Ethics
The study was approved by the Ethics Committee of the Second Xiangya Hospital (Approval No. 2020530, 2020/9/14), and written informed consent was obtained from all participants prior to enrollment. All experimental methods abided by the Helsinki Declaration. All animal studies were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Second Xiangya Hospital Animal Ethical and Welfare Committee (Approval No. 2020489, 2020/9/9).
Patients and Specimens
All HNSCC samples were obtained from the Department of Oral and Maxillofacial Surgery at the Second Xiangya Hospital of Central South University. More specifically, 72 pairs of tumor and adjacent normal tissues were collected from primary HNSCC patients who underwent an initial surgical treatment between July 2017 and June 2019. Patients who received preoperative treatment and had a malignant history were excluded. Clinical parameters were obtained from medical records. Moreover, clinical stage and pathological differentiation were performed according to the World Health Organization Classification of Tumors and the tumor node metastasis staging system from the Union for International Cancer Control.
Cell cultures and reagents
HN6 and HN30 were kindly provided by the University of Maryland Dental School, USA. Cal27 and 293T cells were purchased from the American Type Culture Collection (MD, USA). Cal27, HN6, HN30, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, CA, USA), streptomycin (100 μg/mL), and penicillin (100 units/mL) at 37°C in a humidified 5% CO2 atmosphere. For hypoxia, tumor cells were cultured in a tri-gas incubator (ESCO, Singapore), consisting of 5% CO2, 94% N2, and 1% O2. Hydrogen peroxide (H2O2), CDDP, and N-acetyl-L-cysteine (NAC) were obtained from Sigma-Aldrich (USA).
RNA extraction and real-time PCR analysis
The RNA was extracted from the tumor samples and cells using TRIzol reagent (Takara, Japan), and cDNA was synthesized with a PrimeScript RT Reagent Kit (Takara, Japan). A real-time PCR (RT-PCR) was performed using the SYBR Premix Ex Taq Reagent Kit (Takara, Japan) with the StepOne RT-PCR System (Life Technologies, USA) according to the manufacturer's instructions. The mRNA levels were normalized to β-actin levels. The primer sequences used in the present study are listed in Supplementary Table 1.
MTT assays
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. Tumor cells were seeded in a 96-well plate at a density of 2000 cells per well with 100 μL of medium at 37°C. The cells were incubated with 100 μL of 0.5 mg/mL MTT (Sigma-Aldrich, USA) in Dulbecco’s modified Eagle’s medium for 4 h. Twelve hours later, the cells were cultured with a gradient concentration of CDDP for 48 h. The formazan was solubilized in 150 μL dimethyl sulfoxide. Absorbance was measured using a multi-well plate reader (Bio-Rad Laboratories, Hercules, CA, USA) at 490 nm. The half maximal inhibitory concentration (IC50) was used to evaluate the degree of tumor cell response to CDDP.
Apoptosis analysis
Apoptosis was assessed by flow cytometric analysis and terminal deoxynucleotidyl trans-ferase-mediated dUTP nick end labeling (TUNEL). TUNEL assays were performed using the Click-iTTM Plus TUNEL assay kit (Invitrogen, USA) according to the manufacturer's instructions. Flow cytometric analysis was performed using the Annexin V FITC Apop Dtec Kit (BD Pharmingen, USA) according to the manufacturer's instructions. The cells were trypsinized and centrifuged at 300 × g for 10 min at 4°C. Afterward, the cell pellets were resuspended in binding buffer and stained with Annexin V FITC and propidium iodide for 15 min on ice. Apoptotic cells were analyzed by flow cytometry (BD FACS Calibur, USA).
Western blot analysis
Tumor cells were harvested at the indicated times in SDS lysis buffer (Beyotime, China). Protein samples were separated using 10% polyacrylamide gels and transferred to 0.45-μm poly (vinylidene fluoride) membranes (Merck Millipore, USA). The membranes were soaked in 10% skim milk for 1 h at room temperature and incubated overnight at 4°C with the primary antibody. Subsequently, the membranes were probed with horseradish peroxidase-labeled secondary rabbit antibodies (Beyotime, China) for 1 h. The electrochemiluminescence (New Cell Molecular Biotech, China) developing solution was used to detect signals on a chemiluminometer (Amersham Imager 600). β-Actin was used as a control. The antibodies (1:1000) against Mff, HIF-1α, Mfn2 and β-actin were purchased from Cell Signaling Technology (USA).
