Five human colon cancer cell lines, T84, HT29, HCT116, CaCo2 and SW480 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Next, the expression of YB-1 and the mutational status of RAS/RAF were examined. Based on these results, three cell lines, namely CaCo2, HCT116 and HT29 were selected and used for further experiments. All cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS) (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan), 100 U/ml penicillin and 100 mg/l streptomycin. The cells were maintained in a humidified incubator at 37°C with 5% CO₂ (Sanyo Electric Biomedical Co., Osaka, Japan).

For the detection of KRAS, NRAS and BRAF mutations, genomic DNA was isolated from the cell lines, using an AllPrep DNA/RNA/Protein Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The genomic DNA was subjected to polymerase chain reaction (PCR) amplification using the primers listed in Table I. The primer sets were designed to amplify exons 2, 3 and 4 of KRAS and NRAS and exon 15 of BRAF. The PCR products were treated with ExoSap-IT PCR Product Cleanup reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol to inactivate the free primers and dNTPs, and then were subjected to sequencing using the forward primers and BigDye^® Terminator v3.1 (Thermo Fisher Scientific, Inc.). Each 20-µl PCR reaction consisted of 2 µl 10X LA PCR buffer II, 2 µl 10 mmol/l dNTPs, 0.1 µl AmpliTaq Gold (Thermo Fisher Scientific, Inc.), 2 µl of genomic DNA, 1 µl 100 pmol/µl forward primer, 1 µl 100 pmol/µl reverse primer and 12 µl H₂O. The cycling conditions were 95°C for 12 min; 10 cycles at 94°C for 15 sec, 55°C for 15 sec, and 72°C for 30 sec; 25 cycles at 89°C for 15 sec, 55°C for 15 sec, 72°C for 30 sec; and a final extension at 72°C for 10 min. The PCR products were evaluated by electrophoresis using 2% agarose gels. Sequencing was carried out using an ABI 3100 Genetic Analyzer (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The sequences obtained were aligned to the consensus reference-sequences from the University of California, Santa Cruz (UCSC) Genome Bioinformatics website (http://genome.ucsc.edu/index.html) using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) to identify nucleotide differences.

The small interfering RNAs (siRNAs) for YB-1 (siRNA-1: sense, 5′-GUAAAAUGGUUCAAUGUAAtt-3′ and antisense, 5′-UUACAUUGAACCAUUUUACtg-3′; and siRNA2: sense, 5′-CGAAGGUUUUGGGAACAGUtt-3′ and antisense, 5′-ACUGUUCCCAAAACCUUCGtt-3′) and the negative control siRNAs (siCtr) were purchased from Life Technologies Corp. (Thermo Fisher Scientific, Inc.). The siRNAs were transfected into the cells, using Lipofectamine RNAiMAX Transfection reagent and Opti-MEM medium (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's recommendations. A total of 1×10⁴ cells/well were seeded in 96-well plates (Iwaki Co., Ltd., Tokyo, Japan) and cultured for 24 h before transfection. The adjusted amounts of siRNAs were added to each well. Reverse transcription PCR (RT-PCR) and western blotting were performed 72 h post-transfection, and MTT and migration assays were performed 48 h post-transfection.

Total RNA was extracted using an AllPrep DNA/RNA/Protein Mini kit (Qiagen, Inc.) according to the manufacturer's protocol and reverse-transcribed into complementary DNA (cDNA), using SuperScript IV Reverse Transcriptase (Promega Corporation, Madison, WI, USA). RT-qPCR was performed with TaqMan Gene Expression Assays (Applied Biosystems; Thermo Fisher Scientific, Inc.) for YB-1 (Hs00358903_g1), GAPDH (Hs02758991_g1), Bax (Hs00180269_m1) and Bcl-2 (Hs00608023_m1) using the TaqMan Gene Expression Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The levels of transcripts of the indicated genes were standardized to the corresponding GAPDH transcript levels. All values reported represent the average of at least three independent experiments.

Total cell proteins were extracted using an AllPrep DNA/RNA/Protein Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. Protein determination method is BCA. Mass of protein loaded per lane is 20 µg. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE (4–12%) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% milk in Tris-buffered saline containing Tween-20 (TBST) buffer, the membranes were incubated at 4°C overnight with an anti-YB-1 (dilution 1:1,000; rabbit; cat. no. ab12148; Abcam, Cambridge, MA, USA) or anti-EGFR (dilution 1:1,000; rabbit; cat. no. 4267; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibody. Subsequently, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.). The immunoreactive bands were visualized using Fusion-SL7-400 (Vilber Lourmat, Marne Le-Vallée, France). Immunostaining using an anti-β-actin primary antibody (dilution 1:5,000; mouse; cat. no. ab6276; Abcam) was used as a loading control.

