AMC-HN-8 and OME cells were sourced from the Cell Bank, China Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and incubated in the Thermo forma incubator (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. The cells were grown to 50% confluence. Cells were then transfected with three sets of Jab1-specific siRNAs (Shanghai GenePharma Co., Ltd., Shanghai, China) or negative control siRNA (siCtrl; Shanghai GenePharma Co., Ltd.), using siLentFect Lipid reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The quantity of the siRNAs used for transfection was 200 pmol for each 35 mm culture dish. The target sequences of the Jab1-specific siRNAs used were as follows: Jab1-siRNA1 (si1-Jab1) sense, 5′-CCAGACUAUUCCACUUAAUTT-3′ and antisense, 5′-AUUAAGUGGAAUAGUCUGGTT-3′; Jab1-siRNA2 (si2-Jab1) sense, 5′-GGUGAAACCAUGAUCAUUTT-3′ and antisense, 5′-UAAUGAUCAUGGUUUCACCTT-3′; Jab1-siRNA3 (si3-Jab1) sense, 5′-GGACUAAGGAUCACCAUUATT-3′ and antisense, 5′-UAAUGGUGAUCCUUAGUCCTT-3′.

At 4–6 h after transfection, the culture medium (DMEM) containing the transfection reagent was removed, and fresh medium containing FBS was added. After 48 h of culture, cells were lysed and used for subsequent experiments.

At 48 h after transfection with each si-Jab1, 1 ml of lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) was added to each dish for 30 min at 4°C, with occasional shaking. Then, cell lysates were collected into a 1.5 ml tube followed with centrifugation at 16,000 × g for 15 min at 4°C. The protein concentration was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total proteins were boiled with loading buffer (Vicmed Biotech Co., Ltd., Xuzhou, China) and then loaded (100 µg/lane) and separated by SDS-PAGE (12.5% gel) and transferred onto a nitrocellulose membrane. The membranes were then blocked for 2 h at room temperature in 5% non-fat milk (BD Biosciences, Franklin Lakes, NJ, USA) suspended in tris-buffered saline with Tween-20 (TBST; 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.05% Tween-20). Following blocking, the membranes were incubated with anti-cleaved caspase-3 (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-Jab1, anti-p53, anti-Akt, anti-p-Akt and anti-β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies at 4°C overnight. Membranes were then washed using TBST and incubated with the horseradish peroxidase-conjugated mouse anti-rabbit IgG secondary antibodies (cat no. A2074; 1:20,000; Sigma; Merck KGaA, Darmstadt, Germany) at room temperature for 2 h. The protein bands were visualized with Enhanced Chemiluminescence reagent (Tanon Science and Technology Co., Ltd., Shanghai, China). The densitometric analysis for the quantification of the bands was performed using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).

Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology). In brief, at 48 h post-transfection, 5,000 cells were seeded in 96-well plates with medium containing 10% FBS and incubated for 1, 2, 3 and 4 days according to the manufacturer's protocol. Then, 10 µl CCK-8 solution and 100 µl serum-free culture medium (DMEM) were mixed and added to each well, followed by incubation at 37°C for 2 h. The absorbance was read at 450 nm using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The experiments were performed in triplicate.

AMC-HN-8 cells were plated in 6-well plates and transfected with si1-Jab1. si1-Jab1 was selected for this and subsequent experiments as it yielded the most marked decrease in Jab1 expression of all three Jab1-specific siRNAs. Following transfection, treated cells were collected in a 1.5 ml tube and washed twice with phosphate buffered saline. Following washing, cells were labeled with 200 µl binding buffer (BD Biosciences) containing 5 µl Annexin V-fluorescein isothiocyanate (FITC) and added to 300 µl binding buffer containing 5 µl propidium iodide (PI) at room temperature in darkness for 5 min. Cells were visualized under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The software used for analysis was Olympus cellSens Dimension (version 1.0; Olympus Corporation). A total of 3 independent experiments were conducted and 5 horizons were randomly selected from each group, at a magnification of ×100.

