2.1 Ethics Statement
The protocol for this study was approved by the Institutional Review Board of Nanjing Medical University and all experiments were performed after obtaining written informed consent for OSCC clinical specimens.
2.2 Patient and Tissue Samples
Specimens were obtained from a retrospective cohort of 58 primary human OSCC cases seen from Jan. 2011 to Dec. 2014. 11 samples of normal oral mucosa tissues from other non-cancer surgeries during the same period were collected from patients who underwent surgical resection at the department of oral and maxillofacial surgery, Nanjing Medical University (Nanjing, China). These OSCC patients did not receive any preoperative treatment before surgery. 6 pairs of fresh clinical specimens (for use in RT-PCR and Western blot) were collected from OSCC patients at the same department. Histological examination was performed by senior oral pathologists (according to the diagnosis by Chief Dr Song XL, the senior pathologist at the Affiliated Stomatological Hospital of Nanjing Medical University), and diagnosis made based on carcinoma cell features as seen under a microscope. Tumors were classified according to the International Union Against Cancer (UICC) tumor staging system. All fresh tissue samples were collected and immediately stored at -80 °C until further use.
2.3 Mice
Animal studies were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited SPF animal facility and all protocols approved by the Animal Care and Use Committee of the Animal Research Center, Nanjing Medical University.
2.4 Cell lines and culture
Human oral keratin cells (OKC) and three human HNSCC cell lines: HN4, HN6, SCC25 were purchased from American Type Culture Collection (ATCC). Cancerous cell lines were cultured in DMEM media (Invitrogen) supplemented with 10% FBS (Hyclone) and 100 units/ml penicillin and streptomycin (Sigma) in 5% CO2 at 37 °C.
2.5 Immunohistochemistry
IHC staining was performed using the standard streptavidin–biotin–peroxidase complex method according to the manufacturers’ protocol (Abcam, USA). Briefly, 4 mm tissue sections from representative paraffin blocks were deparaffinized in xylene and rehydrated using an ethanol gradient. Endogenous peroxidases were blocked with 3% hydrogen peroxide. Slides were heated for antigen retrieval in 10 mM sodium citrate retrieval buffer (0.01 M sodium citrate and 0.01 M citric acid, pH 6.0), at 95℃ for 5 minutes. Sections were blocked in 10% normal swine serum for 20 minutes and incubated with monoclonal rabbit primary antibody (anti-SATB2, 1:200 dilution; Abcam, USA), monoclonal rabbit primary antibody (anti-NOX4, 1:400 dilution; Abcam, USA), at 4 ℃ overnight. Specimens incubated with PBS instead of primary antibodies were used as negative controls. Sections were then incubated with secondary antibodies for 45 minutes at room temperature. Reaction products were developed by 3, 3’-diaminobenzidine solution with hydrogen peroxide, followed by hematoxylin counterstaining. Immunoreactivity was semi-quantitatively evaluated according to staining intensity and distribution using the immunoreactive score calculated as: intensity score × proportion score. As previously reported, the intensity score was defined as; 0 (negative), 1 (weak), 2 (moderate), or 3 (strong), and the proportion score was defined as; 0 (negative) 1 (<10%) 2 (11–50%), 3 (51–80%), or 4 (>80%), of total positive cells. The total score ranged from 0 to 12 and the immunoreactivity of each slide was categorized into three subgroups based on the final score: 0 (negative), 1–4 (low expression), and 4–12 (high expression).
2.6 RNA extraction and quantitative real-time PCR
Total RNA was extracted using TRIZOL Reagent (Invitrogen) and cDNA synthesized using a reverse transcription Polymerase Chain Reaction (RT-PCR) Kit (TaKaRa) according to the manufacturer’s instructions. Quantitative real-time Polymerase Chain Reaction (qRT-PCR) was performed using SYBR Premix Ex TaqTM II PCR Kit (TaKaRa) and an ABI 7500HT PCR sequencer (Applied Bio-systems). Primers specificity was verified by dissociation curve analysis. Data was analyzed using ABI SDS v2.4 software (Applied Biosystems). All qRT-PCR reactions were performed in triplicate. The housekeeping gene GAPDH was used as an internal control.
2.7 Western blot analysis
Protein samples were extracted by RIPA (Beyotime, Shanghai, China). Equal amounts of protein lysate were separated by SDS–PAGE, transferred to a polyvinylidenedifluoride (PVDF) membrane (Millipore), then blocked with 3% BSA in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h at room temperature. The blocked membrane was then incubated with polyclonal primary antibody against (SATB2/MOX4) overnight at 4 ℃, washed three times in TBS-T, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Antibody-labelled proteins were detected using an ECL western blot detection kit (Bio-Rad Laboratories, Hercules, CA, USA) and X-ray film (Kodar). Tubulin or GAPDH were used as loading controls.
2.8 Immunofluorescence on HN4 cells
Cells were seeded onto 12-mm coverslips in 24 well plates. After 24 h incubation cells were washed in cold PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, then washed three times with cold PBS. Cells were blocked with 3% BSA and 0.1% Triton-100 for 1 h at 37°C, incubated with a SATB2 primary antibody (Abcam,1:200) overnight at 4 ℃, washed three times with cold PBS, and incubated with FITC-conjugated secondary antibodies for 1 h. Cells were subsequently stained with DAPI for 10 min at room temperature (Roche Diagnostics), sealed with 70% glycerin, and examined using a Nikon fluorescence microscope.
