ml, respectively (11). A total of two groups of cells continued to culture 48 h in drug medium at 37°C. Cell viability was examined using 10% Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocols, and the cells were incubated at 37°C for another 2 h. Subsequently, the optical density (OD) was determined by measuring the absorbance at 450 nm using a plate reader (UV8100D; LabTech, Inc., Hopkinton, MA, USA), and the inhibition ratio was calculated using the following formula: Inhibition ratio=[(OD_control-OD_experiment)/OD_control] ×100%. Each experiment was performed in triplicate.

Total RNA was extracted from the MG63/VCR and MG63 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The quality of total RNA was determined using a NanoDrop 2000 spectrophotometer (1.7<A260/A280<2.2) and the RNA integrity (RIN) was evaluated using an Agilent Bioanalyzer 2100 (RIN≥7.0 and 28S/18S>0.7). The initial amount of total RNA (300–500 ng) was further amplified, labeled and purified by using the Microarray GeneChip 3′IVT Express kit (Affymetrix; Thermo Fisher Scientific, Inc.) According to the standard hybridization procedures and matching kit provided by Affymetrix expression chip. RNA was then subjected to treatment with the GeneChip Hybridization, Wash, and Stain kit reagent (Affymetrix; Thermo Fisher Scientific, Inc.), Rolling hybridization in a Hybridization Oven 645 for 45°C, 16 h, and washed in GeneChip Fluidics Station 450 (Affymetrix; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The results of the chip were scanned with a GeneChip Scanner 3000 system (Affymetrix; Thermo Fisher Scientific, Inc.). Differentially expressed genes in the two cell lines were determined using the fold change (FC) values. The gene expression profile was presented in Excel spreadsheets (Microsoft Corporation, Redmond, WA, USA). Volcano plot, Scatter-plot and Clustergram were used to analyze the differential gene expression. Gene set enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) online software (12,13).

Total RNA was extracted from the MG63/VCR and MG63 cells using TRIzol^® reagent and reverse-transcribed into cDNA using RevertAid First Strand cDNA Synthesis kit (both Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The primers used for RT-qPCR were listed in Table I. qPCR was performed using SYBR^® Premix Ex Taq^™ II (Takara Biotechnology Co., Ltd., Dalian, China) on the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The conditions used for qPCR were as follows: Denaturation at 95°C for 30 sec; annealing at 58–62°C for 30 sec; and 30 cycles of elongation at 95°C for 15 sec, 60°C for 30 sec, and 95°C for 15 sec. The relative expression levels of each gene, normalized to the housekeeping gene GAPDH, was calculated using the 2^−∆∆Cq method (14).

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA), and all data are presented as the mean ± standard deviation. The statistical significance of the differences between the cell lines and treatments was analyzed by a Student's test or one-way analysis of variance and Tukey's test. IC₅₀ was performed using global nonlinear regression analysis. Differential gene expression analysis was performed using a Student's t-test. The result of the chip analysis is the weighted average of repeated probe signals in each group. The threshold for statistical significance was P<0.05, |FC|>1.5. To systematically assign putative functions to the differentially expressed genes, bioinformatics analysis was performed with IPA. The unique statistical index of IPA is the z-score, which represents the direction and multiplier of the molecular changes under the existing experimental conditions. The z-score indicates whether the results are consistent with the references mentioned in the literature; inhibition or activation of the molecular action process was considered when |z|>2. A positive z-score suggests that the molecular interaction is activated, whereas a negative z-score indicates that the molecular interaction is inhibited.

To investigate the chemosensitivity of MG63/VCR and MG63 cells to VCR, the IC₅₀ values in response to treatment with different VCR concentrations were determined. Following 48 h of culture in a VCR-containing medium, the IC50 value for MG63/VCR cells was significantly increased compared with that of MG63 cells (493.175±4.473 vs. 1.407±0.111 ng/ml, P=0.001), suggesting that MG63/VCR cells are more refractory to VCR compared with the parental MG63 cell line (Fig. 1).

In total, the genes that exhibited significantly aberrant expression between the two cell lines in microarray analysis were evaluated by volcano plot filtering (P<0.05, FC≥1.5; Fig. 2A). A total of 602 genes were upregulated and 698 were downregulated in MG63/VCR cells compared with MG63 cells. The scatterplot and cluster analysis (Fig. 2B and C) revealed the differential expression profiles of the genes.

To evaluate the diagnostic potential of differentially expressed genes in VCR-resistant MG63 cells, 45 of the downregulated genes (Table II) and 26 of the upregulated genes (Table III) with statistical significance at P<0.05 and a ≥4-fold difference in expression levels between the two cell lines, were selected for further analysis. Based on their putative functions, all genes (Tables II and III) were categorized into subgroups, including transcription factors (n=2+2), enzymes (n=7+5), transporters (n=2+3), transmembrane receptors (n=1+3) and others (n=33+13).

To systematically assign putative functions to the differentially expressed genes, bioinformatics analysis was performed. Among the 800 signaling pathways identified by IPA, the major signaling pathways regulated by the differentially expressed genes were enriched in B-cell receptor signaling [including early growth response protein 1, mitogen-activated protein kinases 9 (MAPK9), cell division control protein 42 homolog and protein phosphatase 3 catalytic subunit A], ultraviolet A (UVA)-induced MAPK signaling [including phosphoinositide-3-kinase regulatory subunit 1, MAPK9 and epidermal growth factor receptor (EGFR)] and Erb-B2 receptor tyrosine kinase 2/3 (ErbB2/3) signaling pathways (including RAS related 2, signal transducer and activator of transcription 5B and serine/threonine kinase ATM; Table IV). The first two pathways were predicted to be inhibited, while the last pathway was predicted to be promoted by VCR, suggesting that combination therapy with EGFR inhibitor and VCR may be more effective compared with single-drug therapy. The top enriched pathway was UVA-induced MAPK signaling (Fig. 3A and B), with a z-score of −2.309. The data, together with the predicted signaling pathways, may provide novel insight in determining whether the aberrant expression of these molecules, such as EGFR, may contribute to drug resistance.

A total of 10 of the differentially expressed genes identified by microarray analysis were selected for further validation using RT-qPCR, including six upregulated genes [multi-drug resistance gene1 (MDR1), carbonic anhydrase II (CAII), insulin-like growth factor-II binding protein 1 (IGF2BP1), A-kinase anchor protein 12 (AKAP12), roundabout homolog 1 (ROBO1) and collagen A2 (COL1A2)], and four downregulated genes, including slit guidance ligand 2, death-associated protein kinase 1 (DAPK1), fibronectin leucine rich transmembrane protein 3 (FLRT3) and testin LIM domain protein. As presented in Fig. 4, the expression profiles of the 10 genes were consistent with the microarray data; however, the FC values varied to an extent. RT-qPCR revealed that the expression levels of FLRT3 were increased in MG63/VCR cells, which is in controversy with the microarray data. Thus, 9 of the 10 genes were positively determined by RT-qPCR.