The human OSCC-derived cell lines Ca9-22, HSC-2 and the human lung cancer cells A549, were prepared for this study (Human Science Research Resources Bank, Osaka, Japan). The cells were maintained in Dulbecco’s modified Eagle’s medium F-12 HAM (Sigma Chemical Co., St. Louis, MO, USA) and supplemented with 10% heat-inactivated fetal bovine serum and 50 U/ml penicillin and streptomycin. All cultures were grown at 37°C in a humidified atmosphere of 5% CO₂.

The cells were irradiated using X-ray irradiation equipment (MBR-1520R-3, Hitachi, Tokyo, Japan) operated at 150 V and 20 mA with AL filtration at a dose of 2.1 Gy/min.

Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) from irradiated and unirradiated cells 4 h after irradiation, according to the manufacturer’s instructions. The quality of total RNA was determined by Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

Total RNA was extracted using TRIzol reagent. Five micrograms of total RNA from each sample was reverse transcribed to cDNA using Ready-to-Go You-Prime first-strand beads (GE Healthcare, Buckinghamshire, UK) and oligo (dT) primer (Sigma Genosys, Ishikari, Japan), according to the manufacturer’s protocol.

For micro-array analysis, 4 h after irradiation (8 Gy) was selected as the time-point at which to monitor the early response of Ca9-22 cells to X-ray irradiation and to identify differentially expressed early genes that mediate cellular events such as DNA repair and apoptosis. We used Human Genome U133A Array GeneChip oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA). This GeneChip, containing 22,283 probe sets, analyzes the expression level of over 18,400 transcripts and variants, including 14,500 well-characterized human genes. For hybridization, 20 μg of total RNA per sample was prepared according to the manufacturer’s protocol (Affymetrix). Fragmented cRNA (15 μg of each) was hybridized to the Human Genome oligonucleotide arrays. The arrays were stained with phycoerythrin-streptavidin and the signal intensity was amplified by treatment with a biotinconjugated anti-streptavidin antibody, followed by a second staining using phycoerythrin-streptavidin. The arrays stained a second time were scanned using the Affymetrix GeneChip Scanner 3000.

GeneChip analysis was performed based on the Affymetrix GeneChip Manual with Microarray Analysis Suite 5.0, Data Mining Tool 2.0 and Microarray Database software. The genes on the GeneChip were globally normalized and scaled to a signal intensity of 500. The Microarray Analysis Suite software used Wilcoxon’s test to generate detected (present or absent) calls and used the calls to statistically determine if a transcript was expressed or not. After being filtered through a ‘present’ call (P<0.05), the expression data were analyzed using GeneChip Operating Software 1.1 (Affymetrix) and GeneSpring 6.1 (Silicon Genetics, Redwood City, CA, USA). Fold changes were calculated by comparing transcripts between irradiated Ca9-22 cells and unirradiated Ca9-22 cells. We identified 167 genes differentially expressed 2.0-fold or more by X-ray irradiation. The genes, which were identified by microarray analyses, were analyzed for network and gene ontology by Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Mountain View, CA, USA) to identify networks of interacting genes. Gene accession numbers were imported into the IPA software. The genes were categorized based on location, cellular components, and reported or suggested biochemical, biologic and molecular functions using the software. The identified genes were mapped to the genetic networks available in the IPA database and then ranked by score. The score is the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. A score of 3 indicates that there is a 1/1,000 chance that the focus genes are in a network due to random chance. Therefore, scores of 3 or higher have a 99.9% confidence level of not being generated by random chance alone. This score was used as the cut-off for identifying gene networks.

