1. Cell culture
A human laryngeal squamous cancer cell line, Hep2, was purchased from Medical Science Research Center, Zhongnan Hospital of Wuhan University, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100µg/ml streptomycin. Cells were maintained at 37℃ in a humidified incubator with a mixture of 95% air (20% O2) and 5% CO2. 1%O2, 94%N2 and 5%CO2 was chosen for hypoxia experiment. MIF inhibitor ISO-1 (SML0472) was purchased from Sigma-Aldrich, and JAK inhibitor XL019 was purchased from Beyotime Biotechnology.
2. Western blotting
For Western blotting, cells were lysed with RIPA lysis buffer kit (Beyotime, China), supernatants were collected after spin and total proteins were measured using the BCA protein quantification kit (Beyotime, China). Total protein samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the samples were transferred onto 0.22µm PVDF membranes. After blocking with 5% fat-free milk for 1 hour at room temperature, the membranes were incubated with primary antibodies overnight at 4℃. Following three washes with TBST buffer, the membranes were incubated with secondary goat anti-rabbit antibodies conjugated with HRP for 1 hour at room temperature. Signals were visualized using the enhanced chemiluminescence kit (Beyotime, China) and detected by the Imaging system (Baygene Biotech, China). Integrated relative densities of individual bands were quantified using Image J (National Institutes of Health, Bethesda, MD).
3. Quantitative real-time PCR
Total RNA was extracted following the manufacturer's protocol with TRIzol reagent (Invitrogen, CA), dissolved in RNA-free H2O and stored at -80°C. cDNA synthesis was performed from each 1µg RNA sample using the Reverse Transcriptase Kit (Thermo, USA). Then qRT-PCR was performed on a CFX96 Connect (Bio-Rad, CA) using a SYBR Green PCR kit (Vazyme Biotech, China). Expression data were calculated using the 2−ΔΔCt method and normalized by taking GAPDH as an internal reference to control the relative expression levels. Primer sequences were listed in Table 1.
4. ELISA assay
MIF and IL-6 concentrations were detected by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol. Quantitative IL-6 ELISA assay kit was purchased from QuantiCyto (China), Quantitative MIF ELISA assay kit was purchased from Elabscience (USA), and TG, NEFA levels were estimated by assay kits (Nanjing Jiancheng Bioengineering Institute, China). All were used according to the manufacturers’ instructions. All samples were measured in triplicate and average values were determined.
5. Bioinformatics analysis
Hypoxia gene sets and fatty gene sets were downloaded from Gene Set Enrichment Analysis(GSEA, http://www.gsea-msigdb.org/gsea/index.jsp) and Venn diagrams were used to analyze the intersection of the two gene sets. Gene expression profiles of larynx were downloaded from TCGA (https://portal.gdc.cancer.gov/). Gene Set Enrichment Analysis (GSEA) was performed using GSEA software 4.0.3(USA).
Transcriptome sequencing and analysis were performed by the BGI Company (http://bgitechsolutions.com). Total RNA was extracted following the manufacturer's protocol with TRIzol reagent (Invitrogen, CA), dissolved in RNA-free H2O and stored at -80°C. Total RNA was processed by mRNA enrichment: The mRNA with polyA tail was enriched by magnetic beads with OligodT. The RNA obtained was segmented by interrupting buffer, and the random N6 primers were reversely transcribed, and then the cDNA two-strand was synthesized to form double-stranded DNA. The synthetic double-stranded DNA ends are flattened and phosphorylated at the 5' end to form A sticky end protruding an "A" at the 3' end, followed by A bubbling-like connector protruding A "T" at the 3' end. The ligands were amplified by PCR using specific primers. The PCR product was thermally denatured into single strand, and then the single strand DNA was cycled with a bridge primer to obtain a single strand circular DNA library. The constructed library was inspected and sequenced after qualified. The resulting data are referred to as RAW reads or RAW data, and the RAW reads are then subjected to quality control (QC) to determine whether the sequenced data are suitable for subsequent analysis. After quality control, the filtered clean reads were compared to the reference sequence. After alignment, the distribution of alignment rate and reads on the reference sequence was counted to determine whether alignment results passed the second QC of alignment. If passed, gene quantitative analysis was carried out and differential gene expression among screened samples will be conducted.
7. Immunohistochemistry staining
Tissue slides were deparaffinized in xylene and rehydrated in alcohol. Then, antigen retrieval was performed with 0.1 M sodium citrate buffer. Subsequently, the sections were blocked by performing IHC kit(Maixin China) and probed with primary antibodies for 1 h at room temperature. Slides were incubated with poly-HRP secondary antibodies by performing IHC kit, after which sections were counterstained with haematoxylin to visualize nuclei. Images were analyzed by using Image J v1.8.0(National Institutes of Health, USA).
8. Animal experiment
All mice were maintained at Zhongnan Hospital of Wuhan University and all animal experiments were performed in accordance with Zhongnan Hospital animal ethics committee(Wuhan, China). Male BABL/c nude mice (4 weeks old) were purchased from Gempharmatech (Zhejiang, China). Hep2 cells were resuspended at 2×107 cells/mL using saline, and each mouse was subcutaneously injected 200 µL into the right anterior flank. After 8 days, the mice were randomly divided into treatment group and control group(5 mice per group). The treatment group was treated with ISO-1(2.5 mg/kg, intraperitoneally, every day) and the control group was treated with saline(equal volume per weight, intraperitoneally, every day). Tumor volume was measured using a caliper every other day, and volumes were calculated using the standard formula: V = 0.5*length*width2. Finally, mice were euthanized, and tumors were removed, photographed, weighed and collected for ELISA and immunohistochemistry.
9. Statistical analysis
All data were represented as means ± standard deviations (SD). All differences between two independent groups were analyzed using Students’ t test. SPSS 20.0 software (IBM Corporation, USA) was used for statistical analysis. A P-value < 0.05 was considered statistically significant.