Thirty-five pairs of formalin-fixed, paraffin-embedded HSCC tissue specimens were collected. Specimens were obtained from the Department of Pathology, Affiliated Hospital of XXXX University. The whole project was approved by the ethics committee of the affiliated hospital of XXXX University(2017-L052). Following tissue sectioning, the slices were dewaxed and dehydrated with xylene and ethanol. Antigen retrieval was performed in an EDTA buffer (pH = 9.0). Endogenous peroxidase was blocked with 3% H2O2. Non-specific binding was blocked by treatment with blocking reagents, and then slides were incubated with anti-LAMP3 antibody (1:100, Sangon Biotech, China) at 4 °C overnight. Then, at room temperature, the slides followed by incubation with a secondary antibody (Sangon Biotech, China) for 30 minutes. The immunolabel was then displayed using a DAB detection kit (Sangon Biotech, China) to visualize the reaction. LAMP3 protein expression levels were analyzed by using Image-Pro Plus 6.0.
shRNA design and construction of recombinant lentiviral vectors
The small interfering RNA (siRNA) sequence (NM_ 27074) (CCGGTCAGAAGCCTGTTCA) targeting LAMP3 was transformed into short hairpin RNA (shRNA) (stem-loop-stem structure) and cloned into a PGIPZ vector (Asian, Shanghai China). To exclude possible off-target effects of shRNA, comparable results were obtained using two other constructs shRNA-1 (ACGATGGCAGTCAAATGAG) and shRNA-3 (GGAAGCAGACTCTGTATAA) against LAMP3. Interference efficiency was screened for using RT-PCR. Recombinant lentiviral vectors were generated by co-transfecting HEK293T cells with lentiviral expression vectors and packaging plasmid mixtures using Lipofectamine™ 2000. Infectious lentiviral particles were harvested 48 hours after transfection, centrifuged to remove cell debris, and then filtered, centrifuged, and the virus concentrated. The titer of recombinant lentivirus was determined by serial dilution on 293T cells.
Recombinant lentiviral transduction in FaDu cells and screening stable strains
For lentiviral transduction, FaDu cells were subcultured into 6-well culture plates at (1–2 × 106) cells/well. After growth to 70% confluence, cells were transduced with a lentivirus expressing LAMP3 shRNA at a multiplicity of infection (MOI) of 10. An empty vector was used as a control. The cells were harvested after 48 hours and the transduction efficiency was assessed by counting the percentage of GFP-positive cells. Stably transformed strains were screened for drug resistance. The drug for screening drug resistance is puromycin. The cultures were expanded and the interference efficiency was verified by RT-PCR and western blotting.
The human hypopharyngeal carcinoma cell line FaDu was purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). The media used to culture the cells was supplemented with 10% high quality fetal bovine serum (FBS) and 1% Penicillin-Streptomycin solution. The culture conditions were carried out in a 5% CO2、37 OC incubator.
Western blot analysis
The cells were collected and the protein was extracted using a protein lysis buffer (RIPA: PMSF = 100:1) according to the manufacturer’s recommendations. The protein concentration was determined using BCA working solution (Beyotime, Shanghai, China). The protein sample was loaded (20 µg per well) and separated by electrophoresis on a 10% SDS-polyacrylamide gel. Samples were then transferred to a polyvinylidene fluoride film (Millipore, Billerica, MA, USA) and the membrane was blocked in 5% skim milk for two hours. The membrane was incubated with primary antibody at 4 °C overnight (anti-LAMP3 antibody, 1:800, Sangon Biotech, China), (anti-GAPDH antibody, 1:1000, Sangon Biotech, China). After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 1.5 hours. Finally, the signal was developed using a chemiluminescent agent (NCM Biotech, Suzhou, China). Images were captured and Image J software was used to compare and calculate gray values.
Total RNA from cells was extracted using TRIzol reagent (Ambion, Life Technologies, USA) according to the manufacturer's protocol. The concentration of RNA was measured using a SmartSpec Plus spectrophotometer (Bio-Rad). RNA was reverse transcribed into cDNA. Primers were designed as follows: h-LAMP3 sense: 5'-ACTACCCCAGCGACTACAAAA-3' and antisense 5'-CTAGGGCCGACTGTAACTTCA-3'. GAPDH sense: 5’-GGAGCGAGATCCCTCCAAAAT-3’, antisense: 5’-GGCTGTTGTCATACTTCTCATGG-3’. The primers were designed by Asia-Vector Biotechnology (Shanghai) Co., Ltd. GAPDH levels were used to normalize the relative expression levels of LAMP3 mRNA using the DDCt method.
The FaDu cells were seeded in a six-well plate and grown until the cells were at 80% confluence. A scratch was made along the surface of the cells with the tip of a 10 µl pipette. The cells were washed three times with PBS and the culture media was replaced with serum-free basal culture media. Photographs were taken at 0 h and 24 h to measure the width of the scratch.
Cell migration assay
A single cell suspension with a density of 5 × 104 / ml was prepared using serum-free media. The cell suspension (100 µl) was added to the upper chamber of each transwell chamber and 600 µl of 20% FBS medium was added to the lower chamber as a chemical attractant. Three sub-wells were set up in each group. The 24-well plates were incubated at 37 °C for 24 hours in a 5% CO2 incubator. Non-migrating cells on the chamber membrane were removed with a cotton swab. The cells under the chamber were fixed in paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 10 minutes. Photographs were taken (magnification, × 100) and the cells counted.
Cell proliferation assay
To the wells of a 96-well plate, 2000 cells in 100 µl were added and incubated at 37 °C for 24 h. Cell viability was determined using a CCK8 kit (Sangon Biotech, China) according to the manufacturer’s procedures. The number of viable cells was evaluated by measuring the absorbance at 450 nm using a microplate reader at 0.5 h, 1 h, 2 h, and 4 h.
Colony formation assay
FaDu cells (600 per well) were seeded into a six-well plate. After incubation for 14 days at 37 °C, a cell mass visible to the naked eye was formed and removed. The cells were fixed with 4% paraformaldehyde for 30 minutes and stained with crystal violet for 10 minutes. The number of cell pellets was calculated using an optical microscope.
Data are expressed as mean ± standard deviation. Statistical analysis was performed using IBM SPSS Statistics 20.0 software (IBM, Armonk, NY, USA). P < 0.05 was considered statistically significant.