2.9 Lentivirus generation and cell transfection
Lentivirus encoding full-length human SATB2 cDNA was purchased from Shanghai Cyagen biotechnology Co. Ltd. and used to transduct HN4 and HN6 cell lines which had reached 50% confluency. These cells were grown in DMEM medium (Invitrogen) with 10% FBS (Hyclone) for another 48h, after which stable SATB2 –expressing clones were screened by adding 10 μg/ml puromycin (Sigma) and growing for a further 24 hours. This was repeated twice to acquire Lv-SATB2-HN4 cells and N.C-transducted cells were used as controls.
2.10 siRNA Synthesis and Transfection
Specific small interfering RNAs (siRNA) against SATB2 and NOX4 were purchased from Shanghai Genepharma biotechnology Co. Ltd. One day before transfection, 2×105 HN4 cells (in 2ml antibiotic-free growth medium) were seeded at per well in a 6-well plate (Costar) . The second day , each of 5μL siRNA vector (100ng/μL) or 3μL Lipofectamine TM 2000 (Invitrogen) were diluted in 50μL serum-free Opti-MEM medium (GIBCO,USA), mixed gently and incubated at room temperature for 5 min. Then, the diluted siRNA vector and Lipofectamine TM 2000 were combined, gently mixed, and incubated at room temperature for 20 minutes. During this incubation time, the seeded HN4 cells were washed twice with PBS and 1.9ml serum-free medium was added to each well. 100μL of the siRNA- Lipofectamine TM 2000 mixture was added to each well and gently mixed by rocking the plate. The cells were grown in 5% CO2 and 95% air at 37℃ for 6 hours and the serum-free medium was exchanged for serum containing medium. Cells were grown for a further 72 h then collected for assays. The siRNA sequences are listed in Supplementary Table 4.
2.11 Flow Cytometry
HN4 and Lv-SATB2 –HN4 cells were trypsinized and resuspended in PBS. For cell cycle analysis, cells were washed in PBS for 3 times, fixed in 70% ethanol, and then stained with propidium iodide following RNase treatment. The DNA content and cell cycle distributions were analyzed using FACS flow cytometry (BD, USA) and Cell Quest software (BD Biosciences, USA).
2.12 CCK8 assay
Cell proliferation and viability were assessed using a CCK8 assay. 1x103 cells were seeded in the wells of a 96-well plate and incubated overnight at 37°C. 10μl CCK8 was mixed with 90μl DMEM medium (Invitrogen) containing 10% FBS (Hyclone), and added to each well and plates were incubated for another 2-4 h. These steps were conducted in a dark environment. After 2-4 hours’ incubation, we restarted time points (0, 24, 48 and 72 h), the absorbance was measured at 450 nm using an automatic enzyme-linked immune sorbent assay reader (Molecular Devices, San José, CA, USA).
2.13 Cell Migration and Invasion Assays
In wound healing experiments, cells were seeded at 70% confluency in 6-well plates (Costar) filled with DMEM/F -12 containing 10% FBS. 24 hours after seeding, scraped a wound line with a P1000 pipette tip among the confluent monolayers, washed to remove cell debris, and media was replaced. At specific time points, cells were fixed in 3.7% paraformaldehyde and photographed under a phase-contrast microscope. Migration assays were performed using a trans-well chamber (Corning, NY, USA). 1×104 cells (in 200 µL DMEM/F-12) were placed on the upper layer of a cell-permeable membrane and 500 µL DMEM/F-12 containing 10% FBS was placed in the lower chamber. Following an incubation period, the cells that had migrated through the membrane were stained. Matrigel matrix (BD, Billerica, MAUSA) was used to simulate a human basement membrane for the invasion assay. DMEM and Matrigel matrix were mixed and the membrane was also coated with matrigel (1:9 dilution ratio) for testing invasion.
2.14 OSCC tumor xenograft formation in nude mice
All animal protocols used in this study were in accordance with the institutional animal welfare guidelines of Nanjing Medical University. Mice were randomly assigned into four groups, each containing six 4-to-6-week-old male nude mice. For HN4 cell line, each experimental group was subcutaneously injected with a total of 2 × 106 cells of either; HN4, Lv-SATB2-HN4, Lv-SATB2-HN4 with Negative Control, or Lv-SATB2-HN4 with NOX4 knockdown. As for HN6 cell line, each of 5 × 106 HN6 and Lv-SATB2-HN6 cells were subcutaneously injected into the left and right flanks respectively. Tumor sizes and weight were recorded every three days. All extant mice were euthanized at 4 or 8 weeks after injection then opened at injection sites to confirm tumor size and weight. Tumor volumes were measured by caliper and calculated as follows: Volume (mm3) = D × d2 × 0.5, where D is the longest, and d the shortest, diameter of the tumor.
2.14 Statistical analysis
All statistical analysis were performed using Graph Pad Prism 5.01 (La Jolla, CA, USA) or SPSS 18.0 (Armonk, NY, USA). Pearson χ2 test was used to analyze the association between SATB2 expression and clinical pathology parameters. Survival rate analysis was analyzed by Kaplan–Meier plot and log-rank test. Independent Student t-test and ANOVA with post hoc test were used for most other analyses as indicated in figure legends. The data are presented as the mean ±S.D. of at least three independent experiments. The P-values were defined as *P<0.05, **P<0.001 and ***P<0.0001.