Tumor samples were collected from 28 patients with pharyngeal cancer who had undergone surgery at the Department of Otolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Patients recruited to this study had not undergone previous chemotherapy, radiotherapy or immunotherapy. Collected tumor samples were frozen in liquid nitrogen and then stored at −80°C until required. This study was approved by the Ethics Committee of Zhengzhou University, and informed consent was obtained from each patient.

We detected the presence of HPV genes in fresh frozen samples using polymerase chain reaction (PCR) assays followed by reverse dot blots. Using PCR, 28 HPV gene segments were amplified. These were then hybridized to specific probes that were affixed to membranes. The probes we used corresponded with 5 low-risk and 18 high-risk HPV genotypes.

The hypopharyngeal squamous cell carcinoma cell line, FaDu, along with the Hep-2 and 293T cell lines, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco, USA) and grown in a 37°C, 5% CO₂ incubator.

HPV-16 E6-E7 genes were amplified and cloned into the pLV-EGFP-C lentiviral vector, between the HindIII and KpnI sites, to produce the recombinant lentivirus LV-HPV-16-E6-E7. The empty pLV-EGFP-C vector was used as an empty vector control. We co-transfected 5 µg of LV-HPV-16-E6-E7 with 3.75 µg of pH1 and 1.25 µg of pH2 into 293T packaging cells using PolyFect-V (Invitrogen, USA). After incubation at 37°C/5% CO₂ for 48 h, the culture medium was harvested and concentrated 100- to 200-fold by ultrafiltration. Virus titers in the concentrated supernatants were determined on 293T cells based on the expression level of enhanced green fluorescent protein (EGFP). Cells were cultured in DMEM containing 10% FBS, and infected at a multiplicity of infection of 10-30 in the presence of 6 µg/ml Polybrene (Sigma-Aldrich, St. Louis, MO, USA) and 1 mg/ml puromycin. Cell culture medium was changed every 72 h. Positive clones were identified through the expression of EGFP.

RNA was extracted from FaDu and Hep-2 cells using E.Z.N.A.^® Total RNA kit I (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer's instructions. Reverse transcription and PCR amplification were performed using a qRT-PCR quantitation kit (Novland, China). An ABI 7500 HT Sequence Detection system (Applied Biosystems, Foster City, CA, USA) was used to determine the relative levels of E6 and E7 mRNAs in the cells. Primers and probes designed for TaqMan assays were purchased from Applied Biosystems. Amplification was conducted according to the manufacturer's instructions. Results from the qRT-PCR assays were analyzed by the 2^−ΔΔCt method (24).

FaDu cells infected with LV-HPV-16-E6-E7, uninfected FaDu cells, Hep-2 cells, and cells transfected with the empty pLV-EGFP-C vector were lysed, and total proteins were isolated. Total protein concentration was determined using a Bradford assay. We used 30 µg of total protein per sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 12% polyacrylamide gels. Electrophoresed proteins were transferred to nitrocellulose membranes (GE Healthcare, USA), which were subsequently blocked with 5% (w/v) non-fat milk and incubated overnight at 4°C with antibodies against HPV E6 (diluted 1:800; Cell Signaling Technology, Danvers, MA, USA) and HPV E7 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000; Santa Cruz Biotechnology). The intensity of the protein bands was evaluated using a Molecular Dynamics densitometer (Molecular Dynamics, Sunnyvale, CA, USA). We used glycer-aldehyde 3-phosphate dehydrogenase as an internal reference.

Cell proliferation was evaluated using Cell Counting Kit-8 reagents (CCK-8; Dojindo, Japan). Cells in the logarithmic phase of growth were seeded in 96-well plates at a density of 1×10⁴ cells/well. We added 10 µl of CCK-8 to each well on 5 consecutive days, at the same time each day. The optical density at 450 nm in each well was assessed using an EL×800 microplate reader (BioTek, Winooski, VT, USA). All experiments were conducted in triplicate.

Cells in the logarithmic phase of growth were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and fixed with 75% ethanol overnight at 4°C. Cells were then incubated with RNase at 37°C for 30 min, and stained with propidium iodide (PI) for 30 min. We examined 10⁶ events/sample using a BD FACSCalibur™ (BD Biosciences, San Jose, CA, USA). All experiments were performed in triplicate.

