Cell culture and siRNA transfection
CAL27 cells, human normal oral keratinocytes (NOK), human dysplastic oral keratinocytes (DOK), HSC-2 cells and HSC-3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, United States) and 10% fetal bovine serum (FBS) (Gibco, United States). SCC25 cells were cultured in Dulbecco's modified Eagle’s medium/Nutrient mixture F-12 (DMEM/F12) (Gibco, United States) with 10% FBS and 400 ng/ml hydrocortisone (Solarbio, China). All cells were cultured in a 5% CO2 incubator at 37 °C. The CERS1 siRNA (GeneCopoeia, United States) target sequence was AAGGTCCTGTATGCCACCAGT. The ATF4 siRNA target sequence was CUGCUUACGUUGCCAUGAU. For control siRNA, the negative control siRNA from GeneCopoeia was used. CAL27 and SCC25 cells (6 × 105-8 × 105) seeded in 6-well plates (Corning, United States) were transfected with 100 nM of each siRNA using Lipo2000 (Thermo Fisher, United States) and FBS-free medium. After eight hours of transfection, the medium was replaced with fresh FBS-containing medium. Cells were collected 48 hours later.
The cell counting kit-8 method (Dojindo, China) was used to determine cell proliferation and viability. CAL27 and SCC25 cells (5 × 103) were plated in 96-well plates. After siRNA transfection, cells were incubated with 10% CCK-8 for an hour. Then, we used 480 λ to evaluate proliferation (microplate spectrophotometer, Sigma, United States).
The colony formation assay was also used to determine cell proliferation and viability. CAL27 and SCC25 cells (1 × 103) were seeded in 6-well plates. After 10 days of growth, cells were fixed with 4% paraformaldehyde (Solarbio, China) for 20 minutes and stained with 0.2% crystal violet (Solarbio, China) for 5 minutes.
EdU DNA Proliferation in vitro Detection (GeneCopoeia, United States) was used to evaluate cell proliferation. Ten micromolar EdU in DMEM was used for CAL27 and SCC25 cells in 48-well plates (3 × 104) for 2 hours. After fixation and membrane permeabilization, iClick reaction buffer was used to detect EdU. Then, the cells were stained with DAPI (Solarbio, China) for 5 minutes. A Leica microscope was used for imaging (495 nm, 360 nm) and analysis.
Cell migration and invasion assays
A wound healing test was used to test cell migration ability. CAL27 and SCC25 cells (1.5 × 106) were seeded in 6-well plates. After cell adherence, we used 200 µl pipette tips (Thermo QSP, United States) to generate wounds. A Leica microscope was used for imaging at 0 and 48 hours.
Cell invasion was tested by transwell invasion experiments. CAL27 and SCC25 cells (5 × 104) were plated in Matrigel-coated transwell chambers (Corning, 8 µm) and cultured overnight. Then, the medium in the upper chamber was changed to FBS-free medium. The medium in the lower chamber was changed to 20% FBS medium. Twenty-four hours later, the cells that had moved across the membrane were fixed, permeabilized and counted by DAPI (Servicebio) staining. A Leica microscope was used for imaging (360 nm).
Annexin V/PI double staining (GeneCopoeia, United States) was used for apoptosis detection. CAL27 and SCC25 cells (1 × 106) were collected. After labeling with Annexin V and propidine iodide, fluorescent dye solution was added to the cells away from light for 20 minutes. Then, flow cytometry (Beckman) was used for testing (488 λ and 560 λ), and FlowJo VX was used for calculation and analysis.
Human cancer tissues and para-cancer (> 1.5 cm from the tumor margin) normal tissues were collected from 48 OSCC patients in West China hospital of Stomatology, Sichuan University (China). After resection, the tissues were immediately frozen by liquid nitrogen and stored at − 80◦C for quantitative real-time PCR (RT-PCR). Written informed consent were signed by the patients. This study was approved by the Institutional Ethical Committee of West China hospital of Stomatology (WCHSIRB-OT-2016-047).
Both wild-type (CERS1+/+) and CERS1 knockout (CERS1-/-) C57BL/N6 mice were obtained from VITALSTAR (Beijing, China). The animals were housed in specific pathogen-free units at 24 ± 2 °C with 40‑60% humidity in a 14-hour light/10-hour dark cycle with freely accessible food at Sichuan University Animal Center (Chengdu, China). Six- to eight-week-old female mice (CERS1+/+ C57BL/N6 mice, n = 25 and CERS1-/- C57BL/N6 mice, n = 25) were used for the experiments. A stock solution of 4NQO (Sigma, United States) was prepared at 5 mg/ml (in propylene glycol). Two milliliters of stock solution was added to 100 ml of double distilled water to obtain a working concentration of 100 µg/ml. The mice were treated with 4NQO for 16 weeks and then observed for another 8 weeks. At the end of the experimental period, mice were sacrificed. Tongues were collected and then longitudinally bisected. The left half of the tongue was immediately fixed in 10% buffered formalin (Solarbio, China). The right half of the tongue was immediately put into RNAstore (Tiangen, China) and stored at − 80 °C for RT-PCR. All animal experiments were approved by the Subcommittee on Research and Animal Care of Sichuan University (WCHSIRB-D-2017-227).
Tongue tissues from the CERS1-/- group and CERS1+/+ group were processed for hematoxylin and eosin (H&E) staining. After 24 hours of fixation in 10% buffered formalin at room temperature, the tongue tissues were embedded with paraffin. After paraffin sectioning, deparaffinization and rehydration, the sections (4 µm) were stained with H&E (Solarbio, China). Histopathological diagnosis was performed by two experienced oral pathologists in a blinded manner. The tissues were classified into four types: normal epithelium, mild‑moderate dysplasia, severe dysplasia/carcinoma in situ, and carcinoma.