Detection of relative ROS levels
Intracellular ROS levels were detected using 2,7-dichloro-dihydrofluorescein diacetate (Sigma-Aldrich, USA) and dihydroethidium (Sigma-Aldrich, USA) by flow cytometry (BD FACS Calibur, USA). This ROS analysis technique has been described in a previous study.[8]
Mitochondrial staining and analysis of mitochondrial fission
The cells were cultured on coverslips and stained with MitoSpyTM Red CMXRos (BioLegend, USA) for 30 min at 37°C. Afterward, the cells were fixed with 4% paraformaldehyde and stained with 4’-6-diamidino-2-phenylindole (DAPI; Sangon Biotech, China). Cells were viewed using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany). Mitochondrial morphology was then quantified and assessed as described previously.[8]
Analysis of immunofluorescence
The cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% goat serum, and incubated with the Mff antibody (CST, 1:500) and the HIF-1α antibody (CST, 1:500) at 4°C overnight then incubated with the Alexa Fluor 488-cojugated secondary antibody (Invitrogen, USA) or Alexa Fluor 549-cojugated secondary antibody (Invitrogen, USA) at room temperature in the dark. Cells were then stained with DAPI (Sangon Biotech, China) to detect the nuclei and viewed using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany).
Co-immunoprecipitation analysis
A co-immunoprecipitation (Co-IP) analysis was performed in our previous study.[38] In brief, the cells were lysed with IP buffer and incubated with the specific antibodies previously mentioned at 4°C overnight, followed by incubation with protein A/G Magbeads (Merck) for 6 h at 4°C. The proteins were separated from the beads for 10 min at 105°C. The supernatants were then used for subsequent immunoblotting analyses.
Cell transfection
SiRNA (Ribobio, China) was transfected into 293T cells using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer's instructions. Twenty-four hours later, the cells were harvested and the corresponding experiments were performed. The siRNAs used in the present study were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).
Luciferase analysis
In brief, the 293T cells (2×105), which were cultured on a 6-well plate, were co-transfected with Mff response luciferase plasmid and Renilla luciferase vector using the Lipofectamine 3000 reagent (Invitrogen, USA), according to the manufacturer’s instructions. After 48 h, the dual luciferase system (Promega, USA) was used to detect luciferase activity.
Lentiviral transduction and stable strain screening
The Mff interfering lentiviral vector[39] (LV-Mff) and interfering control lentiviral vector (LV-NC), which were constructed by HanYin Biotechnology Co, Ltd (China), conferred puromycin resistance. Lentiviral transduction was performed according to the manufacturer’s instructions. After 72 h of transfection, the culture medium with puromycin at a final concentration of 10 μg/mL was used to screen for stably stained cells.
Xenograft mouse experiments
The animal experiments, using BALB/C nude mice (Shanghai Laboratory Animal Center) aged 4 weeks old, were performed in accordance with the ethical standards and national guidelines. Cal27 cells (1×106) stably expressing LV-Mff or LV-NC were resuspended in 100 μl of phosphate buffered saline and subcutaneously injected into the dorsal flanks of mice (n = 6 in each group). Tumor sizes were measured using a caliper every three days. CDDP and saline were intraperitoneally injected at 5 mg/kg body weight in every 3 days from day 7 to 33. After day 33, the mice were sacrificed to collect tumor samples and measure tumor weights.
Statistical analysis
To compare the differences in expression between HNSCC and matched adjacent normal tissue, paired t-tests were performed. The Kruskal-Wallis tests and Mann-Whitney U tests were used to analyze the association between mRNA (SLC2A1, PDGFB, VEGFA and HIF-1α) levels and clinical parameters. The comparison between the groups was analyzed using one-way ANOVA or Student’s t-test. The correlation between two variables was analyzed using Pearson’s correlation. All statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL, USA). All values were two-sided, and P < 0.05 was considered statistically significant.