Cell culture and treatment
Human lung adenocarcinoma A549 cells are an alveolar epithelial cell line with biological characteristics of AEC II, and were therefore used as an in vitro model of IPF. The A549 cells (ATCCRCRM-CCL-185TM) were purchased from American Type Culture Collection, and cultured in RPMI-1640 culture medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a constant-temperature, constant-humidity incubator maintained at 37°C and 5% CO2. Six-well plates were inoculated with 1 × 106 cells each and cultured under normal or starvation conditions for 24 h. Subsequently, the cells were treated with 0.5 mg/ml TM, 1.0nM 4-PBA (both from Sigma-Aldrich, St. Louis, MO, USA) or 100ng/ml rhIL-32 (from R&D Systems Inc. - Minneapolis, USA. Catalog Number: 6769-IL).
RNA interference
Small interfering RNA (siRNA) targeting IL-32 was designed and provided by Shanghai Genechem Co., Ltd. (Shanghai, China). Lipofectamine 3000 transfection reagent (Invitrogen, CA, USA) was used to transfect A549 cells with plasmids containing IL-32 siRNA. Forty-eight hours after transfection, the cells were observed under an inverted fluorescence microscope to determine the transfection rate. Subsequently, RNA extraction was performed to confirm the interference efficiency via reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
RT-qPCR
Total RNA was extracted from the A549 cells using TRIzol reagent (Takara, Japan) in accordance with the manufacturer’s instructions. Subsequently, the total RNA was reverse-transcribed to cDNA with the reverse transcription kit (TaKaRa). Using the obtained cDNA as the template and β-actin as an internal control, RT-qPCR was performed with the real-time fluorescence-based 2×SYBR Green qPCR mix kits (Solarbio) according to the manufacturer’s instructions. IL-32 primer design and synthesis were performed by Shanghai Genechem Co., Ltd. (Shanghai, China), and the other primers design and synthesis were performed by Sangon Biotech (Shanghai, China) and all of the sequences as follows:
IL-32 forward 5’-CGACTTCAAAGAGGGCTACC-3’ reverse 5’-GATCCTCAACATCCGGGACA-3’
E-Cadherin forward 5’-GGGGTCTGTCATGGAAGGTGC-3’ reverse 5’-GTAAGCGATGGCGGCATTGTA-3’
N-Cadherin forward 5’-CATCATCATCCTGCTTATCCTGT-3’ reverse 5’-GCTCTTCTTCTCCTCCACCTTCTT-3’
Snail forward 5’-CTTCTCCTCTACTTCAGTCTCTTCC-3’ reverse 5’-TGAGGTATTCCTTGTTGCAGTATTT-3’
Vimentin forward 5’-AATCCAAGTTTGCTGACCTCTCTGA-3’ reverse 5’-GACTGCACCTGTCTCCGGTACTC -3’
Zeb-1 forward 5’-TAGATTTTGTGTGGGATTTCCTGTC-3’ reverse 5’-AGTGATTTTAATGATGGCTCGAATA-3’
TNF-α forward 5’-AGGACACCATGAGCACTGAAAGC-3’ reverse 5’-AAGGAGAAGAGGCTGAGGAACAAG-3’
TGF-β1 forward 5’-GAAACCCACAACGAAATCTATGAC-3’ reverse 5’-ACGTGCTGCTCCACTTTTAACT-3’
IL-1β forward 5’-GAAATGATGGCTTATTACAGTGGCA-3’ reverse 5’-GTAGTGGTGGTCGGAGATTCGTAG-3’
IL-6 forward 5’-CCTCCAGAACAGATTTGAGAGTAGT-3’ reverse 5’-GGGTCAGGGGTGGTTATTGC-3’
β-actin forward 5’-CCCATCTATGAGGGTTACGC-3’ reverse 5’- TTTAATGTCACGCACGATTTC-3’
Western blotting
Total protein was extracted from the A549 cells and quantified using the BCA protein assay kit (Solarbio, Beijing, China). A sample containing 20 μg of protein was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Solarbio), transferred to a polyvinylidene fluoride membrane, and incubated with rabbit anti-mouse N-cadherin, GRP78, and α-SMA primary antibodies (Proteintech, Wuhan, China), or β-actin antibody (Bioss, Beijing, China) as a loading control, at room temperature for 2 h. Subsequently, the membrane was washed, incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature, and exposed using the ECL kit. The experiment was repeated three times.
Statistical analysis
SPSS 20.0 software was used for statistical analysis of the experimental data. Quantitative data are expressed as the mean ± standard deviation, n=3, and comparisons between two groups were performed using the t-test. A significance level of α = 0.05 was adopted, with P < 0.05 indicating a statistically significant difference.