Patient characteristics
Tissues were obtained from 30 OSCC patients at the Tianjin First Center Hospital. Written informed consent was obtained from all patients and the study was approved by the ethics committees of the Tianjin First Center Hospital.
Immunohistochemistry
The OSCC tissues were embedded with paraffin and sectioned. The sections were subjected to a routine three-step immunohistochemical (IHC) staining procedure. The slides were incubated with rabbit anti-SEMA3F antibody (1:200 dilution; Sigma) and anti-Ki67 antibody (1:200 dilution; Proteintech Group) at 4 °C overnight. Then, the slides were incubated with horseradish peroxidase-labelled secondary antibody for 30 min at room temperature. The chromogen 3,3′-diaminobenzidine tetrachloride (DAB; Zhongshanjinqiao) was used as a substrate.
The cell nucleus was dyed with Harris hematoxylin solution. Levels of SEMA3F expression were determined based on the extent and intensity of staining with the scoring using this scale: negative for <5%, weak for 5–25%, mid for 25–50%, distinct for 50–75%, and strong for ≥75%. The staining intensity and stained area percentage were multiplied to produce a weighted score. Three independent evaluators assessed the scoring.
Cell culture
We used 4 human OSCC-derived cell lines: SAS, Ca9–22, HSC2 and HSC4. One normal oral keratinocyte strain was obtained from a patient who had undergone dental surgery. This served as the control. The patient provided written informed consent prior to the start of the study. The cell lines were cultured in DMEM (Thermo-Fisher) with 10% fetal bovine serum (FBS) plus 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2.
Plasmid construction
The CDS sequence of SEMA3F was obtained via PCR using specific primers. The PCR products were cloned into the pcDNA3.1(+) vector. The plasmid was named pcDNA-SEMA3F. The primers were: SEMA3F forward: 5’-CTAGCTAGCATGCTTGTCGCCGGTC-3′; SEMA3F reverse: 5’-CTAGTCTAGATCATGTGTCCGGAG-3′.
Cell transfection
Transfection of cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, cells were seeded in 6-well plates at 30–40% confluence 24 h prior to transfection. pcDNA-SEMA3F (2 μg per well) and the control were used for each transfection.
For stable transfection, HSC2 cells were selected in G418 (800 mg/ml). Individual G418-resistant colonies were isolated after 15 days of culture. Cells were treated with G418 (200 mg/ml) and maintained in culture until needed for subcutaneous inoculation into mice.
MTT and colony formation assays
The MTT assay was performed daily over 3 days to evaluate cell proliferation. First, cells were transfected with plasmids (control or pcDNA-SEMA3F). After 24 h, cells were seeded into 96-well plates (3 × 103 cells/well). Next, the cells were incubated with 25 μl of MTT (5 mg/ml, Sigma) at 37 °C for 4 h, the supernatants were removed, and 150 μl methylsulfoxide (DMSO; Sigma) was added to each well. The absorbance value (OD) of each well was measured at 490 nm.
For the colony formation assay, cells (5 × 105 cells per well) were seeded in 6-well plates and transfected with pcDNA-SEMA3F (2 μg per well) or the control for 24 h. The medium was refreshed every 3 days. After 2–3 weeks of culture, the colonies were fixed with methanol, stained with 1.25% crystal violet and counted under a light microscope. All experiments were performed three times and the average results were calculated.
Quantitative RT-PCR
Total RNA was extracted using Trizol reagent (Takara). Then, 1 μg of the RNA was converted to cDNA using Revert Acid Reverse Transcriptase (Fermentas). Real-time PCR was conducted using a Sigma-Aldrich FastStart Universal SYBR Green Master (ROX) Kit according to the manufacturer’s instructions. Double-stranded DNA specific expression was tested by the comparative Ct method using 2-ΔΔCt. The sequence of the primers used were: SEMA3F forward: 5’-CTCTGGGCTTCCCTACTGAC-3′; reverse: 5’-CACTCGCCGTTGACATCC-3′; GAPDH forward: 5’-ATCACCATCTTCCAGGAGCG A-3′; reverse: 5’-CCTTCTCCATGGTGGTGAAGAC-3′. Experiments were performed in triplicate.
Western blot
Cellular proteins were extracted in RIPA buffer (Biomed) after transfection for 48 h. Proteins were separated by gel electrophoresis and transferred to membranes, which were then incubated with primary antibodies. The concentrations and sources of the antibodies were: rabbit polyclonal anti-NRP2 (1:2000; Abclonal), rabbit polyclonal anti-SEMA3F (1:2500; Sigma) and mouse monoclonal antihuman anti-GAPDH (1:2000; Abcam). Treatment with secondary antibodies diluted in PBST was done at room temperature for 1 h. Membranes were washed in PBST and the bound antibody was detected using an enhanced chemiluminescence system. The experiments were repeated three times.
Migration assay
Cells were transfected with pcDNA-SEMA3F and the control. After 48 h, the cells were seeded on the upper chamber of an 8-μm pore size transwell insert (Costar) at 2.5 × 104 cells/insert in DMEM. The lower chamber contained DMEM (600 μl) with 10% FBS. The migrated cells were fixed with 4% paraformaldehyde in PBS and stained in 0.5% crystal violet. The membranes were mounted on a microscope slide. Migrated cells were counted and photographed. The percentage of migrated cells was calculated. The migration index was expressed relative to the control cells. All the experiments were carried out three times and the results were expressed as the means ± SD.
Cell invasion assay
Cell invasion assays were performed in 24-well plates using transwell chambers (8.0 μm pore size, Millipore) were coated with matrigel. The cells were transfected and then seeded in the upper chamber at a density of 2 × 105 cells/ml in 400 μl of medium containing 0.5% FBS. Medium with 10% FBS was added to the lower chamber. Following 24 h incubation at 37 °C with 5% CO2, the invading cells were fixed in 100% methanol and stained with 0.5% crystal violet. Photographs were taken randomly for at least four fields of each membrane. The number of invading cells was expressed as the average number of cells per microscopic field over four fields.
In vivo tumor xenograft model
A total of twelve nude mice (4-week old BALB/c nude mice; Huafukang) were randomly divided into two groups. HSC2 cells stably transfected with pcDNA-SEMA3F and control were inoculated subcutaneously into the flanks of nude mice. The length and width of tumors were taken with Vernier calipers every five days, and the mice were euthanized after thirty days. The volume of the implanted tumor was calculated using the formula: volume = (length × width2)/2. All animals were treated according to the guidelines established by the National Institutes of Health Guide (NIH).
Statistical analysis
Results were analyzed statistically using Student’s t-test for comparisons between two groups. Data are presented as the means ± SD. Analyses were performed using SPSS 20.0. p < 0.05 and p < 0.01 were considered to be statistically significant. Unless indicated, the results shown in the figures are representative. All the experiments were done at least three times.
