Four patients, including two males and two females, between 42 and 78 years old with OSCC at clinical stage III or IV undergoing surgical resection at Linyi People’s Hospital (Linyi, China) between January 1 and July 1, 2013 were recruited, and written informed consent was obtained from all patients. Tumor tissue and adjacent non-tumor tissue samples were obtained from resected tumors and adjacent non-tumor esophageal tissue, respectively. The tissue was confirmed by pathological review. The present study was approved by the Ethics Committee of Linyi People’s Hospital.

The SCC-9, SCC-25, TSCCa and Tca-8113 OSCC cell lines, in addition to tongue epithelial cells (TEC) were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone; Thermo Fisher Scientific, Waltham, MA, USA), at 37°C in an atmosphere containing 5% CO₂ and 95% air.

The TSCCa cells were transfected with lentiviral vectors encoding short hairpin (sh)RNA targeting human AGK for AGK knockdown [AGK/RNA interference (RNAi)], or with a scrambled shRNA as a control (Scramble) (Sigma-Aldrich, St. Louis, MO, USA). The multiplicity of infection was 10, and the cells were cultured for 72 h post-transfection, and were grown to 80% confluency in 60-mm dishes. TSCCa cells were seeded in six-well plates at a density of 3×10⁵ cells/well in DMEM at 37°C overnight. A total of 10 ng AGK plasmid (ExAGK) or empty plasmid (ExControl) (Sigma-Aldrich) was transfected into the cells using Lipofectamine^® 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were cultured for 48 h post-transfection in DMEM containing with 10% FBS, at 37°C in an atmosphere containing 5% CO₂.

Nuclear or total protein was extracted from the OSCC cells and tissue samples using radioimmuno-precipitation assay buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). For immunoblotting, equal quantities of proteins (20 µg) were separated by 5–8% SDS-PAGE (Beyotime Institute of Biotechnology, Haimen, China) and were electrophoretically transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline-0.5% Tween 20 (TBST; Beyotime Institute of Biotechnology) supplemented with 5% milk (Beyotime Institute of Biotechnology) for 2 h at room temperature, prior to incubation with primary antibodies (Beyotime Institute of Biotechnology) overnight at 4°C. The following rabbit antibodies were used: Polyclonal anti-AGK (HPA020959; Sigma-Aldrich), polyclonal anti-cyclin D1 (#2922; Cell Signaling Technology, Inc.), polyclonal anti-p21 (SAB4500065; Sigma-Aldrich), polyclonal anti-retinoblastoma (Rb) (#9969; Cell Signaling Technology, Inc.) and monoclonal anti-phosphorylated (p)-Rb (#8180; Cell Signaling Technology, Inc.). All antibodies were used at dilutions of 1:1,000 unless stated. To control sample loading, the membranes were incubated with an anti-β-actin antibody (1:5,000; Sigma-Aldrich). The membranes were subsequently washed with TBST and incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (MFCD00162786; Sigma-Aldrich) for 2 h at room temperature. Immunocomplexes were visualized using the ECL Advance western blotting detection kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), according to the manufacturer’s instructions.

Total RNA was extracted from the OSCC cells using TRIzol (Qiagen China Co., Ltd., Shanghai, China). cDNA synthesis was performed according to the manufacturer’s instructions using the QuantiNova SYBR Green PCR kit (Qiagen China Co., Ltd.). qPCR was performed using the SYBR PCR kit (Qiagen China Co., Ltd) on a 7500 Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA, USA). The PCR reaction conditions for all assays were as follows: 95°C for 30 sec, followed by 40 cycles of amplification (95°C for 5 sec, 59°C for 30 sec and 72°C for 30 sec). Target RNA expression was normalized against β-actin mRNA expression. The primers were synthesized by GeneCopoeia Co. (Rockville, MD, USA). The primer sequences were as follows: AGK forward, 5′-CGA AGG CTT GCG TCC TAC TG-3′ and reverse, 5′-TGG TGG ACA GCT GCA CAT CT-3′ (4); cyclin D1 forward, 5′-AAC TAC CTG GAC GCT TCC T-3′ and reverse, 5′-CCA CTT GAG CTT GTT CAC CA-3′; p21 forward, 5′-CAT GGG TTC TGA CGG ACA T-3′ and reverse, 5′-AGT CAG TTC CTT GTG GAG CC-3′; and β-actin forward, 5′-CCA TGT ACG TTG CTA TCC AGG-3′ and reverse, 5′-TCT CCT TAA TGT CAC GCA CGA-3′. Expression data were normalized to the geometric mean of β-actin to control the variability in expression levels and were calculated as 2-[(Ct of AGK, CyclinD1 and p21) – (Ct of β-actin)].