The Annexin V-FITC Apoptosis Detection kit (Abcam, USA) was used to detect and quantify apoptosis by flow cytometry. Briefly, cells in the logarithmic phase of growth were harvested using cold PBS and centrifuged (5 min at 1,000 × g). The cells were resuspended in binding buffer at a density of 1×10⁶ cells/ml, stained with FITC-labeled Annexin V for 5 min, and subjected to flow cytometry on a BD FACSCalibur™. Samples were tested in triplicate and analyzed with CellQuest software (BD Biosciences).

Cell invasion assays were performed using Transwell chambers with 8.0-µm pores (Costar, Cambridge, NY, USA). Basement membrane matrix was added to the top chambers and allowed to solidify for 30 min at 37°C. We added 500 µl of culture medium containing chemotactic factor into the lower chamber. Cells were then seeded into the top chambers at a density of 5×10⁵ cells/well and allowed to incubate at 37°C for 24 h. Cells were then fixed with paraformaldehyde, stained with 0.1% crystal violet, and quantified. Experiments were independently repeated six times, in quadruplicate.

We isolated miRNAs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For reverse transcription and qPCR assays, we used miR-155, miR-363, miR-15A or U6 as primers (Table I). Assays were independently repeated three or more times.

All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software. Student's t-test was used to compare the mean between two samples. Multiple comparisons between parental and control vector groups were made using Tukey's honest significant difference test. The expression levels of miRNAs in cells and tissues were analyzed using the Wilcoxon signed-rank test. Values are presented as the mean ± SD. A p-value <0.05 was considered statistically significant.

We observed indicators of HPV infection in 25% (7/28) of the hypopharyngeal squamous cell carcinoma cases (Table II and Fig. 1). The criteria used to define heavy smoking were: an individual that smoked for more than 20 years; and smoked not less than one pack per day. The criteria used to define heavy drinking were: an individual that had regularly consumed alcohol for more than 20 years; and drank not less than 150 g of alcohol per day. Patients were separated into two groups for statistical analyses: HPV-positive and HPV-negative. There was a significant difference between the two groups when heavy smoking was considered as a variable (P<0.05, Table III). Differences between the two groups of patients with respect to age, gender, pathological type, and tumor T stage were not significantly different (P>0.05, Table III).

Positive clones were identified through the expression of EGFP. We observed E6-E7 expression in stably transfected FaDu cells at the mRNA and protein levels. The relative E6-E7 mRNA levels in the HPV-16 E6-E7 FaDu cells (2.6±0.22, 1.8±0.12) were higher than these levels in the empty vector control cells (0.003±0.0001, 0.003±0.0002, P<0.05) and blank control cells (FaDu cells) (0.002±0.0002, 0.005±0.0001, P<0.05), while consistent with the Hep-2 cells (Table IV and Fig. 2).

We observed that HPV-16 E6-E7 promoted the proliferation of FaDu cells in vitro (Fig. 3), and that these effects were dependent on time. Proliferation levels were maximal after 5 days.

Apoptosis was determined using flow cytometry and caspase-3- and caspase-9-specific enzyme-linked immunosorbent assays (ELISAs). We observed a significant decrease in the number of Annexin V⁺ apoptotic FaDu cells that were stably transfected compared with the numbers in the cells containing the empty vector (7.246±0.815 vs. 13.464±0.609%; P<0.05) or blank control (7.246±0.815 vs. 13.298±1.324%; P<0.05). According to our ELISA results, no significant difference was noted between the blank and empty vector control (P>0.05), while there was a significant difference with the HPV-16 E6-E7 group (Fig. 4).

The proportions of FaDu cells in the G0/G1 phase of the cell cycle were 53.816±1.665, 62.284±1.609, and 62.262±2.139% for those that were stably transfected, those transfected with the empty vector, and the blank control, respectively (Fig. 5).

Our in vitro cell invasion assay results showed that HPV-16 E6-E7 promoted the invasive ability of the FaDu cells when compared with the control cells (Fig. 6). These results demonstrate that HPV-16 E6-E7 promotes the invasive ability of FaDu cells in vitro.

Relative expression levels of miR-363 and miR-15a were significantly higher in the HPV-positive specimens than these levels in the HPV-negative specimens. We did not observe a significant difference in miR-155 levels for specimens that were HPV-positive/negative and in FaDu cells that stably expressed HPV-16 E6-E7 (Fig. 7).