Thirty pairs of LSCC and corresponding adjacent normal tissues, used for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, were collected from the Ear, Nose and Throat department of The 463 Hospital of PLA (Shenyang, China) between January 2009 and December 2014, following receipt of written informed consent. Tissue samples were obtained from 24 males and 6 females (mean age, 64.72 years; range, 45–83 years) and included 6 cases of stage I LSCC, 6 cases of stage II LSCC, 8 cases of stage III LSCC and 10 cases of stage IV LSCC. Tumors were staged according to the International Union Against Cancer TNM classification for malignant tumors (22). All tissue samples, including cancer tissues and matched adjacent normal tissues (typically removed from areas 4–15 mm from the tumors), were obtained during surgery. All specimens were frozen and stored at −80°C prior to use. Approval for this study was obtained from the Ethics Committee of China Medical University (Shenyang, China).

The Hep2 human laryngeal carcinoma cell line, was obtained from the Shanghai Institute for Biochemistry, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 (Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, 100 µg/ml penicillin and 100 µg/ml streptomycin (all obtained from GE Healthcare Life Sciences, Logan, UT, USA) in humidified 5% CO₂ at 37°C. Trypsin solution (0.25%; GE Healthcare Life Sciences) was used to detach cells from the culture flask.

Three siRNAs targeting human FOXQ1 and a negative control siRNA (FOXQ1-NC), were designed and obtained from GenePharma Co., Ltd. (Shanghai, China). The siRNAs and FOXQ1-NC siRNA (FOXQ1-siRNA1, 5′-CGCGGACTTTGCACTTTGA-3′; FOXQ1-siRNA2, 5′-AGGGAACCTTTCCACACTA-3′; FOXQ1-siRNA3, 5′-CCATCAAACGTGCCTTAAA-3′; and FOXQ1-NC siRNA, 5′-TTCTCCGAACGTGTCACGT-3′) were used to inhibit the expression of FOXQ1. Preliminary experiments indicated that FOXQ1-siRNA1 most effectively down-regulated FOXQ1 expression. This sequence was therefore selected for subsequent experiments. Hep2 cell were seeded in 6-well plates at a density of 0.5×10⁶ cells/well. FOXQ1-siRNA, FOXQ1-NC and mock group (blank control ± transfection reagent) were transfected into Hep2 cells using lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. Following transfection for 72 h, cells were collected for subsequent experiments.

Total RNA was extracted using TRIzol™ reagent (Invitrogen Life Technologies) for analysis of FOXQ1 and GAPDH mRNA expression, according to the manufacturer's instructions. RNA was reverse transcribed, using the Reverse Transcription PCR kit with Oligo-dT primers and RT-qPCR was conducted, using SYBR-Premix Ex Taq™ (Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. For detection of FOXQ1-mRNA expression, qPCR was performed under the following conditions: Denaturation at 95°C for 30 sec, followed by 40 cycles of amplification (annealing at 95°C for 5 sec and elongation at 60°C for 30 sec). GAPDH was used to normalize FOXQ1-mRNA expression levels using the 2^−ΔΔCt method. The following primers were used: Forward, 5′-ATTTCTTGCTATTGACCGATGC-3′ and reverse, 5′-CCCAAGGAGACCACAGTTAGAG-3′ for FOXQ1 and forward, 5′-GGAAGATGGTGATGGGATT-3′ and reverse, 5′-GGATTTGGTCGTATTGGG-3′ for GAPDH. All primers were purchased from Takara Bio, Inc.

Western blot analysis was performed according to standard procedures. In brief, protein was isolated from tissue samples or cells. Protein concentration was determined using a bicinchoninic acid Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins were fractionated using SDS-PAGE (Invitrogen Life Technologies) and transferred to PVDF membranes (Beyotime Institute of Biotechnology, Haimen, China). After blocking with 5% milk in Tris-buffered saline with Tween-20 (TBST; Invitrogen Life Technologies), membranes were incubated with a polyclonal rabbit anti-human FOXQ1 antibody (cat. no. sc-134549; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution over 3 h. The membranes were then washed thrice with TBST, and incubated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit (cat. no. KC-MM-095) or goat anti-mouse (cat. no. KC-MM-035) secondary antibodies (KangCheng, Shanghai, China) at a 1:2,000 dilution for 2 h at room temperature. The membranes were also stripped and blotted with a monoclonal mouse anti-human β-actin antibody (cat. no. A5316; Sigma-Aldrich, St. Louis, MO, USA) at a 1:1,000 dilution, as a loading control. Blots were developed with enhanced chemiluminescence and chemiluminescence detection film (Beyotime Institute of Biotechnology).