Cell growth was assessed by MTT assay. The TSCCa cells were seeded in 96-well plates in DMEM supplemented with 10% FBS at ~2×10³ cells/well. For quantification of cell viability, the cultures were stained following 1, 2, 3, 4 and 5 days of incubation. Briefly, 20 µl 5 mg/ml MTT solution (Sigma-Aldrich) was added to each well and incubated at 37°C for 4 h. The medium was then removed from each well and the resulting MTT formazan crystals were solubilized in 150 µl dimethyl sulfoxide (Sigma-Aldrich). The absorbance was measured at a wavelength of 490 nm using a Multiskan plate reader (Thermo Fisher Scientific).

For the colony formation assay, TSCCa cells were plated in three 6-cm cell culture dishes (1×10³ cells/dish) and incubated for 12 days in medium supplemented with 10% FBS at 37°C in an atmosphere containing 5% CO₂. The plates were then washed with phosphate-buffered saline (PBS) and stained with Giemsa (Beyotime Institute of Biotechnology). The number of colonies containing >50 cells was manually counted in 10 fields using a Motic AE30 inverted fluorescence microscope (Microscope Systems Limited, Glasgow, UK) at magnification, x100.

Flow cytometric analysis was performed as previously described (8). Briefly, 48 h post-transfection, the TSCCa cells were harvested, washed with PBS and fixed with 70% ethanol. The fixed cells were subsequently treated with 20 µg/ml RNase A and 50 µg/ml propidium iodide (Sigma-Aldrich) for 30 min. The stained cells were immediately analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

All statistical analyses were performed using SPSS version 19.0 (IBM, Armonk, NY, USA). Continuous data were measured using Student’s t-test, and differences in measurements were compared using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To determine whether AGK contributed to the pathogenesis of OSCC, the present study examined the expression levels of AGK in OSCC cell lines and human OSCC tissue samples. The protein and mRNA expression levels of AGK were differentially upregulated in all four OSCC cell lines, as compared with normal TECs, and in all four OSCC patient tissue samples, as compared with the paired adjacent non-tumor tissue samples (Fig. 1). These results indicated that AGK may be overexpressed in OSCC. AGK expression levels were lower in the TSCCa cells than those in the SCC-9 and Tca-8113 cells, but were higher than those in the SCC-25 cells. Therefore, TSCCa appeared to be the best-fit cellular model for evaluating the up- and downregulation of the expression levels of AGK.

To determine whether AGK promoted the proliferation of OSCC cells, plasmid transfection and shRNA knockdown were performed in TSCCa cells. Transfection with the AGK plasmid significantly upregulated the protein and mRNA expression levels of AGK, whereas transfection with the AGK shRNA decreased AGK protein and mRNA expression levels in the TSCCa cells (Fig. 2A and B). The proliferation rate of the TSCCa cells was determined using an MTT assay. AGK upregulation promoted cell growth, whereas a significant decrease in proliferation was observed in the TSCCa cells transfected with the AGK shRNA, as compared with the negative control (P<0.05; Fig. 2C). Consistent with the results of the MTT assay, the colony formation assay demonstrated that AGK overexpression in TSCCa cells induced an increase in foci numbers, whereas AGK knockdown decreased the colony forming ability of the cells (P<0.05; Fig. 2D).

To investigate the mechanisms underlying cell proliferation, the effects of AGK on cell cycle progression, and the expression of cell cycle-associated proteins cyclin D1, p21, Rb and p-Rb were evaluated. CDK inhibitor p21 and cyclin D1 are two proteins targeted by AGK, which negatively and positively modulate cell cycle progression and proliferation, respectively (6). Cyclin D1 exerts its effects by binding the CDK subunits 4 and 6, resulting in the formation of a complex, which induces successive phosphorylation of the Rb protein (9). The results of the present study demonstrated that the overexpression of AGK resulted in upregulation of the protein and mRNA expression levels of cyclin D1, and increased the expression levels of p-Rb; while p21 expression levels were downregulated (Fig. 3). Conversely, knockdown of AGK resulted in reduced mRNA and protein expression levels of cyclin D1 and p-Rb, whereas the expression levels of p21 were increased (Fig. 3). These results suggested that AGK influenced p21 and cyclin D1 expression in way that means increases in AGK expression may be able to accelerate cell cycle progression and cell proliferation. In order to evaluate this hypothesis, the present study aimed to analyze cell cycle distribution. The percentage of cells in the G₀/G₁ phase was demonstrated to be decreased in those cells overexpressing AGK; whereas it was increased in the AGK knockdown cells. Conversely, the percentage of cells in the S phase was increased in the cells transfected with AGK plasmid and decreased in the cells transfected with AGK shRNA (Fig. 4). These results suggested that AGK expression may promote malignant cancer growth by regulating the expression of cyclin D1 and p21, during the G₁-S phase transition.