Fresh SCC and adjacent normal tissues were obtained from 20 patients (12 males and 8 females; age range, 32–58 years) who underwent skin biopsies between 2013 and 2014 at the Department of Dermatologic Surgery of The Second Affiliated Hospital of the Medical College of Xi'an Jiaotong University (Xi'an, China). All SCC cases were clinically and pathologically verified. Standard protocols established by the Hospital's Protection of Human Subjects Committee were followed in this study and informed consent was provided from all patients in the present study. SCC lesions were obtained from the center of SCC tissues. The uninvolved tissues surrounding the SCC were obtained from the normal tissues surrounding the SCC tissues. Normal skin tissues were obtained from cosmetic surgeries.

A human squamous cell carcinoma cell line (A431) (Shanghai Xiang Shi Biotechnology, Co., Ltd., Shanghai, China) was cultured in F12 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (both Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells were cultured at 37°C with 5% CO₂.

Normal skin tissues, uninvolved tissues surrounding the SCC and SCC lesions were analyzed by immunohistochemistry. Paraffin-embedded tissue sections (4-µm thick) were dewaxed in xylene and re-hydrated in gradient alcohol, then incubated with rabbit anti-human YB-1 monoclonal antibody (1:50; ab76149; Abcam, Cambridge, UK) and the slides were placed in a humid environment at 4°C overnight before being incubated with goat anti-rabbit antibody (1:1,000; ab6721; Abcam) at 37°C for 30 min. Next, the sections were stained with 3,3′-diaminobenzidine (Sigma-Aldrich; Merck KGaA) and observed using an optical microscope (magnification, ×200). Normal skin tissue samples were used as the negative controls.

The following siRNA oligonucleotide sequences were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) in order to select the most effective one: YB-1 siRNA 1: 5′-GCAGACCGUAACCAUUAUATT-3′ (sense) and 5′-UAUAAUGGUUACGGUCUGCTT-3′ (antisense); YB-1 siRNA 2: 5′-CGGCAAUGAAGAAGAUAAATT-3′ (sense) and 5′-UUUAUCUUCUUCAUUGCCGTT-3′ (antisense); YB-1 siRNA 3: 5′-CUGCCAUAAAGAAGAAUAATT-3′ (sense) and 5′-UUAUUCUUCUUUAUGGCAGTT-3′ (antisense). The negative control duplexes of siRNA were random sequences and did not target any known mammalian gene according to GenBank searches (National Center for Biotechnology Information, Bethesda, MD, USA). A431 cells were transfected with siRNAs according to the recommended procedures for Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA). A431 cells were seeded in 6-well plates at a density of 10⁵ cells/well. When the fusion reached 70–80%, the cells were treated with serum-free F12 medium, according to the manufacturer's protocols. The YB-1 inhibition rate was analyzed using western blot analysis (described below).

Transfected cells, which were transfected with siRNA1, the most effective siRNA of the 3 siRNA sequences, were seeded at a density of 3×10³ cells/well and cultured in serum-free F12 medium for 24 h. MTT (5 mg/ml; 10 µl; Sigma-Aldrich; Merck KGaA) was added to each well and left to incubate at 37°C for 4 h. Subsequently, 150 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) was added and the absorbance was measured at 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell invasion was evaluated using a polycarbonate membrane with a pore size of 8 µm that was pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and Transwell chambers (Costar; Corning, Inc., Corning NY, USA). The transfected cells were starved without serum for 12 h, then re-suspended in serum-free medium at a density of 2.5×10⁵ cells/ml. A 200-µl cell suspension was added to each Transwell chamber and 500 µl culture medium with 10% FBS was added to the lower chamber. After culturing for 24 h at 37°C, the cells that invaded the lower chamber were stained with a staining solution (0.1% crystal violet ethanol). Three representative fields were randomly selected and the average number of invaded cells was calculated.

