2.1: Data Download༚
Human head and neck squamous cell carcinoma and normal tissue microarray data were obtained from the geo database(https://www.ncbi.nlm.nih.gov/geo/), Down loaded GSE6631 and GSE13398 datasets,GSE6631was uploaded by M A Kuriakoseet.al, which contains paired HNSCC tumor and normal samples from 22 patients were evaluated for differential gene expression on Affymetrix U95A chips. GSE13398 was uploaded by Christopher R Cabanski et.al, contains 8 HNSCC tumor samples and 8 normal tonsil samples.Human Protein Atlas (http://www.proteinatl as.org) was used to validate expression levels of FTH1.
2.2 DEG analysis:
The R language limma package (version 3.4.2) (22) (http://www.bioconductor.org/packages/
release/bioc/html/limma.html) was used to analyze theDEGs among samples. The adjusted P<0.05 and |log2fold change|>1 were taken as the cut-off values. The DEG-related PPI network was established using the STRING database (https://www.string-db.org/) followed using visualization by Cytoscape software (version 3.8.2)(http://manual.cytoscape.org)
2.3 FTH1 gene and HNSCC correlations analysis:
Human Protein Atlas (http://www.proteinatl as.org) was used to validate FTH1 relations with HNSCC and further display corralations between FTH1 and tumor stage. The Kaplan-Meier (K–M) survival curves and log-rank test were generated to evaluate the difference in FTH1 high expression group and Low expression group in total TCGA HNSCC cohort.
2.4 Cell acquisition
This work was approved by Ethics Committee of Shanghai General Hospital. HNSCCs were purchased from procell Life Science & Technology Co., Ltd. The obtained cells were cultured and passaged as primary HSCC cell line, Cells were cultured in DMEM medium composed of 10 % fetal bovine serum and 1 % double antibody (penicillin-streptomycin mixture) in 37℃ and 5 % CO2 incubator, the third-generation cells were used in this experiment.
Observation of FTH1 by immunohistochemistry and Immunofluorescence staining
FTH1 expression was visualized using immunohistochemistry (IHC) method. Fadu cells were fixed in 4% paraformaldehyde (Beyotime, Beijing, China) for 15 minutes at 37°C, permeabilized using 0.1% Triton X-100 (Beyotime) for 20 minutes, blocked using 5%bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO) for 1 hour at 37°C, and immunostained using the following antibodie for 2 hours at 37°C, perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol: FTH1:(ab65080) 1/400. One hundred microliters of reaction enhancer from a 2-step IHC kit (Zhongshanjinqiao, Beijing,China) was added to the samples, which were incubated at 37°C for 20 minutes. The samples were then incubated with HRP labeled broad spectrum secondary antibodies (1:1000) for 30 minutes at 37°C, after which the nuclei were counterstained with diaminobenzidine (1:1000; Beyotime) for 5 to 8 minutes. Washed with tap water, samples were stained with hematoxylin for 20 seconds (Beyotime), then dehydrated with different concentrations of ethanol and clarified using a dimethylbenzene solution. Images were captured with an inverted phase-contrast microscope.
IF assays were conducted to identify FTH1 protein expression location.The pre-treatment procedure was the same as that used for IHC. IF was performed with FTH1:( 5 µg/ml.,ab65080) antibody for 12 h in the dark, followed by incubation with 1:1000 DyLight 594 Conjugated, Goat Anti-Human IgG (Abbkine Inc., North Chicago,IL). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a fluorescence microscope (Leica DMi8, Wetzlar, Germany).
2.5 Cell survivals:
Cell survivals were measured by CCK-8 method. TanIIA (0.24 mg/L, 0.5 mg/L, 1.0 mg/L) intervention for 24 h,48h,72h served as the treatment group. Grouped cells were grown in 96-well plates and cell survival was measured after 24 hours following the manufacturer’s protocol. CCK-8 and serum-free medium were mixed at a volume ratio of 1:10 and incubated in a 5 % CO2 at 37 ºC for 1 hour. Absorbance at 450 nm wavelength (OD value) was measured by a microplate reader.
2.6 Tumor invasive ability
BD Matrigel frozen in a - 80°C freezer at 4 degrees overnight for 24 h, after which 300ul serum-free medium was added 60ul Matrigel mix (4°C operation), 100ul each of the upper chamber was added (3 chambers). Put into the 37°C incubator, for 5h, serum-free medium and Matrigel were diluted at 1:5, and 50ul was added to each well, incubated 2h in the 37 ° C. The cells were digested, washed 3 times with serum-free medium, counted, and a cell suspension was made. Matrigel was washed 1 time with serum-free medium, 100ul of cell suspension was added to each well; 500ul of conditioned medium containing 20% FBS was then added to the lower chamber. Incubation in 37 ℃ incubator for 24h. Transwell chambers were removed and washed 2 times with PBS, fixed with 5% glutaraldehyde at 4 ℃. Add crystal violet (0.1%) to stain for 5-10 min, leave at room temperature for 0.5 h, wash with PBS twice, wipe off the upper surface cells with cotton ball, then observe under microscope.
2.7 Western-Blot assay
Total proteins were extracted using One Step Animal Tissue/Cell Active Protein Extraction buffer (RIPA; Thermo Fisher Scientific). The cells were seeded in 6-well plates and incubated overnight in 37℃ and 5 % CO2 incubator. All groups were starved for 48 hours in FBS-free medium to achieve synchronization. TanIIA group was intervened with 0.5mg / L concentration. Cells cultured in drug-free medium represented the control group. Samples were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad Electrophoresis System. The proteins were transferred to a nitrocellulose membrane gel electrophoresis separation. Blocking with 5 % skim milk for 1 hour, Tris-buffered saline with Tween 20 (TBST) was used to wash the membranes. Overnight incubation of the membranes with primary antibodies: FTH1: (1µg/ml, Abcam, ab65080,21kDa); GAPDH (1:500, Abcam, ab8245,36kDa) was done at 4°C. This followed another incubation with a 1:10000 dilution of HRP labeled secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 hour. A Bio-Rad ChemiDoc MP was used to expose the membranes, and analysis was performed using Image Lab software (Bio-Rad).
2.8 Statistical analysis
All experiments were repeated in triplicate and the results were presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 21.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8 (La Jolla, CA, USA). Statistically significant differences were determined at P<0.05.