Immunohistochemical methods were used on sections (4 µm) of tongue tissues from hyperplastic lesions. The different groups of tongue tissues were deparaffinized and rehydrated in a graded ethanol series and distilled water. The slides were immersed in 0.01 M sodium citrate buffer (pH 6.0) and heated in a water bath at 95 °C for 30 minutes. Activities of endogenous peroxidases were inhibited by using 3% hydrogen peroxide. The sections were blocked with 3% BSA (Solarbio, China) for 20 minutes. Then, the sections were incubated overnight at 4 °C with anti-BAX antibody (1:1000), anti-BCL2 antibody (1:100), anti-MMP2 antibody (1:1000), anti-MMP9 antibody (1:800), anti-PCNA antibody (1:500) and anti-VEGFA antibody (1:500). All antibodies for immunohistochemistry were from Servicebio (China).The slides were rinsed with PBS 3 times and incubated with biotinylated anti‑mouse/rabbit IgG (Servicebio, China) for 50 minutes at room temperature. Then, we used diaminobenzidine to visualize the slices. Finally, nuclei were counterstained with hematoxylin for 3 minutes at room temperature. PBS was used as a negative control instead of the primary antibody.
Five high power (40x) fields of each slides were randomly selected and evaluated under a light microscope (Leica, Germany). The assessment of IHC staining was performed by evaluating the staining intensity and the percentage of positive cells. The intensity was graded as 0 (no staining), 1 (mild staining), 2 (moderate staining), or 3 (strong staining). The proportion of stained cells was graded as 0 (negative), 1 (0‑10% positive), 2 (10‑30% positive) or 3 (> 30% positive). The index score ranged from 0 to 9.
After treatment with siRNA, cells were collected, extracted with RIPA buffer (Beyotime, China) and boiled at 100 °C for 10 minutes. Then, the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%, Bio-Rad, China) and transferred to PVDF membranes (0.22 µm, Millipore). After blocking with 5% BSA, the membrane was incubated with the primary antibody and then secondary antibody. Equivalent protein loading was confirmed using anti-GAPDH (#2118, Cell Signaling Technology, United States). Anti-CERS1 (#ab98062, Abcam, United States), anti-ATF4 (#11815, Cell Signaling Technology, United States), anti-VEGFA (#ab46154, Abcam, United States), anti-BIP (#ER40402, Huabio, China), anti-CHOP (#5554, Cell Signaling Technology, United States), anti-BAX (#5023, Cell Signaling Technology, United States) and anti-BCL2 (#4223, Cell Signaling Technology, United States) were used. A chemifluorescence kit (Bio-Rad, China) and imaging system (Bio-Rad, China) were used for visualization of blots. ImageQuant 5.2 (GE Healthcare) was used for quantification.
RNA extraction and quantitative real-time PCR
The cells were first treated with siRNA and then collected using RNAiso Plus (Takara, United States). After 20% volume of chloroform was added, and the cells were subjected to high-speed centrifugation, RNA was present in the aqueous phase. Then, an equivalent volume of isopropyl alcohol (Solarbio, China) promotes RNA degradation. After washing with 75% ethanol and dissolving in RNase-free water, RNA was transcribed into cDNA by a RevertAid RT Kit (Thermo, United States). Primers were designed by BLAST and synthesized by Sangon Biotech. SYBR Premix Ex Taq II (Takara, United States) and ABI Q7 were used for RT-PCR.
Construction of VEGFA reporter plasmids
A 2.369 kb fragment containing a 5’ VEGFA sequence from − 2304 to + 65 relative to the transcription initiation site was amplified by PCR using Q5 High-Fidelity DNA Polymerase (NEB, United States). The forward primer with a SalI site was 5’-GATGTCGACTTGCTGGGTACCACCATGGA-3’, and the reverse primer, which had a XbaI site, was 5’-GATTCTAGACAGAGCGCTGGTGCTAGCC-3’. After digestion by QuickCut restriction endonucleases (Takara, United States), a DNA Ligation Kit (Takara, United States) was used to insert the PCR sequences into the SalI and XbaI sites of pEZX-FR01 (GeneCopoeia, United States), which contains a Rinella luciferase (Rluc) coding sequence with a CMV promoter and a promoter-less firefly luciferase (HLuc) coding sequence.
These recombinant plasmids were transfected into DH5α cells (TSINGKE, China) and amplified in nutrition agar plate (Solarbio, China) culture with kanamycin monosulfate (50 µg/ml, Solarbio, China) to select different monoclonal bacterial colonies. Then, the selected single clones were cultured in LB medium (Solarbio, China) with kanamycin. The recombinant plasmids were purified by a plasmid extraction kit (TIANGEN, China). All plasmid constructs were verified by direct sequencing (Sangon Biotech, Chengdu). The reporter plasmid was designated pEZX-VEGFA.
Plasmid transfection and luciferase assay
CAL27 cells (3 × 104) were plated in 96-well plates. After cell attachment, cells were transfected with the pEZX-VEGFA plasmid using Lipo2000 for 12 hours. Then, transfection media was replaced with the appropriate growth media. Next, the cells were divided into 4 groups based on the different treatments: control group, siNC; ATF4 knockdown group, siATF4; CERS1 knockdown group, siCERS1; and CERS1 + ATF4 knockdown group, siCERS1 + siATF4. After transfection with siRNA, a Luc-Pair Duo-Luciferase HS Assay kit (GeneCopoeia, United States) and microplate spectrophotometer were used to determine the relative luciferase activity.
IBM SPSS Statistics 20.0 was used for data statistics and analysis. Each experiment was performed independently at least three times with similar results. Data from one representative experiment are presented. The statistical methods are noted in the figure legends. p < 0.05 was deemed significant.