Patients and specimens
Between February 2016 and March 2018, 47 patients with hypopharyngeal squamous carcinoma underwent tumor resection at the First Hospital of Jilin University and were included in this study. The clinical staging of these patients was classified according to the 8th edition of the Union for International Cancer Control stage classification. The diagnosis was made by chest radiography, nasopharyngeal and neck magnetic resonance imaging, nasopharyngeal fibroscopy, Epstein-Barr virus serological examination, and histopathological examination. The patient received no chemoradiotherapy before surgery. The tissue obtained from each patient contained both tumor tissues and corresponding adjacent nontumor tissues. We conducted all experiments according to the relevant guidelines and regulations. This study was approved by the institutional ethics committees of the First Hospital of Jilin University (Changchun, PR China). Written informed consent was obtained from each participant.
Cell culture
The human hypopharyngeal squamous cancer cell line FaDu and monocyte leukemia cell line THP-1 were obtained from Shanghai Zhongqiaoxinzhou Biotech Co., Ltd. (Shanghai, China). All the cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT, USA), 1% penicillin, and 1% streptomycin and were maintained at 37°C in a humidified incubator with 5% CO2.
Preparation of FaDu cells with LGR5 over-expression
FaDu cells were transfected with the LGR5 plasmid or control plasmid, which was purchased from GeneCopoeia (EX-Q0041-M03, EX-NEG-M03; Guangzhou, China). The LGR5 recombinant plasmid was constructed using pReceiver-M03 as the backbone plasmid and was inserted into the LGR5 cDNA coding sequence (NM_003667, ORF length: 2,724 bp). FaDu cells transfected with the LGR5 plasmid were cultured in medium containing G418 for 14 d. The LGR5+ FaDu cells were collected to identify LGR5 over-expression using quantitative polymerase chain reaction (qPCR) and western blot assay.
Short interfering RNA (siRNA) transfection
FaDu cells were stimulated with inflammatory factors and then were transfected with siRNA oligo targeting the LGR5 gene, with a scrambled sequence as the control. Eight hours after transfection, the cell culture medium was replaced with RPMI 1640 containing 10% FBS and the cells were continued to incubate at 37°C for another 40 hours before the next step. An siRNA interference oligo sequence targeting the human LGR5 gene sequence (LGR5-homo-883) was designed and synthesized by Shanghai GenePharma Co., Ltd. The target sequence was UAAUAAGAG AAG GGUUGCCTT.
Cell proliferation assay
The proliferation ability of cells was measured by the CCK-8 assay using Cell Counting Kit (Yeasen, Shanghai). Different groups of cells were inoculated into 96-well plates at the density of 1,000 cells per well. After 24 hours, the CCK-8 reagent was added, followed by incubation at 37°C for 1 hour. The absorbance at 450 nm was recorded using a microplate reader.
Immunofluorescence staining
Cells to be observed were inoculated on glass slides and rinsed three times with phosphate-buffered saline (PBS) 24 hours later, and then the cells were fixed in 4% paraformaldehyde at room temperature for 30 minutes. The cells were washed three times with PBS and then were incubated with 0.1% Triton X-100, blocked in 5% goat serum for 1 hour, and then were incubated overnight with 1:100 diluted primary antibody at 4°C. Next, the cells were washed with PBS and then were incubated with 1:400 diluted secondary antibody in the dark for 1 hour at room temperature, followed by washing three times with PBS. The slides were sealed with glycerine at a concentration of 50% and then were observed and photographed with a laser confocal microscope (Fluo-View FV1000; Olympus, Japan).
Wound healing assay
FaDu cells of different groups were inoculated in 24-well plates and scratched with a 200-µl pipette tip when the plate was completely covered. PBS solution was used to wash away floating cell debris. Next, photos of the freshly scratched surfaces were taken. Thereafter, the cells were cultured at 37°C and 5% CO2 for 24 hours and then were photographed again to compare the healing of each “wound”. The scratch spacing at each time point was measured by Image J software [RM = (W2-W1)/W2×100% (RM=relative mobility, W1=initial cell covering rate, W2=final cell covering rate)]. The relative mobility of the cells at each time point was calculated with 0-hour scratch spacing as a reference, and the experimental results were analyzed using SPSS17.0 statistical software. The counting data were expressed as a percentage, while the measurement data were expressed as ±S. One-way analysis of variance was used for comparison of multi-group mean values.