Hep2 cells were transfected with mock, FOXQ1-NC and FOXQ1-siRNA, and cells were seeded in 96-well plates at 4,000 cells per well. The proliferating cells were measured using a Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology), at 2, 4, 6 and 8 days following transfection. Cells were incubated at 37°C for 2 h following the addition of 10 µl CCK-8 to each well and the absorbance at 450 nm was detected using a microplate reader (MK3; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

For analysis of cell cycle progression and apoptosis, mock and transfected cells were fixed in 70% cold ethanol for 30 min. After washing with cold phosphate-buffered saline (PBS) 3 times, the samples were centrifuged at 500 × g for 5 min. The pellets were then suspended and stained with 10 mg/l propidium iodide and 100 mg/l RNase for 20 min. The distribution of cells in each phase of the cell cycle and the proportion of apoptotic cells were analyzed using FACScan cytometry (Becton Dickinson, San Jose, CA, USA).

Following transfection for 24 h, 2×10⁵ Hep2 cells were suspended in culture medium with 1% FBS and plated in the upper chamber of the Transwell plate with matrigel-coated membrane (Becton Dickinson). Cells were incubated for 36 h, following which, cells that had not invaded through the filter were removed. Cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min, then washed with PBS and stained using hematoxylin and eosin, according to the manufacturer's instructions. The number of cells on the membrane were counted under a microscope (CX31; Olympus Corporation, Tokyo, Japan). The number of migrated cells was expressed as the mean value of five randomly-selected fields. Each experiment was repeated three times.

All values in the present study are reported as the mean ± standard deviation of three independent experiments. The paired samples t-test was used to compare the expression of FOXQ1 mRNA and protein between LSCC and adjacent tissues, while one-way analysis of variance and Student's t-test were used to compare values between the experimental and control groups, using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Total RNA was extracted from 30 pairs of LSCC tissues and adjacent normal tissues and subjected to RT-qPCR in order to measure the expression of FOXQ1 mRNA. Following normalization to GAPDH, the mean expression of FOXQ1 mRNA in LSCC tissues was significantly higher than that in adjacent normal tissues (1.54±0.66 vs. 0.75±0.28; P<0.05; Fig. 1A). FOXQ1 protein expression was also measured by western blotting in the same samples in which FOXQ1 mRNA expression was measured. The results demonstrated that FOXQ1 protein expression was increased in 19 of 30 LSCC tissues (~63%), compared with matched adjacent normal tissues. FOXQ1 protein expression was higher in LSCC tissues than that in adjacent normal tissues (Fig. 1B; P<0.05). These findings were in accordance with the FOXQ1 mRNA expression data. By contrast, analysis of the association of FOXQ1 expression with characteristics, such as patient age, gender and tumor stage, revealed no significant associations between these variable (data not shown).

Following transfection of Hep2 cell with FOXQ1 siRNA for 72 h, the expression of FOXQ1 mRNA and protein was detected by RT-qPCR and western blotting. Cells were also transfected with FOXQ1-NC as a negative control. The results are shown in (Fig. 2). Following transfection with FOXQ1 siRNA, Hep2 cells exhibited significant downregulation of FOXQ1 expression at the mRNA and protein levels (Fig. 2A and B; P<0.05).

Cell proliferation was determined using a CCK-8 assay. The results demonstrated that downregulation of FOXQ1 in Hep2 cells resulted in a significant reduction in cellular proliferation at 4, 6 and 8 d after transfection (P<0.05). This indicates that suppression of FOXQ1 correlates with decreased proliferation of Hep2 cells (Fig. 3).

Flow cytometric analysis of the cell cycle demonstrated that inhibition of FOXQ1 in Hep2 cells reduced the proportion of cells in the S and G2/M phases, and more cells were arrested in the G0/G1 phase compared with cells in the control group (Table I). Furthermore, apoptosis of FOXQ1-NC- and FOXQ1-siRNA-transfected cells was examined using flow cytometry. As shown in Table I, after 4 days, 2.42% and 2.84% of control cells and FOXQ1-NC cells were apoptotic, respectively, while 2.95% of FOXQ1-siRNA cells were apoptotic. No significant difference in the level of apoptosis in Hep2 cells was detected among these groups.

The results of the matrigel invasion assay demonstrated that the number of migrating cells was significantly decreased in the FOXQ1-siRNA transfection group, compared with that in the control group. The numbers of invading cells in the mock and FOXQ1-NC groups were 21.46±3.35 and 19.29±3.16, respectively, which were significantly higher than the number in the FOXQ1-siRNA group (10.24±2.52; P<0.01; Fig. 4).