Real-time qRT-PCR was performed to validate mRNA expression with a single method using a LightCycler FastStart DNA Master SYBR-Green 1 kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the procedure provided by the manufacturer. Oligonucleotides used as primers were 5′-GCTGACTTC GGAACTAAAGGAGAA-3′ and 5′-GGGAAGACGATGTG GTTTTCA-3′ for LCN2 mRNA and 5′-CATCTCTGCCCCC TCTGCTGA-3′ and 5′-GGATGACCTTGCCCACAGCCT-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Using LightCycler (Roche Diagnostics GmbH) apparatus, the experiment was carried out in a final volume of 20 μl of reaction mixture consisting of 2 μl of FirstStart DNA Master SYBR-Green I mix, 3 mM MgCl₂ and 1 μl of the primers, according to the manufacturer’s instructions. Subsequently, the reaction mixture was loaded into glass capillary tubes and subjected to an initial denaturation at 95°C for 10 min, followed by 45 rounds of amplification at 95°C (10 sec) for denaturation, 62 to 64°C (10 sec) for annealing, and 72°C for extension. The transcript amount for the genes differentially expressed in the microarray analysis was estimated from the respective standard curves and normalized to the GAPDH transcript amount determined in corresponding samples.

SMARTpool siRNA targeting LCN2 consists of four siRNAs targeting multiple sites on LCN2 (LCN2-siRNAs). The sequences for LCN2-siRNAs are 5′-UGG GCAACAUUAAGAGUUAUU-3′ (sense), 5′-PUAACUCUU AAUGUUGCCCAUU-3′ (antisense), 5′-GAGCUGACUUC GGAACUAAUU-3′ (sense), 5′-PUUAGUUCCGAAGUCAGC UCUU-3′ (antisense), 5′-GAAGACAAAGACCCGCAAAUU-3′ (sense), 5′-PUUUGCGGGUCUUUGUCUUCUU-3′ (antisense), 5′-GAAGACAAGAGCUACAAUGUU-3′ (sense) and 5′-PCAU UGUAGCUCUUGUCUUCUU-3′ (antisense) (On-Target plus SMARTpool, L-003679-00-0005, Human LCN2, NM005564). Positive and negative control siRNAs were purchased (Dharmacon, Lafayette, CO, USA). Two negative controls were used, vehicle control and siControl non-targeting siRNA pool (D-001206-13-20; siNT). Cyclo philin B (siControl cyclophilin B, siCyclo) was used as a positive silencing control to ascertain the transfection efficiency in each experiment. Cells were transfected with siRNAs using DharmaFECT1 reagent (Dharmacon). Cells were plated in antibiotic-free Dulbecco’s modified Eagle’s medium F-12 HAM at a density of 200,000 cells/4 ml in 60-mm dishes. After 24 h, the cells were transfected with 100 nmol/l siRNA in DharmaFECT1 reagent, according to the manufacturer’s instructions. Briefly, 8 μl DharmaFECT1 was diluted in 392 μl of serum-free medium and incubated at room temperature for 5 min. In a separate tube, 200 μl of 2 μmol/l siRNA was diluted in 200 μl of serum-free medium at room temperature for 5 min. Diluted DharmaFECT1 (400 μl) was added to the diluted siRNA and the complex was incubated for 20 min at room temperature. The cells were washed with antibiotic-free Dulbecco’s modified Eagle’s medium F-12 HAM and 3.2 ml antibiotic-free Dulbecco’s modified Eagle’s medium F-12 HAM was added to each dish. siRNA + DharmaFECT1 complex (800 μl) was added gently to the dish. The final concentration of siRNA was 100 nmol/l. Control cells were treated with medium only, the 100 nmol/l non-targeted siRNA (siNT negative control), and the 100 nmol/l cyclophilin B siRNA (positive silencing control). After 4 h of transfection, the medium of cells treated with LCN2-siRNA (siLCN2) and control cells was replaced with fresh medium, and cells were incubated at 37°C in 5% CO₂ for 120 h before the experiments.

Cells were lysed in buffer [10 mM Tris base (pH 8.0), 400 mM NaCl, 3 mM MgCl₂, 0.5% Nonidet P-40 (Sigma), 100 mM phenylmethylsulfonyl fluoride and 0.01% protease inhibitor cocktail (Sigma)] at 4°C for 10 min. Protein extracts were electrophoresed on 11% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Immunoblot PVDF membranes were washed with 0.1% Tween-20 in TBS, and affinity-purified mouse anti-human LCN2 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added at 1:100 and incubated overnight at room temperature. PVDF membranes were washed again and incubated with a 1:5,000 of horseradish peroxidase-conjugated anti-mouse IgG Envision+ (Dako Japan Inc., Kyoto, Japan) as a secondary antibody for 2 h at room temperature. Finally, membranes were incubated with enhanced chemiluminescence (ECL)+ horseradish peroxidase substrate solution included in the ECL+ kit (GE Healthcare) and immunoblotting was visualized by exposing the membrane to Hyperfilm (GE Healthcare).

