Analysis of IRF-1 expression in silico
GEPIA (25) (http://gepia.cancer-pku.cn/index.html) was utilized to explore the expression of IRF-1 in different tumors in the cancer genome atlas (TCGA) database. Scatter plots and histograms were drawn, and survival analyses were conducted. Expression data and corresponding clinical information for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) in TCGA database were acquired using UCSC Xena (http://xena.ucsc.edu/) (26). The original count values were converted to transcripts per million (TPM) using TPM standardization. The extracted IRF-1 values were used for further analysis. IRF-1 protein expression levels in LUAD and LUSC were verified using the Human Protein Atlas (HPA) (27), which is designed for the investigation of protein expression in various human tissues and organs.
Reagents and antibodies
Cisplatin, methyl thiazolyl tetrazolium (MTT), and LDH-dependent cytotoxic nonradioactive cytotoxicity assays were obtained from Promega (Madison, WI, USA). The LC3B antibody was purchased from Novus Biologicals (Littleton, CO, USA). N-acetylcysteine (NAC), β-actin, and 2,7-dichloro-fluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). An anti-IRF-1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Caspase-3 Fluorometric Assay kit was obtained from BioVision (Milpitas, CA, USA). Goat anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was obtained from Invitrogen (Carlsbad, CA, USA). Assay kits for ATP, superoxide dismutase (SOD), and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Bioengineering Institute. (Nanjing, China). The FITC-Annexin V/7-AAD apoptosis detection kit was obtained from BD Biosciences (USA).
Clinical sample collection
This study included lung cancer patients who underwent surgery at Hunan Cancer Hospital (Changsha, Hunan, China) between August 2014 and August 2015. All diagnoses were confirmed from histopathological reports. The Ethics Committee of Hunan Cancer Hospital approved this study, and informed consent was obtained from all study patients.
Cell culture
The human lung cancer cell lines A549, SK-MES-1, H1299, H460, H358, and H1975 were obtained from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in PRMI-1640 or DMEM supplemented with 10% FBS (Biowest, South America Origin), 100 U/ml penicillin sodium, and 100 mg/mL streptomycin sulfate at 37°C in a humidified atmosphere containing 5% CO2, as previously described (28).
Cell transfection
IRF-1 shRNA and overexpression lentiviral vectors (Hanyin, Shanghai, China) were prepared at a titer of 1 × 109 TU/mL. These vectors were transfected into cells using 5 µg/mL polyamine in RMI-1640 medium at a multiplicity of infection (MOI) of 20:1. After 4 h of transfection, the cells were cultured for 48 h in fresh culture medium. The efficiency of IRF-1 knockdown and overexpression was assessed by western blotting.
Western blotting
Following treatment with cisplatin (20 µM), cytoplasmic and nuclear proteins were extracted according to the manufacturer’s instructions. Then, the proteins were separated by electrophoresis and transferred to Trans-Blot nitrocellulose membranes (Bio-Rad Laboratories). After blocking with 5% nonfat milk for 1 h at room temperature and incubation with the primary antibodies and the HRP-conjugated secondary antibody, the color reaction was initiated using the Super Signal West Pico chemiluminescent kit (Pierce Chemical Co.), and the blot was exposed to film (Eastman Kodak). The data were analyzed with densitometry using Image J software (Bethesda, MD, USA). Histone was used as an internal loading control.
Mitochondrial membrane potential and reactive oxygen species detection
As described in a previous study (14), mitochondrial membrane potential was measured using JC-1 staining, and ROS was detected using DCFH-DA staining.
Detection of intracellular ATP, SOD, and MDA
Intracellular ATP, MDA, and SOD levels were detected using corresponding assay kits according to the manufacturer’s instructions.
Cell viability detection and lactate dehydrogenase release
The MTT assay was conducted to measure cell viability, and lactate dehydrogenase (LDH) was determined using the LDH-dependent Cytotoxic Non-Radioactive Cytotoxicity Assay kit, as described by Chen et al. (29).
Caspase3 activity assay and apoptotic cell death detection by flow cytometry
Caspase-3 activity was measured using the Caspase-3 Fluorometric Assay kit according to the manufacturer’s instructions. Apoptotic cells were detected using the FITC-Annexin V/7-AAD apoptosis detection kit according to the manufacturer’s instructions.
Statistical analysis
Data were processed using SPSS software (version 23.0). Quantitative data are shown as the mean ± standard deviation of three replicates as determined with Student’s t-test or one-way analysis of variance (ANOVA) between two or more groups. Repeated measures ANOVA followed by an LSD test was used to analyze differences within groups. Samples from TCGA were categorized into high- and low-expression groups based on the upper and lower quartiles of IFR-1 expression. Kaplan-Meier curves and log-rank tests were used to analyze the correlation between IRF-1 expression and patient survival. Clinical phenotypes between groups were compared using the Chi-square test. Univariate Cox regression analysis was used to screen survival-related factors. A P value less than 0.05 was considered statistically significant.