Hep-2 and LSC-1 human laryngeal cancer cell lines (Wanleibio, Shenyang, China) were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA). The cells were passaged by trypsinization when they reached 80–90% confluence.

The following primers were designed for amplification of the coding region of the ECRG4 gene: ECRG4-F, 5′-ATAGAAGCTTGCCCCTCGCCCTC-3′ (HindIII restriction site underlined); and ECRG4-R, 5′-CCGGATCCTCAGAAACCAAGTAGTG-3′ (BamHI restriction site underlined). The human cDNA preserved in the laboratory was utilized as the template. The amplified ECRG4 gene was ligated into the pcDNA3.1 plasmid (Invitrogen Life Technologies, Carlsbad, CA, USA). The recombinant plasmid, pcDNA3.1-ECRG4, was digested with HindIII and BamHI, according to the manufacturer's instructions (Fermentas, Ontario, Canada). The recombinant plasmid was sequence-analyzed by Sangon Biotech (Shanghai, China). After the double restriction analysis and sequence analysis, the correct recombinant plasmid was termed pcDNA3.1-ECRG4. Logarithmically growing Hep-2 cells were seeded into 6-well plates. Upon reaching ~80% confluence, the cells were transfected with pcDNA3.1-ECRG4 or negative control (pcDNA3.1 plasmid; Invitrogen Life Technologies) using Attractene Transfection Reagent (Qiagen, Shanghai, China) according to the manufacturer's instructions. At 24 h after transfection, the cells were subjected to G418 selection (400 µg/ml, Invitrogen Life Technologies). After 7–14 days, positive clones were selected and ECRG4 expression was examined.

Total RNA was extracted from all groups of cells using the total RNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA and then subjected to quantitative fluorescence analysis on the Exicycler™ 96 quantitative fluorescence analyzer (Bioneer, Daejeon, Korea) using the SYBR Green MasterMix (Solarbio, Beijing, China). RT-qPCR was performed in a volume of 20 µl, containing 0.5 µl each primer (10 µM), 1 µl cDNA template, 10 µl SYBR GREEN master mix and 8 µl ddH₂O. Thermal cycling conditions were as follows: Initial denaturation at 95°C for 10 min; amplification for 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. The mixture was then cooled at 4°C for 5 min. The internal control was β-actin and each sample was tested in triplicate. The 2^−ΔΔCt method (17) was used for relative quantification of gene expression. The sequences of the primers are listed in Table I.

The total protein was extracted from all groups of cells, and the protein concentration was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal quantities of protein were loaded onto each lane, separated using 10 or 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated first with the following primary antibodies: Rabbit anti-human anti-ECRG4 polyclonal antibody (1:200; cat. no. sc-135139; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human anti-cleaved-caspase-3 polyclonal antibody (1:500; cat. no. bs-0081R; Bioss, Beijing, China), rabbit anti-human anti-cleaved-poly ADP-ribose polymerase (PARP) polyclonal antibody (1:200; cat. no. sc-23461-R; Santa Cruz Biotechnology, Inc.), rabbit anti-human anti-Bcl-2-associated X protein (Bax) polyclonal antibody (1:400; cat. no. BA0315; Boster, Wuhan, China) and rabbit anti-human anti-B-cell lymphoma 2 (Bcl-2) polyclonal antibody (1:400; cat. no. BA0412; Boster) at 4°C overnight. The membranes were washed four times with Tween-20 in Tris-buffered saline for 5 min. They were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000 dilution, Beyotime Institute of Biotechnology) for 45 min at 37°C. After addition of the enhanced chemiluminescence (ECL) reagents (Qihai Biotec, Shanghai, China), the target proteins were visualized and scanned using Gel-Pro Analyzer software 4.0 (Media. Cybernetics, Inc., Bethesda, MD, USA). The detected proteins were normalized to β-actin.

Cells grown on coverslips were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 30 min and incubated with rabbit anti-human anti-ECRG4 polyclonal antibody (1:100; cat. no. sc-135139; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The cells were then incubated with Cy3-labeled goat anti-rabbit secondary antibody (1:100 dilution; Beyotime Institute of Biotechnology) for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (Biosharp, Hefei, China) was added drop-wise to completely cover the cells for nuclei staining. The coverslip with cells was inversely placed and mounted on a slide with anti-fluorescence quenching agent (Solarbio). The staining results were observed under a laser scanning confocal microscope and imaged (FV1000S-SIM/IX81; Olympus, Tokyo, Japan).

Cells from each experimental group were plated in 96-well plates at a density of 2×10³ cells/well. Five replica wells were set up for each experimental group. Blank control wells were also included. At days 0, 1, 2, 3 and 4 after cell inoculation, MTT solution (final concentration of 0.2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added to each well containing cells. After incubation at 37°C for 5 h, the supernatant was removed, and 200 µl dimethylsulfoxide (Sigma-Aldrich) was added to each well to dissolve the purple crystals. The optical density at 490 nm (OD₄₉₀) was determined using a microplate reader (ELX-800; Bio-TEK Instruments Inc, Winooski, VT, USA), and cell growth curves were constructed.

Cells from each group were seeded at ~300 cells per 60 mm Petri dish. The Petri dishes were incubated at 37°C and 5% CO₂ until visible colonies were formed. The colonies were fixed in 4% paraformaldehyde, stained with Wright-Giemsa staining reagent (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and observed under a microscope (AE31; Motic Electric, Xiamen, China). A cell cluster containing at least 50 cells was counted as a colony. The colony formation rate was calculated based on the following formula: Colony formation rate (%) = (number of colonies/number of seeded cells) × 100.