Cells were transfected as described previously with the vehicle, siNT and siLCN2. At 96 h after transfection, the cells were trypsinized, counted and the appropriate number of cells was plated in 60-mm dishes and allowed to attach for 24 h. After 24 h, the cells were irradiated (2, 4, 6, 8 Gy) and incubated for 8 to 10 days. The colonies were stained with crystal violet (Sigma Chemical Co.) and colonies of 50 cells or greater were counted. Clonogenic fractions of irradiated cells were normalized to the plating efficiency of unirradiated controls.

To determine the effect of siLCN2 transfection on cell proliferation, Ca9-22 cells transfected with non-targeting or LCN2 siRNA were seeded in 12-well plates at a density of 1×10⁴ viable cells per well. At the indicated time point, cells were trypsinized and counted using a hemocytometer in triplicate samples.

The gene expression profile in the irradiated OSCC-derived cell line Ca9-22, was analyzed with DNA microarray analysis. A total of 167 genes were overexpressed after X-ray irradiation (8 Gy) by at least 2-fold when compared with unirradiated Ca9-22 cells, while 14 genes were upregulated more than 5-fold (Table I).

We then investigated whether the 167 genes that were overexpressed by at least 2-fold interacted biologically by performing genetic network analysis with the IPA tool. Among these genes, 82 genes were mapped to six genetic networks in which functional relationships between gene products have been reported (Fig. 1). The six networks were highly significant and contained some common biological functions, such as cancer, cell death, cellular growth and proliferation (Table II). LCN2, which was mapped to network 2, had the greatest increase in expression after X-ray irradiation (Table I).

To verify the gene expression identified in the DNA microarray analysis, real-time qRT-PCR was performed by using the same RNA that was used in the DNA microarray analysis. Consistent with the results of DNA microarray analysis, there was a significant increase in the expression levels of LCN2 in X-ray irradiated Ca9-22 cells as compared with unirradiated Ca9-22 cells (Fig. 2). The data are expressed as the mean ± standard deviation (SD) of two independent experiments with samples in triplicate.

To determine whether LCN2 silencing contributes to radiation sensitivity, cells were transfected with siRNAs and screened for their ability to downregulate target protein expression. To ascertain that RNA inhibition conditions were optimal and transfection efficiency was satisfactory, cyclophilin B siRNA was used as a positive control in each experiment. In Ca9-22, HSC-2 and A549 cells transfected with cyclophilin B siRNA (siCyclo), the cyclophilin B protein level was reduced significantly as compared to the vehicle or siNT controls (siNT) (Fig. 3). LCN2 protein expression was examined by western blot analysis in Ca9-22, HSC-2 and A549 cells 120 h after transfection with siRNAs (Figs. 4–6). LCN2 protein levels in vehicle and siNT-transfected cells were comparable to that of LCN2 in non-treated cells. In addition, in cells transfected with 100 nmol/l siLCN2, the LCN2 protein level was reduced significantly as compared with the positive and negative control cells. These transfected cells were subjected to functional analysis to reveal the effect of LCN2 gene silencing in radiation response. Survival of Ca9-22, HSC-2 and A549 cells transfected with siLCN2 at 120 h decreased significantly (P<0.01, Student’s t-test) after 2, 4, 6 and 8 Gy of irradiation compared with that of corresponding cells treated with siNT (Figs. 4–6). To determine the effect of siLCN2 transfection on cell proliferation, cellular proliferation assay was performed. The data showed no significant effect by siLCN2 transfection on cellular proliferation in Ca9-22 cells (Fig. 7).