Total 60 HNSCC and 8 normal specimens were collected from the Hospital of Stomatology, Wuhan University. Our research was permitted by the Ethics Committee of Wuhan University. Written informed consent was obtained from each participant.
Cell lines and culture
The CAL27, SCC25, and HN4 cell lines were cultivated with culture medium containing 10 % fetal bovine serum (FBS, Natocor, Córdoba, Argentina). Above cell lines were obtained from China Center for Type Culture Collection (Shanghai, China). Human Immortalized Oral Epithelial Cell (HIOEC) was generously donated by Professor Chengzhang Li and cultivated in KGM-goldTM keratinocyte cell basal medium (Lonza, Walkersville, MD) with its supporting growth factors. All cells were cultured at 37 °C in moist circumstances with 5% CO2.
Recombinant lentivirus (Genechem, Shanghai, China) was used for FOXD1 overexpression. Short hairpin RNAs (shRNAs; Genechem, Shanghai, China) were used to knockdown FOXD1 expression (Additional file 1: Table S1). Small interference RNAs (siRNAs; Hanbio, Shanghai, China) were applied to silence SNAI2 expression (Additional file 1: Table S2). Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA) was used to transfect siRNAs and shRNAs. To screen out stable FOXD1 overexpression cell lines, the transfected cells were cultured with 2 μg/mL puromycin for 7 days.
Real-Time PCR (RT-PCR)
We conducted the experiments as already described . Primers that we employed in this research were shown in Table S3 (Additional file 1: Table S3).
The experiment was carried out following our previous protocol . Anti-FOXD1 antibody was purchased from Genetex (1:1000; CA, USA). Anti-E-cadherin, anti-N-cadherin, anti-Vimentin antibodies were purchased from Cell Signaling Technology (1:1000; MA, USA); anti-CD44, anti-ALDH1A1, anti-BMI1, anti-SNAI2 antibodies were purchased from Proteintech (1:1000; Wuhan, China).
Wound healing and Matrigel invasion assays
The experiments were processed in the light of previously designated . The images of migration were acquired at 0 and 24 h after scratch. The results of invasion were acquired 24 h after the inoculation. The migration areas and the number of invasion cells were measured by Image-ProPlus 6.0 (Media Cybernetics, Inc., USA).
Sphere forming and colony formation assays
400 cells were sown into each well of 12-well plates and then cultured for 10 days to observed colony formation ability. Then the colonies were fixed and stained with crystal violet. Colonies were photographed and counted under a light microscope.
The 6-well culture plates were pretreated with polyHEMA like previously described to construct low adhesion dishes . Cells were seeded into the pretreated plate (1000 cells/well) and cultivated with a standard cancer stem cells medium. After 12 days’ cultivation, the quality of spheres was observed under a microscope.
The experiment was conducted as previously described . In a few words, cells were sowed into 96-well plates and incubated with the mixture of 100 μl medium and 10 μl Cell Counting Kit‐8 (CCK‐8, Biosharp, Hefei, China) for 24, 48, and 72 h. After that, the proliferation ability of the cells was determined by the optical density (OD) at 450 nm.
EdU incorporation assay
EdU detection was performed using BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Shanghai, China). The HNSCC cells (1 × 104) were evenly sown into each well of 24-well culture plates and then cultivated with Edu reagents. 2 h later, the cells were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China). And then, cell staining was progressed following the manufacturer’s explanatory memorandum. Finally, images were attained with the fluorescent microscope (Biozero BZ-8000, Keyence, Osaka, Japan).
Luciferase reporter assay
The pGL4-basic plasmids containing SNAI2 promoter, pGL4-basic luciferase plasmids, phRL-TK plasmids were bought from Miaolingbio (Wuhan, China). The pGL4-basic plasmids containing SNAI2 promoter and phRL-TK plasmids were used to co-transfect into cells with Lipofectamine™ 3000. The cells transfected with phRL-TK plasmid and pGL4-basic luciferase plasmid were used as negative control. 48 h later, luciferase activity was determined by the Luciferase Assay System Kit (Promega, USA) according to the specification.
Paraffin‐embedded HNSCC tissues, normal mucosae, and xenograft tumors sections were IHC stained using anti-FOXD1 (1:100), anti-E-cadherin (1:400), anti-CD44 (1:200), anti-SNAI2 (1:400) antibodies. The IHC process was carried out as our previous study . The IHC score was determined by Image-ProPlus 6.0.
All the animal experiments were approved by the Ethics Committee of the Hospital of Stomatology at Wuhan University. The BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). CAL27 cells transfected with FOXD1 overexpression lentivirus or empty vectors were used for this assay. 45 days later, the tumors were removed and analyzed.
Student t-tests and one‐way analysis of variance were used in this research. All statistical analyses were performed using GraphPad Prism 6 software (San Diego, CA, USA). Experimental results were presented as the mean ± standard error of the mean. The statistical results were showed as: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.