TRIzol reagent (Sigma-Aldrich; Merck KGaA) was used to extract total RNA from cells. Total RNA (3 µg) was then reverse transcribed to cDNA in a total volume of 20 µl using a reverse transcription reaction kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Then, qPCR was performed using an Mx 3000P qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd., Dalian, China) was used as a DNA-specific fluorescent dye. PCR was performed for 50 cycles of 95°C for 10 sec and 60°C for 30 sec. Primer sequences for detection of mRNA expression were synthesized as follows: YB-1 specific primers, 5-CAGAATAGTGAGAGTGGGG-3 (forward) and 5-ATGTAGTAAGGTGGGAACC-3 (reverse); human β-actin primers, 5-TTCCATATCGTCCCAGTTGGT-3 (forward) and 5-CCAGGGCGTTATGGTAGGCA-3 (reverse). The YB-1 transcriptional level was normalized against the transcriptional level of β-actin. All values are from the results of at least three independent experiments. Relative mRNA expression levels were calculated using the 2⁻∆∆^Cq method (25).

After placing a monolayer of cells on the climbing film, LL-37 (Sigma-Aldrich; Merck KGaA) of the appropriate concentration (0, 0.05, 0.5 or 5 µg/ml) was added for 48 h. Then, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, followed by treatment with 5% Triton X-100 for 15 min at room temperature. They were then sealed with 1% goat serum (200 µl; Thermo Fisher Scientific, Inc.) at 37°C for 30 min. Cells were incubated with a 1:50 diluted rabbit anti-human YB-1 monoclonal antibody overnight at 4°C. Then, they were incubated with a fluorescein isothiocyanate-labeled goat anti-rabbit antibody (1:2,000) at 37°C for 1 h. Finally, the cells were stained with DAPI for 1 min and the staining intensity was observed using a fluorescence inverted microscope (magnification, ×400; Zeiss LSM 700; Zeiss AG, Oberkochen, Germany).

LL-37-stimulated cells were treated with 100–400 µl of a mixture comprising PBS, 5 mM EDTA, 0.5% Triton X-100, 20 mM NaF, 1 mM orthovanadate, 1 mM pyrophosphate and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin, 25 µg/ml aprotinin) for 30 min at 4°C, then the cell lysates were separated via centrifugation (13,523 × g at 4°C for 20 min). Approximately 10 µg of the protein isolates were separated with 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk and 0.1% PBS with Tween-20 at room temperature for 2 h and then treated with rabbit anti-human monoclonal antibody YB-1 (1:500) at 4°C overnight. Subsequently, the membrane was hybridized with goat anti-rabbit secondary antibody (1:5,000) for 1 h. The results were detected using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA). The sample protein expression was presented relative to β-actin expression.

SCC cells were seeded at 10⁵ cells/well and treated with the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059 (10 µM; Abcam), the p38/mitogen-activated protein kinase (MAPK) inhibitor, SB203580 (10 µM; Abcam) or the NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 1 µM; Abcam) for 30 min. Then, 0.5 µM LL-37 was added and incubated at 37°C for 24 h. The protein was then extracted for western blot analysis (described above).

The cells were seeded at a density of 3×10³ cells/well with serum-free medium for 24 h. In order to evaluate the effect of dosage, LL-37 at concentrations of 0, 0.05, 0.5 or 5 µg/ml was used to stimulate the cells for 24 h. In order to evaluate the effect of treatment duration, 0.5 µg/ml LL-37 was used to stimulate the cells for 0, 24, 48 or 72 h. Then, MTT (5 mg/ml; 10 µl) was added and the cells were cultured for 4 h at 37°C. DMSO (150 µl) was added for 10 min. The absorbance was measured at 490 nm using a microplate reader.

The migration assay was conducted using a polycarbonate membrane with a pore size of 8 µm and Transwell chambers (EMD Millipore, Billerica, MA, USA). The cells (2.5×10⁵ cells/ml) were suspended in serum-free F12 culture medium. A 200 µl cell suspension was added to each Transwell chamber and 500 µl culture medium with 10% FBS was added to the lower chamber. In the upper chamber, a specific concentration of 0, 0.05, 0.05 or 5 µg/ml LL-37 was added. After 24 h, the residual cells on the surface were gently wiped out using a cotton swab. The cells that invaded the lower chamber were stained with staining solution (0.1% crystal violet ethanol) and observed under a light microscope (magnification, ×200). Three representative fields were randomly selected and the average number of invaded cells was calculated. With the pre-coated Matrigel (BD Biosciences) Transwell chambers, the invasion experiment was performed similar to the migration assay.

