30 OSCC specimens containing adjacent noncancerous areas and 28 normal oral mucous tissues for immunohistochemical (IHC) analysis were collected from the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Sichuan University. Demographic data and other variables, including dates of diagnoses, site and size of primary tumor, local regional recurrence, and distant metastasis were retrieved from the database provided by the oncology registry. The cancerous or noncancerous areas were identified by two pathologists independently, according to the IHC staining. The pathologists were blinded to patient clinical information. If the evaluations were controversial, the samples were re-evaluated and classified based on the assessment given most frequently by the pathologists. All the samples were obtained with patient's informed consent. The protocol of the study was approved by the Institutional Ethics Committee of West China Center, Sichuan University, China.
Immunohistochemistry Anti-FGF8 rabbit monoclonal antibody (ab81384, 1:200) was purchased from Abcam (Cambridge, MA, USA). Immunohistochemistry was detected on a slide carrying 4-mm–thick tissue from paraffin-embedded tumor species. After baked in a 37℃ oven overnight, all slides were dewaxed in xylene and then rehydrated in ascending series of ethanol. Antigen retrieval was conducted by citrate antigen retrieval solution in an autoclave for 5min. Three percent of hydrogen peroxide was incubated for 15min and normal goat serum working fluid incubated for 15min at 37°C after washing for 5min twice. Then the sections were exposed to the primary antibodies at 4°C in the wet box for one night. The slide tissues were washed in PBS for 5min three times and incubated secondary antibody for 15min at 37°C. DAB chromogenic reagents were used to detect the reaction of antigen and antibody and the slides were counterstained in hematoxylin, dehydrated in gradient alcohol, cleared in xylene.
To estimate the score of each section, eight individual fields were chosen by 2 dependent observers, and 100 cancer cells were counted for each field. We quantitatively scored the tissue sections according to the percentage of positively stained cells and staining intensity as described previously, with minor modifications. We assigned the following proportion scores: 0 if 0% of the tumor cells with positive staining, 1 if 0% to 10%, 2 if 11% to 30%, 3 if 31% to 70%, and 4 if 71% to 100%. We also rated the intensity of staining on a scale of 0 to 3: 0, negative; 1, weak; 2, moderate; 3, strong and 4, very strong. We then multiplied the proportion score by the intensity score to obtain a total score (range: 0-16). Scores were compared with overall survival duration, which was defined as the time from the date of diagnosis to death or the last known date of follow-up.
- Bioinformatics analysis
Bioinformatics analysis of FGF8-assocaited proteins were performed following previous reports. The protein-protein interaction (PPI) network was conducted based on the identified proteins, and biological evidence was collected from PrePPI to obtain the correlation of protein localization, the correlation of expression, the mutual binding, the upstream and downstream related proteins. Identified FGF8-associated proteins were classified according to the GO (Gene Ontology) Annotation clustering. The network group analysis was conducted via DAVID database (http://david.abcc.ncifcrf.gov/).
- Cell culture.
The HSC-3 and HSC-4 cell lines were provided by State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Hyclone, USA), penicillin (107 U/L) and streptomycin (10 mg/L) at 37°C in a humidified chamber containing 5% CO2.
Total RNA of OSCC cell lines was isolated by TRIzol reagent (Invitrogen) and reverse transcript to cDNA with 1μg RNA in a volume of 20 μl by ExScript TM reagent kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The primers detailed sequences were as follows: FGF8: Forward Primer: 5’-CGC AAA GCT CAT TGT GGA GA-3’, Reverser Primer: 5’ ACA CGC AGT CCT TGC CTT TG-3’; GAPDH: Forward Primer: 5’-GAG TCA ACG GAT TTG GTC GT-3’, Reverser Primer: 5’-TTG ATT TTG GAG GGA TCT CG-3’. Gene expression level was assessed by SYBR green qPCR SuperMix (Applied Biosystems Life Technologies, Foster, CA) and GAPDH served as an internal reference. The fold-change in the expression of each target mRNA relative to GAPDH was calculated using the CT (2 −ΔΔCT) method. Each experiment was conducted in triplicate.
