A total of 45 pairs of primary hypopharyngeal squamous cell carcinoma (HSCC) and corresponding non-malignant tissue specimens were obtained from patients with HSCC at the Affiliated Hospital of Nantong University and General Hospital of Jinan Military Region between July 2003 and June 2011. Clinical characteristics of HSCC patients were extracted from their medical records, including: gender, age, histological type, differentiation grade and tumor stage. None of the cancer patients received any type of treatment (radiation therapy, chemotherapy or immunotherapy) before surgery. Specimens were collected immediately after tumor excision during surgery. All specimens were subjected to histological diagnosis by a pathologist. The study protocol was approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University and General Hospital of Jinan Military Region, and all patients provided written informed consent prior to participation in the study.

The human HSCC cell line, FaDu cells, was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). FaDu cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C in a humidified incubator with 5% CO₂.

Total RNA was extracted from FaDu cells, human HSCC tissues, and corresponding non-malignant tissues from the same patients. Extraction was done using with TRIzol reagent, as described by the manufacturer (Invitrogen). One microgram of DNase I-treated (Fermentas, Vilnius, Lithuania) total RNA were converted to cDNA using a Reverse Transcription System kit (Fermentas, Burlington, ON, Canada). The GRK6 mRNA expression level was determined by following Maxima SYBR Green/ROX qPCR Master Mix kit (Fermentas) with the following primer specific either for GRK6 or the housekeeping gene GAPDH (internal control). GRK6: 5′-AAAACACCTTCAGGCAATACCG-3′ (sense) and 5′-AGGCCAAGCTCACTACAAACCTA-3′ (antisense); GADPH: 5′-CATGAGAAGTATGACAACAGCCT-3′ (sense) and 5′-AGTCCTTCCACGATACCAAAGT-3′ (antisense). As a negative control we used DEPC-treated water to replace cDNA templates for every PCR. The relative quantitative value was expressed by the 2^−ΔΔCt method.

Human HSCC tissues and their corresponding non-malignant tissues were homogenized in RIPA lysis buffer, and the lysates were cleared by centrifugation (12,000 rpm) at 4°C for 20 min, and denatured by boiling for 5 min. Equal amounts of protein (30 µg) were electrophoresed in 10% polyacrylamide gels. Subsequently, the proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk in TBST buffer for 1 h at room temperature and incubated with a rabbit polyclonal anti-human GRK6 antibody (1:300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or an anti-GAPDH (internal control) at 4°C overnight at room temperature. The next day, the membranes were washed three times with TBST, after which they were incubated for 2 h with a 1:2,000 dilution of secondary antibodies. After washing, immunoreactive protein bands were visualized using an electrochemiluminescence (ECL) detection kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein bands were scanned and quantified using a Gel Image Analysis System. Each experiment was repeated three times. The protein levels were normalized to that of GAPDH.

Genomic DNA was extracted from FaDu cell line and primary HSCC tissues through SDS/proteinase K treatment, followed by phenol-chloroform extraction and ethanol precipitation. Bisulfite treatment was performed using the EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA, USA), according to the manufacturer's instructions. http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi was used to analyze the 3000 bp region of the GRK6 gene promoter region (−2000 bp to +1000 bp) for the presence of CpG islands. After prediction analysis, we found two CpG islands (−855 to −318 and −310 to +475). The GRK6 methylation-specific primers used were 5′-TGGGTGGAGAGAAATTATAATATTC-3′ (sense) and 5′-TATAACCGTATTCCCTTAAATCGAC-3′ (antisense), 167 bp; and the unmethylation-specific primers used were 5′-GGTGGAGAGAAATTATAATATTTGG-3′ (sense) and 5′-TATAACCATATTCCCTTAAATCAAC-3′ (antisense), 165 bp. The reaction mixture contained 1 µl DNA in a volume of 50 µl containing 1 µl of each primer, 2X GC buffer I, 2.5 mM dNTP Mix and 2.5 U LA Taq (Takara). The MSP conditions were as follows: 94°C for 5 min, 40 cycles of 94°C for 30 sec, 54°C for 30 sec, 72°C for 45 sec and 72°C for 10 min. Water was run as a negative control in every MSP reaction. All procedures were repeated three times. The PCR products were subjected to 2% agarose gel electrophoresis at 100 V for 30 min.

HSCC cells (FaDu) were seeded at a density of 5×10⁵ cells/well in 6-well culture plates in DMEM (Invitrogen) containing 10% FBS, and incubated in a humidified atmosphere of 5% CO₂ at 37°C. After overnight culture, cells were incubated with medium containing 0, 5 and 10 µmol/l 5-Aza-2′-deoxycytidine (5-Aza-dC) (Sigma, St. Louis, MO, USA) for 3 days. Total RNA was isolated after treatment, and mRNA was analyzed by qPCR as described above.