Cells were trypsinized, harvested and fixed in precooled 70% ethanol at 4°C for 2 h. The fixed cells were centrifuged at 252 × g, resuspended in 500 µl staining buffer and mixed thoroughly with 25 µl propidium iodide (PI) staining solution according to the instructions of the kit (Beyotime Institute of Biotechnology). Subsequently, 10 µl RNase A (Beyotime Institute of Biotechnology) was added to the cell suspension. After incubation at 37°C for 30 min in the dark, the cells were processed for flow cytometry (FACSCalibur; Becton, Dickinson and Company, USA).

Cells from each experimental group were seeded onto coverslips in 12-well plates at a density of 3×10⁴ cells/well and cultured for 48 h in an incubator at 37°C and 5% CO₂. The cells were then fixed, stained with Hoechst solution (Beyotime Institute of Biotechnology), covered with drops of anti-quenching mounting solution and mounted onto microscope slides. The slides were observed under a fluorescence microscope (BX61; Olympus) and photographed.

Cells were trypsinized, harvested and resuspended in 500 µl Binding Buffer according to the instructions of the apoptosis detection kit (Wanleibio). A total of 5 µl Annexin V-fluorescein isothiocyanate was added to the cell suspension and mixed thoroughly. Subsequently, 5 µl PI was added. After incubation for 15 min at room temperature in the dark, the cells were analyzed by flow cytometry (FACSCalibur; Becton, Dickinson and Company) for determination of apoptosis rate.

Experimental data are expressed as the mean ± standard deviation. Comparisons between the experimental groups were conducted using one-way analysis of variance. Multiple comparisons were conducted using the Bonferroni post hoc test. Data analysis and image processing were performed using the Graphpad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To select a suitable human laryngeal cancer cell line for stable transfection and subsequent ECRG4 gene-related experiment, the total protein from Hep-2 and LSC-1 cells was extracted. The expression levels of ECRG4 in the two cell lines were examined using western blot analysis. The results showed that the ECRG4 expression level in Hep-2 cells was significantly lower than that in LSC-1 cells (Fig. 1, P<0.05). To eliminate the effect of basal expression of ECRG4, Hep-2 cells were selected for subsequent experiments.

To investigate the function of the ECRG4 gene, pcDNA3.1-ECRG4 was transfected into Hep-2 cells, and ECRG4 expression in positive cells was analyzed by western blot analysis and RT-qPCR. Cells transfected with empty pcDNA3.1 vector and parental cells were used as controls. The results showed that the expression levels of ECRG4 protein and mRNA in the pcDNA3.1-ECRG4 group were increased by 3.05 (Fig. 2A, P<0.01) and 3.07-fold (Fig. 2B, P<0.01), respectively, compared with the pcDNA3.1 group. The immunofluorescence staining results showed that ECRG4 expression was obviously elevated in the pcDNA3.1-ECRG4 group compared with the other two groups (Fig. 2C), which were consistent with the western blot analysis and RT-qPCR results. Thus, a laryngeal cancer cell line stably overexpressing ECRG4 was established.

To investigate the effect of ECRG4 overexpression on the proliferative capability of laryngeal cancer cells, cell proliferative capability was examined using the MTT assay and colony formation assay. The results of the MTT assay showed that at day 2, day 3 and day 4, the proliferative capability was severely impaired in the pcDNA3.1-ECRG4 group compared with the pcDNA3.1 group (Fig. 3A; day 2, P<0.05; days 3 and 4, P<0.01). The effect of ECRG4 on the clonogenic capacity of laryngeal cancer cells was further examined using the colony formation assay. The results showed that the colony formation rate in the pcDNA3.1-ECRG4 group was 45.27±7.19%, which was lower than that in the pcDNA3.1 group (83.07±7.51%). The results indicated that ECRG4 significantly reduced the colony formation ability of laryngeal cancer cells (Fig. 3B, P<0.01). This study further investigated the cell-cycle phase distribution in all three groups of cells using flow cytometry. As shown in Fig. 3C, the percentage of cells in the G0/G1 phase was significantly increased in the pcDNA3.1-ECRG4 group compared with that in the pcDNA3.1 group (71.7 vs. 39.41%, P<0.01). By contrast, the percentage of cells in the S phase and G2/M phase were decreased markedly in the pcDNA3.1-ECRG4 group (S phase, 20.14 vs. 44.83%, P<0.01; and G2/M phase, 8.17 vs. 15.76%, P<0.05, respectively). These results indicated that overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and arrested cells in the G0/G1 phase of the cell cycle.

Cell apoptosis was measured by flow cytometry and fluorescence microscopy using Annexin V/PI and Hoechst staining, as well as the analysis of the expression of apoptosis-related factors. The results of flow cytometric analysis showed that the apoptotic rate was significantly elevated in the pcDNA3.1-ECRG4 group compared with the pcDNA3.1 group (19.37±0.67 vs. 0.66±0.09%; Fig. 4A, P<0.01). Hoechst staining showed that compared with cells in the parental group and the pcDNA3.1 group, cells in the pcDNA3.1-ECRG4 group exhibited significantly increased chromatin condensation and more densely stained nuclei (Fig. 4B). To determine whether ECRG4-induced apoptosis of laryngeal cancer cells affected the expression of apoptosis-related factors, western blot analysis was performed to examine the expression of Bax, Bcl-2, cleaved-caspase-3 and cleaved-PARP. The results showed that the expression levels of cleaved-PARP, cleaved-caspase-3 and Bax were significantly elevated in cells from the pcDNA3.1-ECRG4 group compared with that from the pcDNA3.1 group (Fig. 4C, P<0.01). By contrast, the expression level of Bcl-2 was markedly decreased (P<0.01). These results demonstrated that overexpression of ECRG4 significantly induced apoptosis in laryngeal cancer cells.