- Wound healing assay
When the cells cultured in 6-well plates reached approximately 100%, the wells were gently scratched with a 100 μL pipette tip to create a uniform linear scratch. Then the cells were cultured in serum-free culture medium, and observed and photographed at 0h, 12h, and 36h. Cell migration was assessed by percent of wound closure through using Image-Pro Plus Analysis software (Media Cybernetics company, Rockville, MD). All experiments were conducted for three times to obtain the average value.
- Transwell invasion assay
Invasion assays were carried out using 24-well culture plates containing the transwell chamber covered with Matrigel (1:4, BD, USA). 1×10 5 HSC-3 and 2×10 5 HSC-4 cells suspended in serum-free medium were placed in the upper chamber. 500μL medium containing 10% FBS were placed in the lower chamber. Cells remaining on the upper chamber were removed using a cotton swab after being incubated at 37°C for 12-36h, while cells traversed to reverse face of the membrane were fixed in 4% paraformaldehyde, stained with 1% Crystal Violet, washed three times with PBS, then air dried. The chamber was inverted on a microslide and observed under a microscope. Five fields per chamber were randomly selected for counting the number of invasive cells, and images were taken. Each experiment was conducted for three times.
- In vivo tumor metastasis
All animals were humanely treated under the guidelines of the Institutional Animal Care and Treatment Committee of Sichuan University. 5×10 6 OSCC-FGF8 or OSCC-mock cells were injected into female athymic nude mice (10 mice per group) through the tail vein. Animals were sacrificed 28 days after injection. The lungs were excised and fixed in formalin for standard hematoxylin and eosin (H&E) staining.
- Western blotting
After FGF8 treatment, total proteins of HSC-3 cells were extracted in RIPA buffer (50 mM Tris base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1% cocktail) and quantified by coomassie brilliant G-250 (Bio-Rad). Samples were separated on 12% or 15% SDS- PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST for 1h at 37°C and probed with primary antibody overnight at 4°C. After washing with TBST membranes were incubated with secondary antibody (1:5000 dilution; Santa Cruz Biotechnology) conjugated to horseradish peroxidase for 1h at 37°C. Finally, the proteins were detected by electro-chemiluminescence (ECL) Western blotting reagents. The following primary antibodies were used according to the manufacturer's instructions: anti-E cadherin mouse monoclonal antibody (ab1416, 1:1000), anti-Vimentin rabbit polyclonal antibody (ab137321, 1:1000), Anti-Snail rabbit polyclonal antibody (ab82846, 1:800), and anti-GAPDH (ab8245, 1:1000, Abcam).
- Immunofluorescence staining
The OSCC cells were cultured in 24-well cell culture plates, fixed for 15min with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 min. After blocking with normal goat serum working fluid for 1h at 37°C, primary antibody was incubated overnight at 4°C, and then staining was detected with fluorescein-conjugated secondary antibodies (PeproTech; 1:200) for 1h in dark condition. Finally, cells were stained with 4,6-diamidino-2-phenylindole (DAPI; blue) to show the nuclear position for 5min. Immunofluorescence signals were examined using a fluorescence microscope (Leica, Bensheim, Germany).
- Lentiviral transduction
Expression of FGF8 were established using a pCDH Lentivector Expression System (System Biosciences, Mountain View, CA) according to the manufacturer’s instructions. Briefly, the indicated shRNAs or cDNAs were cloned into pCDH lentiviral vector. Lentiviruses were produced by co-transfecting 293T cells with one of the expression plasmids and three packaging plasmids (pLP1, pLP2, pLP/VSVG). Infectious lentiviruses were harvested 72 h after transfection, centrifuged to remove cell debris, and filtered through 0.45 µm filter (Millipore, Bedford, MA).