For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) reduction assays, the three colorectal cell lines were used as either untreated (CaCo2, HT29 and HCT116), treated with siYB-1 (CaCo2-siYB-1, HT29-siYB-1 and HCT116-siYB-1), or treated with siCtr (CaCo2-siCtr, HT29-siCr and HCT116-siCtr). The cells were added to 96-well plates (1×10⁴ cells/well) and incubated for 2 days in a humidified incubator at 37°C with 5% CO₂. MTT Cell Proliferation Assay Reagent (Cell Biolabs, Inc., San Diego, CA USA) was added (10 µl) to each well, and the cells were incubated for 3 h at 37°C. Thereafter, the detergent solution provided with the assay kit was added to each well (100 µl) and the cells were incubated for 3 h at room temperature while being protected from the light. The absorbance of each sample for each well was measured at 540 nm using a microplate reader (VersaMax ELISA; Molecular Devices Japan K.K., Tokyo, Japan).

A total of 1×10⁴ CaCo2, CaCo2-siYB-1, CaCo2-siCtr, HT29, HT29-siYB-1, HT29-siCtr, HCT116, HCT116-siYB-1, or HCT116-siCtr cells/well were seeded into 96-well Collagen I coated plates (Platypus Technologies, LCC, Fitchburg, WI, USA) and incubated at 37°C to allow for cell attachment. After 24 h, all silicon stoppers at the center of each well were removed, and any unattached cells were removed by washing with 100 µl of sterile phosphate-buffered saline (PBS). After 72 h, the number of migrated cells was quantified using ImageJ software (ver. 1.51, NIH; National Institutes of Health, Bethesda, MD, USA).

Each assay was independently performed three times. The data were analyzed using JMP software (version 13.0.0; SAS Institute Inc., Cary, NC, USA). The values were presented as the means ± standard deviation (SD). The comparisons between groups were analyzed using the Kruskal-Wallis test. P<0.05 was considered to indicate a statistically significant difference.

In colorectal cancer, the mutational status of RAS markedly affects the characteristics of the cancer cells. Therefore, in order to confirm the molecular background of each of the five colon-cancer cell lines, we determined the genetic mutation of RAS/RAF by direct sequence evaluation. A summary of the KRAS/NRAS/BRAF gene mutations is presented in Table II. The detected KRAS mutations were G12V (GGT→GTT) and G13D (GGC→GAC), and the BRAF mutation was V600E (GTG→GAG). The genotypes of the mutant KRAS and mutant BRAF are displayed in Fig. 1A.

Subsequently, western blot analysis was performed to investigate the levels of protein expression of YB-1 in the five colon cancer cell lines. As revealed in Fig. 1B, the expression of YB-1 was confirmed in all five cell lines. HCT116 demonstrated the highest level of YB-1 expression among the cell lines. Notably, the expression level of YB-1 tended to be higher in the cell lines harboring the RAS/RAF mutation compared to that in the cell line harboring wild-type RAS. To clarify the biological function of YB-1 in colorectal cancer with respect to the mutational status of the RAS, the CaCo2, HCT116 and HT29 cell lines were selected for further examination.

Two siRNAs (siRNA-1 and siRNA-2) were transfected into the cell lines HCT116, HT29 and CaCo2 in order to knockdown YB-1. The suppression of YB-1 mRNA levels and protein levels were confirmed by RT-qPCR and western blot analyses, respectively. As revealed in Fig. 2A, the expression level of YB-1 was significantly decreased in both siRNA-1-transfected and siRNA-2-transfected cells compared to that in siCtr-transfected cells (P<0.05). According to the RT-qPCR analysis, the reduced level of YB-1 expression was not significantly different between the siRNA-1-transfected and siRNA-2-transfected groups. However, western blot analysis revealed that the reduced level of YB-1 expression was higher in the siRNA-1-transfected group compared to that in the siRNA-2-transfected group (Fig. 2B). Based on these results, siRNA-1 was used in subsequent in vitro experiments.

To investigate the role of YB-1 in cell proliferation and migration, MTT assays and migration assays were performed, respectively. In the MTT assays, proliferation potency was significantly suppressed in all the cell lines transfected with siRNA-1 compared to that in the siCtr-transfected group (Fig. 3A). Moreover, in the migration assays, the number of cells transfected with si-RNA-1 that migrated into the center of the well was significantly less than that of the siCtr-transfected group (Fig. 3B). Similar results were obtained from siRNA knockdown experiments using the other cell lines. Overall, the effects of YB-1 on cell proliferation and cell migration were not affected by the presence or absence of mutations in KRAS or BRAF.

Since there are few studies regarding the relationship between YB-1 and apoptosis, the effect of YB-1 on apoptosis was analyzed by evaluating alterations in the expression of apoptosis-related genes Bax and Bcl-2 in siRNA-1-treated cells compared to that in siCtr-treated cells, by RT-qPCR analysis. There were no significant gene expression differences between the siRNA-1-treated and siCtr-treated groups for either HCT116 or HT29 cells. In contrast, Bcl-2 expression levels were significantly decreased (P=0.018) and Bax levels were significantly increased (P=0.021) in siRNA-1-treated CaCo2 cells compared to that in siCtr-treated CaCo2 cells (Fig. 4).

EGFR protein expression was compared between the siRNA-1-treated and siCtr-treated groups by western blot analysis. There were no significant differences in EGFR expression between the siRNA-1-treated and siCtr-treated groups for either HCT116 or HT29 cells. However, EGFR expression was significantly decreased in the siRNA-1-treated group of CaCo2 cells compared to that in siCtr-treated CaCo2 cells (Fig. 5).