The target sequence of GRK6 siRNA sequence (5′-GCCGACUACCUCGACAGCAUCUACU-3′) was used as previously described (13), a scrambled sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as negative control for RNA interference (RNAi), which had no significant homology to any human gene sequences. Inverted and self-complementary hairpin DNA oligos targeting GRK6 mRNA were obtained from Genchem Biotechnology Co. (Shanghai, China). The stem-loop oligonucleotides were synthesized and cloned into a lentivirus-based vector pGCSIL-GFP and resulting plasmids were named as GRK6-shRNA and scrambled shRNA respectively. Lentiviral vector production was performed according to standard Invitrogen protocol. Then the lentiviral titers of GRK6-shRNA and scrambled shRNA were identified. Experiments for lentivector transduction were carried out as follows: human HSCC cell line FaDu were plated at 5×10⁵ cells in 6-well culture plates, and transducted with scrambled shRNA, GRK6-shRNA lentivirus at a multiplicity of infection (MOI) of 20 in the presence of 8 mg/ml of polybrene. After 24 h, the medium was replaced with fresh DMEM containing 10% FBS and the cells were cultured for another 48 h. Cells were harvested at 72 h after infection and the knockdown efficiency of GRK6 was evaluated by quantitative real-time PCR and western blot analysis.

The human HSCC cell line, FaDu cells, incubated with 5-Aza-dC for 3 days or transfected with GRK6-shRNA lentivirus were separately detached from the culture plates, then seeded onto Matrigel-coated 8 µm pore size Transwell filters (Corning Life Sciences, Corning, NY, USA) in the upper well at the density of 5×10⁴ per in 200 µl of culture medium (DMEM containing 1% fetal calf serum), in the lower chamber, 500 µl DMEM containing 10% FBS was added as a chemoattractant. After 24 h of incubation, non-invading cells on the upper wells were removed with a cotton-tipped swab. Then, the lower side of the filters were fixed in 4% paraformaldehyde for 10 min, and stained with 0.1% crystal violet for 30 min. The invaded cells were viewed under a microscope and counted in five fields of view at ×200 magnification. The invasion ability of the cancer cells was expressed as the mean number of cells in five fields. The assay was performed in three independent experiments.

Statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). We used the χ² test for clinicopathological features, and the Student's t-test or one-way ANOVA test for continuous variables. Data are expressed as mean ± SD. P<0.05 was considered statistically significant.

We investigated GRK6 protein and mRNA levels in the hypopharyngeal squamous cell carcinoma (HSCC) specimens and corresponding non-malignant tissues by western blotting and quantitative real-time PCR. Western blot analysis showed that the expression level of GRK6 protein was significantly decreased in 18 of 25 paired HSCC tissues compared with the corresponding adjacent non-malignant tissues, and ten cases of the representative western blotting results are shown in Fig. 1A. Moreover, the average GRK6 protein level in HSCC tissues was significantly lower than that of GRK6 in adjacent non-malignant tissues (P=0.012; Fig. 1B). In addition, the results of quantitative RT-PCR revealed that GRK6 mRNA expression was downregulated in 32 of 45 paired HSCC samples compared with corresponding non-malignant tissues. The mean expression value of mRNA in HSCC tissues was sig nificantly lower than the value in paired adjacent nonmalignant tissues (P=0.029; Fig. 1C).

To identify whether low GRK6 expression was due to DNA methylation, MSP was used to examine DNA methylation status of GRK6 gene promoter CpG islands in 45 paired HSCC specimens and corresponding non-malignant tissues. DNA methylation occurred in 77.8% (35/45) of HSCC tissues and 42.2% (19/45) of non-malignant tissues. No methylation was observed in 22.2% HSCC tissues and 57.8% non-malignant tissues. The difference in methylation between the primary HSCC and the non-malignant tissue specimens was significant (Fig. 2; P=0.001). Furthermore, DNA methylation of GRK6 was related to TNM stage and lymph node metastasis (Table I). We also examined the DNA methylation status of GRK6 gene promoter in hypopharyngeal cancer cell lines. Data from MSP analysis showed hypermethylation of GRK6 gene promoter in FaDu (Fig. 3A).

After treatment with the demethylating agent 5-Aza-dC, we first assessed GRK6 mRNA expression in HSCC cell lines and found that the expression of GRK6 mRNA was significantly increased in FaDu, with the highest expression occurring at a concentration of 10 µmol/l (Fig. 3B). We further detected the expression of GRK6 at the protein level after treatment with 5-Aza-dC, and found that GRK6 protein was also significantly elevated, and reached the peak at a concentration of 10 µmol/l (Fig. 3C and D).

To examine whether the reactivation of GRK6 expression can regulate HSCC invasion, we analyzed the invasion capability of the FaDu cells using the Matrigel invasion assay as described above. Briefly, after treatment with 10 µmol/l 5-Aza-dC for 3 days, FaDu cells were seeded the upper well of Matrigel-coated Transwell filters. After 24 h of incubation, cells in the lower side of the filters were fixed, stained and counted. The Matrigel invasion assay showed that the number of invading FaDu cells was significantly reduced after 5-Aza-dC treatment compared to the control group, and this reduction was significant (P<0.05; Fig. 3E and F).

We used the GRK6-shRNA lentivirus to knock down GRK6 expression in the FaDu cell lines. Real-time quantitative PCR and western blot results confirmed GRK6 was significantly downregulated after 72-h transfection (Fig. 4A and B). Matrigel invasion assay showed the number of invading FaDu cells was significantly increased after transfection with GRK6-shRNA (312±34) compared with the scramble shRNA transfected group (189±14) and the control group (201±22) (P<0.05; Fig. 4C and D).