Statistical analysis was conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation. Comparisons between two groups were analysed using a two-tailed t-test. Comparisons among three or more groups were evaluated using analysis of variance and Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

In normal skin tissues and uninvolved skin tissues adjacent to SCC, YB-1 protein was strongly expressed in the basal layer and weakly expressed in the lower layers of the stratum spinosum (Fig. 1A and B). However, in SCC tissues, YB-1 was strongly expressed in nearly all tumour cells (Fig. 1C).

Total protein was extracted from siRNA-transfected cells and analysed by western blot analysis. Compared with the control group, the YB-1 siRNA 1 significantly inhibited the expression of YB-1 (P<0.05; Fig. 2A). Based on this result, the expression of YB-1 in A431 cells was then inhibited by transfection with YB-1 siRNA 1. Compared with the control group, the viability of the cells significantly decreased (P<0.05; Fig. 2B), which indicated that the viability of SCC cells was at least partly dependent on YB-1. The invasion ability of the A431 cells was also evaluated. Compared with the control group, the proportion of invaded cells significantly decreased (P<0.05; Fig. 2C), which suggested that the invasiveness of SCC cells was at least partly dependent on YB-1. Thus, the inhibition of YB-1 may reduce the viability and invasiveness of A431 cells.

A431 cells were stimulated with LL-37 (0, 0.05, 0.5, 5 or 20 µg/ml) and the effect on cell viability was observed at different time intervals (24, 48 or 72 h). Treatment with 0.5 µg/ml LL-37 significantly increased cell viability at both 24 and 48 h compared with the respective control groups (P<0.05 for both; Fig. 2D).

The effect of different LL-37 concentrations (0, 0.05, 0.5 or 5 µg/ml) on the migration of A431 cells was investigated. Compared with the control group, 0.05 µg/ml LL-37 significantly increased the migration of A431 cells (P<0.05; Fig. 3A). Furthermore, the effect of different LL-37 concentrations (0, 0.05, 0.5 or 5 µg/ml) on the invasiveness of A431 cells was investigated. Compared with the control group, 0.05 µg/ml LL-37 significantly enhanced the invasiveness of A431 cells (P<0.05; Fig. 3B).

qPCR was used to evaluate the effect of LL-37 treatment at different concentrations (0, 0.05, 0.5 or 5 µg/ml) and durations (0, 12, 24, 36 or 48 h) on the expression of YB-1 in SCC cells. Compared with the control group, stimulation of A431 cells with LL-37 for 24 or 36 h significantly increased the mRNA expression of YB-1 (P<0.05; Fig. 3C). When different concentrations of LL-37 treatment were evaluated, 0.05 µg/ml LL-37 resulted in a significant increase in YB-1 mRNA expression (P<0.05; Fig. 3C).

Western blot analysis indicated that treatment with LL-37 for 24 h and at a concentration of 0.5 µg/ml significantly increased the protein expression of YB-1 compared with the controls (P<0.05; Fig. 4A and B). Furthermore, stimulating A431 cells with various concentrations of LL-37 (0.05, 0.5 or 5 µg/ml) increased the fluorescence intensity of YB-1 immunostaining, and 0.5 µg/ml LL-37 was observed to cause the greatest increase (Fig. 4C). These results suggested that LL-37 promoted the expression of YB-1 protein in A431 cells.

In order to investigate the signaling pathway of LL-37 that induced the expression of YB-1, inhibition experiments were performed. A431 cells were pre-treated with the MEK inhibitor, PD98059, the p38/MAPK inhibitor, SB203580, and the NF-κB inhibitor, PDTC. Treatment with PDTC significantly inhibited the LL-37-induced expression of YB-1 in the A431 cells (P<0.05; Fig. 4D). This indicated that the increased expression of YB-1 by treatment with LL-37 involved the NF-κB signaling pathway.