The human OSCC cell lines (Ho-1-u-1, KOSC2, HSC-4, HSC-3 and HSC-2) were purchased from the Human Science Research Resources Bank, Osaka, Japan. Primary cultured human normal oral keratinocytes (HNOKs) served as normal controls (8–17). All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 Ham (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 50 U/ml penicillin and streptomycin (Sigma) in a humidified 5% CO₂/air atmosphere at 37°C (18–20).

One hundred and twenty-eight pairs of primary OSCC samples and corresponding normal oral epithelial tissues were obtained intraoperatively at Chiba University Hospital, Chiba, Japan. All patients provided informed consent for use of the protocol, which the institutional review board of Chiba University reviewed and approved. The resected tissues were divided into two parts, one of which was frozen immediately and stored at −80°C until RNA isolation and the second was fixed in 20% buffered formaldehyde solution for pathologic diagnosis and immunohistochemistry (IHC). Histopathological diagnosis of each tissue was performed according to the World Health Organization criteria by the Department of Pathology, Chiba University Hospital. Clinicopathological staging was determined according to the tumor-node-metastases classification of the International Union against Cancer. All OSCC samples were confirmed histologically and checked to ensure the presence of tumor in >90% of specimens.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was generated from 5 μg of total RNA using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Buckinghamshire, UK) and oligo(dT) primer (Hokkaido System Science, Sapporo, Japan) according to the manufacturer’s instructions.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to evaluate the expression level of POFUT1 mRNA in the OSCC-derived cell lines and HNOKs. Primary OSCCs and paired specimens of normal oral tissues obtained from 128 patients also were evaluated. The expression levels were determined using primers and probes that were designed using the Universal Probe Library (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s recommendations. The primer sequences used for qRT-PCR were: POFUT1, forward 5′-CTGATGACCCGATGGTAAGC-3′; reverse 5′-AAG CCTCCTTTCACCAACCT-3′; and universal probe no. 78. All qRT-PCR analyses were performed using a LightCycler^® 480 PCR system (Roche Diagnostics). Amplifications were initiated by a 10-min pre-incubation at 95°C, followed by 45 cycles of 10 sec at 95°C for template denaturation, 30 sec at 55°C for primer annealing/extension and cooling for 30 sec at 40°C. The transcript amount for POFUT1 was estimated from the respective standard curves and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5′-CAT CTCTGCCCCCTCTGCTGA-3′; reverse, 5′-GGATGACCTT GCCCACAGCCT-3′; and universal probe no. 60) transcript amount determined in corresponding samples.

The cells were washed twice with cold phosphate buffered saline (PBS) and centrifuged at 500 × g. The cell pellets were incubated at 4°C for 30 min in a lysis buffer [7 M urea, 2 M thiourea, 4% w/v CHAPS and 10 mM Tris (pH 7.4)] with the proteinase inhibitor cocktail (Roche Diagnostics). The protein concentration was measured with a protein assay (Bio-Rad Laboratories, Hercules, CA, USA).

Cytoplasmic and nuclear proteins (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4–12% gel, transferred to nitrocellulose membranes and blocked for 1 h at room temperature in Blocking One (Nacalai Tesque, Tokyo, Japan). The membranes were incubated with anti-POFUT1 polyclonal antibody (OriGene Technologies, Rockville, MD, USA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies for 4 h at room temperature. The membranes were washed with 0.1% Tween-20 in Tris-buffered saline, incubated with secondary antibodies and coupled to horseradish peroxidase-conjugated anti-rabbit IgG (Promega, Madison, WI, USA) in Blocking One for 1 h at room temperature. The proteins were detected by SuperSignal Chemiluminescent substrate (Thermo, Waltham, MA, USA). Finally, immunoblotting results were visualized by exposing the membrane to a cooled CCD camera system (Light Capture^®, ATTO, Tokyo, Japan). Signal intensities were quantitated using the CS Analyzer^® version 3.0 (ATTO).

IHC was performed on 4-μm sections of paraffin-embedded specimens using rabbit anti-POFUT1 polyclonal antibody (OriGene Technologies). Briefly, after deparaffinization and hydration, the endogenous peroxidase activity was quenched by 30-min incubation in a mixture of 0.3% hydrogen peroxide solution in 100% methanol. The sections were blocked for 2 h at room temperature with 1.5% blocking serum (Santa Cruz Biotechnology) in PBS before reacting with anti-POFUT1 antibody (1:1,000 dilution) at 4°C in a moist chamber overnight. Upon incubation with the primary antibody, the specimens were washed three times in PBS and treated with Envision reagent (Dako, Carpinteria, CA, USA) followed by color development in 3,3′-diaminobenzidine tetrahydrochloride (Dako). Finally, the slides were counterstained lightly with hematoxylin, dehydrated with ethanol, cleaned with xylene and mounted. Non-specific binding of the antibody to proteins other than the antigen sometimes occurred. As a negative control, triplicate sections were immunostained without exposure to primary antibody, which confirmed the staining specificity. To quantify the state of POFUT1 protein expression in those components, we used IHC scoring systems described previously (6,8–17). The intensity of the POFUT1 immunoreaction was scored as follows: 1+, weak; 2+, moderate; and 3+, intense. The cell numbers and the staining intensity then were multiplied to produce the POFUT1 IHC score. Cases with a POFUT1 IHC score exceeding 71.5 [the maximal score within +3 standard deviations (SD) of the mean of normal tissues] were defined as POFUT1-positive. The ±3 SD cutoff, which statistically is just 0.2% of the measurement and expected to fall outside this range, was used because it was unlikely to be affected by a random experimental error produced by sample manipulation (21). Two independent pathologists, neither of whom had knowledge of the patients’ clinical status, made these judgments.