Cell migration assay
Cell invasion ability was determined by the Transwell assay. Cell suspensions with concentrations of 5×105/ml were prepared with 0.1% FBS in RPMI 1640 medium. Next, 5×104 cells were placed in the upper chamber (pore size: 8 µm; Millipore, Billerica, MA, USA) precoated with 1:4 diluted Matrigel (Yeasen, Shanghai, China). The lower chamber was filled with RPMI 1640 medium containing 20% FBS as an inducer. After incubation for 24 hours, the cells in the upper chamber and Matrigel were erased with cotton swabs, and the lower cells were stained with 0.5% crystal violet. Cell invasion was observed under an optical microscope (magnification, ×40).
Real-time quantitative PCR (qPCR) detection system
Total RNA was extracted from each group using Trizol reagent, and the RNA integrity was verified by agarose gel electrophoresis. The RNA was reversed transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Cat.04897030001). Additionally, cDNA was amplified using FastStart Essential DNA Green Master, Cat.06924204001, (Roche, Indianapolis, IN, USA). The specific primer designs are shown in Table 1. The real-time PCR protocol was performed according to our previous studies[14]. The target gene mRNA level was normalized to that in β-actin, and the relative mRNA levels were calculated using the formula: Fold change = 2−△△CT.
Western blot analysis
The proteins were extracted with cell lysate buffer (T-PER Tissue Protein Extraction Reagent containing phosphatase inhibitor (Halt Protease and Single-Use Inhibitor Cocktail; 78442; Thermo). The proteins were separated using 10% SDS-polyacrylamide gel electrophoresis and then were transferred to PVDF membranes. The membrane was blocked with 5% non-fat milk powder for 1 hour and then was incubated with antibodies overnight at 4°C, washed three times with TBST, and then incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1.5 hours. The primary antibodies were as follows: LGR5 Polyclonal antibody (Cat# A10545), TWIST1 Polyclonal antibody (Cat# A7314), YAP Polyclonal antibody (Cat# A1001), pYAP Polyclonal antibody (Cat# AP0489), and SNAIL Polyclonal antibody (Cat# A5243), all from ABclonal Biotechnology Co., Ltd, China. β-Actin monoclonal antibody (Cat# ab8226; Cambridge, MA, USA) was used as a working internal control. ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize protein bands, and images were obtained using a Dolphin-C hemi Mini image system (Wealtec Corp., Sparks, NV, USA). Gray values for each protein band were analyzed using Image J analysis software, and relative protein levels were calculated using β-actin as an internal control.
Immunohistochemistry
The tissue sections were dewaxed and hydrated, rinsed with 0.01 mol/L of PBS, and heated in 0.01 mol/L of citrate buffer (pH=6.0) for 10 minutes for antigen repair. After washing with PBS, 3% hydrogen peroxide was added and then the sections were incubated for 15–20 minutes to remove endogenous peroxidase activity. The sections were blocked with goat serum for 15–20 minutes and then were incubated with LGR5 monoclonal antibody (1:300) at 4°C overnight. The biotinylated secondary antibody IgG was added dropwise onto the sections, followed by incubation at temperature for 15–20 minutes, incubation with streptomycin-labeled horseradish enzyme for another 15–20 minutes, application of DAB (diaminobiphenyl; Bioss, Beijing, China) for color development, and re-staining with hematoxylin for 1 minute. The LGR5 staining intensity (PI) = cell staining intensity (SI) × percentage of positive cells (PP) and was scored as follows: 0~5 points, negative; 6~12 points, positive. The SI results were scored as follows: 0, no staining; 1, yellow staining; 2, brown staining; 3, brown staining. The PP result judgment was scored as follows: 0%, 0 points; < 15%, 1 point; 16%~50%, 2 points; 51%~75%, 3 points; > 75%, 4 points.
Statistical analysis
All the data were collected from at least three independent experiments. Results were analyzed using SPSS17.0 statistical software and are expressed as means ± standard deviation (SD). Student’s t-test, one-way analysis of variance, and multivariate analysis of variance were used to analyze data as appropriate. A value of P < 0.05 was considered statistically significant. Chi-squared test was used to evaluate the correlation between LGR5 and clinicopathological features.