Hep2, the human laryngocarcinoma cell line, was obtained from the Medical Science Research Center, Zhongnan Hospital of Wuhan University, and maintained in DMEM/HIGH GLUCOSE supplemented with 10% fetal bovine serum, plus the 1X penicillin–streptomycin solution (Bioshrp, Hefei, China). Cells were cultured in a humidified incubator at 37℃, and 5% CO2. 1% O2 and 5% CO2 gas mixture was chosen for hypoxia experiment. MIF inhibitor ISO-1 (SML0472) was purchased from Sigma-Aldrich(Sigma-Aldrich, Shanghai, China), and JAK inhibitor XL019 was purchased from Beyotime Biotechnology (Beyotime, Shanghai, China). Drug concentrations were 25 µM(ISO-1) and 100 nM(XL019).
Cell samples were lysed with RIPA buffer protease inhibitor cocktail (Beyotime, Shanghai, China). Protein was separated by 10% SDS-PAGE, transferred onto 0.45 μm PVDF membranes, blocked and immunoprobed. Finally, chemiluminescence kit (Beyotime, Shanghai, China) was applied to detect the signals.
Quantitative real-time PCR
TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA, and cDNA synthesis was performed following the protocol of Transcriptase Kit (Thermo, Waltham, MA). And qRT-PCR was conducted on a CFX96 Connect (Bio-Rad, Hercules, CA) using a SYBR Green PCR kit (Vazyme Biotech, Nanjing, China). The mRNA expression levels were calculated by using the 2−ΔΔCt method. Results are shown as fold-change relative to the normal group or the DMSO group. Primer sequences were listed in Supplementary Data.
CCK-8 cell viability assay
Hep2 cells treated with hypoxia and ISO-1 and control cells were seeded into 96-well plates at 2000 cells per well. Cell viability was evaluated by replacing with fresh medium containing 10% Cell Counting Kit-8 (CCK8) reagent (Biosharp, Hefei, China) with incubation at 37℃for 2 h and plates were measured at 450 nm by a microplate reader (Thermo Fisher, Waltham, MA). Each group was measured every 24 h for 7 consecutive days.
Colony formation assay
Hep2 cells treated with hypoxia and ISO-1 and control cells were seeded at 500 cells into 6-well plates per well. The cell colonies were fixed and stained with 0.3% crystal violet in ethanol for 15 min until the colonies were obviously visible to the naked eye. Finally, pictures of the whole well were taken.
MIF and IL-6 concentrations were tested by ELISA kits based on the manufacturer’s protocol. Quantitative IL-6 ELISA assay kit was purchased from QuantiCyto (QuantiCyto, Shenzhen, China), Quantitative MIF ELISA assay kit was purchased from Elabscience (Thermo, Waltham, USA), and TG, NEFA levels were assayed by assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
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/). GSEA was performed using GSEA software 4.0.3(UC San Diego and Broad Institute, La Jolla, CA).
Hep2 cells maintained for 24 h in normoxia as the control group and cultured for 24 h in hypoxia as the treatment group were used for transcriptome sequencing at BGI (BGI, Shenzhen, China) (three independent replicates per group). The relative analysis was carried out on the website(https://biosys.bgi.com).
Xenograft tumour tissues were fixed, and dehydrated using gradient ethanol. Then, IHC staining was conducted following the manufacturer's instructions for the IHC kit(Maixin, Fuzhou, China). Anti-Ki-67 antibodies (dilution, 1:500; cat. no. ab15580; Abcam, Cambridge, UK) were incubated overnight at 4˚C. Finally, Image J v1.8.0 (National Institutes of Health, Bethesda, MA)was used for the analysis.
All mice were maintained at Zhongnan Hospital of Wuhan University and all animal experiments were conducted in accordance with Zhongnan Hospital animal ethics committee(NO. ZN2021049, June 8, 2021). Male BABL/c nude mice (4 weeks old) were purchased from Gempharmatech (Nanjing, China). Hep2 cells were collected by trypsinization and washed three times with pre-chilled PBS. Then cells were resuspended at 2 × 107 cells/mL using pre-chilled saline, and each mouse was subcutaneously injected 200 μL into the right anterior flank. After 8 days, the mice were randomly divided into the treatment group and the 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). Tumour volume was measured using a caliper every other day. Finally, mice were sacrificed, and tumours were removed, photographed, weighed and collected for ELISA and immunohistochemistry.
All cellular experiments were repeated for at least three independent times. All data were presented as the mean ± SD. SPSS 20.0 software (IBM Corporation, Armonk, USA) was used for statistical analysis. T-test and ANOVA were used for continuous variables, and Chi-square test was used to analyze categorical variables. A difference of P < 0.05 was considered statistically significant.