The OSCC cell lines, KOSC2 and HSC-2, in which POFUT1 protein expression was higher than in the other cell lines, were stably transfected with POFUT1 shRNA (shPOFUT1) or control shRNA (mock) (Santa Cruz Biotechnology) using Lipofectamine LTX and Plus Reagents (Invitrogen). The stable transfectants were isolated by the culture medium containing 1 mg/ml puromycin (Invitrogen). Two to 3 weeks after transfection, viable colonies were picked up and transferred to new dishes. shPOFUT1- and mock-transfected cells were used for further experiments.

To investigate the effect of POFUT1 knockdown on cellular proliferation, shPOFUT1- and mock-transfected cells were seeded in 6-well plates at a density of 1×10⁴ viable cells/well. At the indicated time-points, the cells were trypsinized and counted in triplicate using a hemocytometer and Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) (22). Images were obtained every 12 h using a Leica LCD microscope (Leica Microsystems, Wetzlar, Germany) (original magnification, ×200) and were representative of three independent experiments.

To evaluate the effect of POFUT1 knockdown on migration, we performed the wound-healing assay described previously (17). Briefly, after uniform wounds were made in confluent culture of the shPOFUT1 and mock cells, the extent of closure was monitored visually every 4 h for 24 h. The results were visualized by measuring the wound spaces using Lenalaf220 software (available at http://www.vector.co.jp/download/file/win95/art/fh442375.html). The mean value was calculated from data obtained from three separate chambers.

To evaluate the effect of POFUT1 knockdown on invasiveness, we performed an invasiveness assay (23). A total of 2.5×10⁵ cells were seeded on a polyethylene terephthalate membrane insert (pore size, 3 μm) in a transwell apparatus (Becton-Dickinson, Franklin Lakes, NJ, USA). In the lower chamber, 1 ml of DMEM with 10% FBS and puromycin (Invitrogen) were added as chemoattractants. After the cells were incubated for 22 h at 37°C, the insert was washed with PBS and the cells on the top of the insert surface were removed with a cotton swab. Cells adhering to the lower surface of the membrane were fixed with methanol and stained with crystal violet; the numbers of cells invading the pores in five random fields were counted using a light microscope at ×100 magnification.

The significance of the POFUT1 expression levels was evaluated using Fisher’s exact test or the Mann-Whitney U test. P<0.05 was considered statistically significant. The data are expressed as the mean ± standard error of the mean (SEM).

We examined POFUT1 mRNA and protein expression in five OSCC-derived cell lines (Ho-1-u-1, KOSC2, HSC-4, HSC-3 and HSC-2) and HNOKs using qRT-PCR and western blot analysis. Significant upregulation of POFUT1 mRNA occurred in all OSCC cell lines compared with the HNOKs (Fig. 1A, P<0.05).

Fig. 1B shows representative results of the western blot analyses. The values obtained from densitometric analysis of POFUT1 protein were normalized to the GAPDH levels and then expressed as a percentage of the HNOK values. A significant increase in POFUT1 protein expression was seen in all OSCC cell lines compared with the HNOKs (Fig. 1B).

We evaluated POFUT1 mRNA expression in the normal oral tissues and paired primary OSCCs from 128 patients. Similar to the data obtained from the OSCC-derived cell lines, qRT-PCR showed that POFUT1 mRNA expression was upregulated in 104 (81.2%) of 128 primary OSCCs compared with the matched normal oral tissues (data not shown).

Representative IHC results for POFUT1 protein in normal oral tissue and primary OSCC are shown in Fig. 2A. The POFUT1 IHC scores of the cytoplasm in primary OSCCs were significantly (P<0.05) higher than in normal tissues. The IHC scores in the normal oral tissues and OSCCs ranged from 76.2 to 180.0 (median, 165.8) and 112.5 to 225.5 (median, 192.4), respectively (Fig. 2B). The POFUT1 IHC scores of T3/T4 were significantly (P<0.05) higher than that of T1/T2. The IHC scores for T1/T2 and T3/T4 ranged from 77.8 to 215 (median, 189.5) and 149.8 to 240 (median, 204.5), respectively (Fig. 2C). Table I shows the correlations between the clinicopathologic characteristics of the patients with OSCC and the status of POFUT1 protein expression using the IHC scoring system. Among the clinical classifications, POFUT1-positive OSCCs were correlated with primary tumor size (P=0.048) (Table I).

Since POFUT1 expression was upregulated in the OSCC cell lines, we assumed that POFUT1 might play an important role in OSCCs. To assess the POFUT1 functions in vitro, an shRNA experiment was performed using the KOSC2 and HSC-2 cells. Expressions of POFUT1 mRNA and protein in the shPOFUT1-transfected cells were significantly (P<0.05) lower than those in the mock-transfected cells (KOSC2 and HSC-2 derived transfectant cells) (Fig. 3).

To evaluate the effect of POFUT1 knockdown on cellular growth, we performed a cellular proliferation assay. shPOFUT1- and mock-transfected cells were seeded in 6-well plates at a density of 1×10⁴ viable cells/well counted on 7 consecutive days. There was a significant (P<0.05) decrease in cellular growth of the shPOFUT1-transfected cells compared with the mock-transfected cells (KOSC2 and HSC-2 derived transfectant cells) (Fig. 4A). Photomicrographs (Fig. 4B) showed the cytostatic effect (P<0.05) of cell line-transfected shPOFUT1 and mock cells. In the migration assay, when we visually monitored the uniform wound area in confluent cell culture, the wound areas in POFUT1 knockdown cells were the same as that in the mock cells. In the invasiveness assay, the number of penetrating POFUT1 knockdown cells was similar to that of the mock cells (data not shown).