Effects of HPV-16 infection on hypopharyngeal squamous cell carcinoma and FaDu cells

Hypopharyngeal squamous cell carcinoma is a common type of malignant tumor among head and neck squamous cell carcinomas (HNSCCs). Heavy smoking and/ or drinking is associated with the development of HNSCC. However, HNSCC also occurs in individuals that do not drink or smoke, possibly due to infection with the human papilloma virus (HPV). HPV-16 has been shown to be closely associated with the occurrence of several types of cancers. However, its role in hypopharyngeal squamous cell carcinoma remains unclear. In the present study, we investigated the effects of HPV-16 on hypopharyngeal squamous cell carcinoma and FaDu cells. Lentiviral vectors were used to establish FaDu cells that expressed the E6 and E7 proteins of HPV-16. We used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays and western blotting to detect and determine the levels of expression for E6-E7 mRNAs and proteins. Cell Counting Kit-8 (CCK-8) assays, enzyme-linked immunosorbent assays (ELISA), Transwell assays, and flow cytometry were used to assess the effects of HPV-16 E6-E7 on the proliferation, invasion, metastasis and apoptosis of FaDu cells. Expression of microRNAs was analyzed by qRT-PCR. We found that the expression levels of HPV-16 E6-E7 were increased in FaDu cells transfected with the lentiviral vector compared with that observed in the control cells. In addition, the rates of apoptosis were decreased in the transfected cells, while proliferation was increased. The average numbers of cells penetrating the Matrigel were significantly higher than those for the controls. We detected miR-363 and miR-15a, and their expression levels were significantly increased in the HPV-16-positive patients and in FaDu cells expressing HPV-16 E6-E7. We found that HPV-16 E6-E7 appeared to inhibit apoptosis, and to increase cell proliferation, invasion and metastasis. Furthermore, miR-363 and miR-15a were overexpressed in the hypopharyngeal squamous cell carcinoma samples infected with HPV-16, and in FaDu cells stably expressing HPV-16 E6-E7. These findings may provide a new clue of the mechanisms involved in the pathogenesis of HPV-16-positive hypopharyngeal squamous cell carcinoma.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world (1).It is characterized by phenotypical, etiological, biological and clinical heterogeneity.Despite surgery, radiation therapy, and chemotherapy, approximately half of all patients die (2)(3)(4).Laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinoma are two common malignancies of HNSCC that mainly occur in middle-aged men.Tobacco smoking and alcohol abuse are predominant risk factors in HNSCC.A subset of oropharyngeal carcinoma cases are strongly associated with the infection of high-risk human papilloma virus (HPV), predominantly HPV-16 (5)(6)(7).The oncogenicity of high-risk HPV is dependent on the constitutive expression of oncogenes, such as E6 and E7 (8)(9)(10)(11)(12)(13)(14)(15).
The E6 and E7 genes of the HPV-16 genome encode the oncoproteins E6 and E7, respectively (16,17).Lentiviral vectors can be used to transfect cells with high efficiency, allowing for the stable integration of genes into cells.MicroRNAs are involved in almost all biological processes in the human body, including cell proliferation, differentiation, apoptosis, invasion and migration (18)(19)(20).Abnormal expression of miRNAs is associated with the occurrence and development of many types of tumors (21)(22)(23).
In the present study, we used a lentiviral vector to transfect and integrate the HPV-16 E6-E7 genes into the hypopharyngeal squamous cell carcinoma cell line, FaDu.We then observed the effects of E6-E7 expression on these cells.We also sought to determine any effects of HPV-related miRNAs on HNSCC by examining miRNA expression levels in hypopharyngeal squamous cell carcinoma tissues.

Materials and methods
Patients and tumor samples.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.
HPV DNA detection and typing.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.
Cell culture.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% CO 2 incubator.
Integration, transfection, and expression of HPV-16 E6-E7 genes.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% CO 2 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 isolation and quantitative reverse transcription-PCR (qRT-PCR) assays.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).

Western blot analysis. 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 Table I. miR-155, miR-363, miR-15A and U6 reverse transcription primers.

Gene name
RT primers PCR primers (Molecular Dynamics, Sunnyvale, CA, USA).We used glyceraldehyde 3-phosphate dehydrogenase as an internal reference.
Cell proliferation assays.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 1x10 4 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 ELx800 microplate reader (BioTek, Winooski, VT, USA).All experiments were conducted in triplicate.
Cell cycle analysis.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 10 6 events/sample using a BD FACSCalibur™ (BD Biosciences, San Jose, CA, USA).All experiments were performed in triplicate.
Apoptosis assays.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 x g).The cells were resuspended in binding buffer at a density of 1x10 6 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).
Transwell assays.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 5x10 5 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.
miRNA expression assays.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.
Statistical analysis.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.