AMC-HN-8 cells were plated in 6-well plates and transfected with si1-Jab1 for 48 h. The cells were collected and labeled using an Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences), according to the manufacturer's protocol. The apoptotic cell fraction was detected using a FACScan flow cytometer (BD Biosciences). Data were analyzed using ModFit LT 3.0 software (Verity Software House, Inc., Topsham, ME, USA). Living cells (Annexin V⁻FITC⁻/PI⁻), early apoptotic cells (Annexin V⁻FITC⁺/PI⁻), late apoptotic cells (Annexin V⁻FITC⁺/PI⁺) and necrotic cells (Annexin V⁻FITC⁻/PI⁺) were enumerated.

Data were analyzed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). The relevant data are expressed as the mean ± standard deviation (SD). Results were analyzed using Student's t-test when only 2 groups were compared, or one-way analysis of variance when >2 groups were compared. The post hoc test used was the Student-Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

Western blot analysis was used to detect the expression of Jab1 in normal human oral mucosal epithelial (OME) and AMC-HN-8 cells. As presented in Fig. 1, the expression level of Jab1 protein was increased in AMC-HN-8 cells compared with OME cells, suggesting that, although Jab1 can be expressed in normal cells, its levels are increased in laryngeal cancer cells.

Jab1 expression was knocked down in AMC-HN-8 cells using three different sets of siRNAs targeting Jab1 (si1-Jab1, si2-Jab1 and si3-Jab1). Jab1 protein expression was evaluated using western blot analysis and it was identified that Jab1 was significantly downregulated in si1-Jab1 and si2-Jab1 groups compared with the siCtrl group (Fig. 2). However, si1-Jab1 exhibited the most marked effect in decreasing the expression levels of Jab1 and was therefore used in subsequent experiments.

The effect of Jab1 on carcinoma cell proliferation was investigated. Following transfection of AMC-HN-8 cells with Jab1 siRNA, a CCK-8 assay was performed and a significant decrease in cellular proliferation was identified on days 3 and 4 (Fig. 3). This suggests that loss of Jab1 expression has an inhibitory effect on the proliferation of laryngeal cancer cells.

On the basis of the cell proliferation results, it was investigated whether the loss of Jab1 expression may also have an effect on the apoptosis of laryngeal cancer cells. An Annexin V-FITC binding assay and fluorescence microscopy were used to analyze changes in the total apoptotic cells including early apoptotic cells (Annexin V⁻FITC⁺/PI⁻) and late apoptotic or necrotic cells (Annexin V⁻FITC⁺/PI⁺). It was identified that the si1-Jab1-treated cells exhibited enhanced apoptosis at prophase (Fig. 4A and B) and anaphase (Fig. 4C and D) compared with the control group. Apoptosis was additionally analyzed between the two groups by flow cytometry. According to the results, it was observed that si1-Jab1 treatment increased the apoptotic cell fraction in AMC-HN-8 cells (Fig. 4E and F). In particular, the Annexin V⁻FITC⁺/PI⁻ early apoptotic cell fraction accounted for 4.4% of the cells in the control and 7.3% of the cells in the si1-Jab1-treated group. Annexin V⁻FITC⁺/PI⁺ late apoptotic cells accounted for 2.9% of the cells in the control and 6.5% of the cells in the si1-Jab1 group, 48 h post-transfection. These results suggest that decreasing the expression of Jab1 protein facilitates the apoptosis of laryngeal cancer cells.

To understand the mechanisms involved in Jab1-mediated apoptosis and proliferation in carcinoma cells, the effect of Jab1 deficiency on certain key factors involved in anti-apoptotic pathways and the apoptotic cascade, including Akt, p-Akt, c-caspase-3 and p53, were examined. Western blot analysis revealed that the level of p-Akt expression was downregulated, whereas c-caspase-3 and p53 were upregulated in the Jab1-knockdown group in comparison with the control group (Fig. 5). These results indicate that these proteins may be involved in Jab1-mediated proliferation and apoptosis in AMC-HN-8 cells.