Results
Presence of HPV in the specimens and clinical features of the hypopharyngeal squamous cell carcinoma cases.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).
HPV-16 E6-E7 promotes FaDu cell proliferation.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.
HPV-16 E6-E7 inhibits the apoptosis of FaDu cells.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±0.815vs. 13.464±0.609%;P<0.05) or blank control (7.246±0.815vs. 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).

HPV-16 E6-E7 reduces G0/G1 arrest in the FaDu cells and promotes progression of the cell cycle and cell proliferation.
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).
HPV-16 E6-E7 increases the invasive ability of the FaDu cells.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.
miR-363 and miR-15a are overexpressed in the HPV-positive hypopharyngeal squamous cell carcinoma samples.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).

Discussion
It is estimated that HNSCC affects 600,000 individuals per year worldwide (25).Smoking has been implicated in the increased occurrence of HNSCC in developing countries.The role of HPV has emerged as an important factor in the increase in the incidence of oropharyngeal tumors affecting non-smokers in developed countries (26).In comparison with environment-related HNSCC, patients with HPV-related malignancies display a better response to treatment and a lower risk of death and tumor progression (27,2,(28)(29)(30).Therefore, we investigated the effects of  HPV-16 infection on the behavior of hypopharyngeal squamous cell carcinoma.Of the 28 frozen hypopharyngeal squamous cell carcinoma tissues we examined, 7 were positive for the presence of HPV, with HPV-16 as the predominant genotype.We generated the LV-HPV-16-E6-E7 lentivirus to establish a FaDu cell line that stably expressed HPV-16 E6-E7.Our findings indicate that the E6-E7 proteins of HPV-16 inhibited apoptosis and increased the levels of proliferation, invasion and metastasis in the transfected FaDu cells.
In addition, we investigated miRNA expression levels in hypopharyngeal squamous cell carcinoma tissues and the  generated FaDu cell line.Results from previous studies have demonstrated that the miRNA expression profiles are altered in HNSCC and that these changes can be attributed to HPV infection (31,32).We found that expression levels of miR-363, miR-33 and miR-497 were upregulated in the HPV-16-positive HNSCC cases.Expression levels of miR-181a, miR-181b, miR-29a and miR-218 were downregulated, and this was significant for miR-363 and miR-155.
Results from another study showed that miR-15a expression was upregulated in HPV-positive HNSCC.In the present study, we found that miR-15a was upregulated in the hypopharyngeal squamous cell carcinoma tissues and in LV-HPV-16-E6-E7-infected FaDu cells.This particular miRNA plays an important role as a tumor suppressor, and may be associated with a favorable prognosis in HPV-related HNSCC.It is possible that miR-15a could be used in the development of miRNA-based therapies for hypopharyngeal squamous cell carcinoma.We failed to observe any significant changes in miR-155 expression levels for HPV-positive/ negative hypopharyngeal squamous cell carcinoma tissues and LV-HPV-16-E6-E7-infected FaDu cells.Findings from previous studies have shown that miR-155 expression was significantly downregulated in HNSCC cells that were positive for HPV-16.We speculate that these contrasting results may be due to inconsistencies between tumor tissues and tumorderived cells, and since different detection methods were used.Future studies to assess the roles of miR-363, miR-15a, and miR-155 in hypopharyngeal squamous cell carcinoma are warranted.
Figure 7. miR-363 and miR-15a are overexpressed in the HPV-positive pharyngeal squamous carcinoma tissues.The relative expression of miR-363 and miR-15a were significantly higher in the HPV-positive tissues than the expression in the HPV-negative tissues while there was no significant change of miR-155 in both HPV positive/negative pharyngeal squamous carcinoma tissues and HPV-16 E6-E7-infected FaDu cells.

Figure 1 .
Figure 1.Sample test of 7 cases of HPV-positive pharyngeal squamous carcinoma tissues.Five cases had a single type of HPV infection, of which 3 cases presented with type HPV-16, 1 case presented with type HPV-18, and 1 case presented with type HPV-52.Two cases presented with two types of HPV infections; 1 case had types HPV-16 and -33, and one case had types HPV-16 and -52.

Figure 3 .
Figure 3. Cell proliferation of the FaDu cells transfected with the LV-HPV-16-E6-E7 lentivirus was assessed using the CCK-8 assay.Data are presented as the mean of triplicate experiments.(A) Comparison of the status of cell growth at 48 h.(B) The growth promoting effect of the HPV-16 E6-E7 lentivirus was time-dependent, with a maximum effect detected at day 5 achieving a significant difference (P<0.05).

Figure 2 .
Figure 2. E6-E7 is overexpressed in the stably transfected FaDu cells.(A) Positive clones were identified by positive EGFP expression.(B) Expression levels of E6-E7 mRNA in the three groups of FaDu cells and in the Hep-2 cells.(C) Expression levels of E6-E7 protein in the three groups of FaDu cells and in the Hep-2 cells.β-actin served as a loading control to verify that equal amounts of protein were present in each lane ( * P<0.05).

Table III .
Analysis of the HPV status and the laryngopharyngeal carcinoma clinical characteristics of the cases.