Sample collecting and preparation
5 ml peripheral blood samples were obtained from 65 patients (20 non-smokers, 20 smokers without COPD and 25 smokers with COPD) at Qilu Hospital of Shandong University. Thereafter, PBMCs were separated using isolation fluids (TBD sciences, Tianjin, China). All patients underwent lung function tests. The inclusion criteria of COPD were: forced expiratory volume in 1second / forced vital capacity (FEV1/ FVC) < 0.7 after bronchodilator treatment, 40 to 80 years old, and smoking for at least 20 pack-years. Non-smokers and smokers without COPD had matched age and gender with COPD patients. The procedure of study was approved by the Medical Ethics Committee of Qilu Hospital of Shandong University and consisted with the Declaration of Helsinki (as revised in 2013). All patients signed informed consent forms.
Preparation of the CSE
CSE was prepared according to a previous method[25]. One cigarette without filter was prepared, and a vacuum pump was used to draw the smoke into a glass container filled with 10 ml serum-free RMPI-1640 medium (Gibco, USA). Then, the CSE was filtered, and the pH was titrated to 7.4. The absorbance of the CSE at 320-nm wavelength was standardized to optical density (OD) of 0.74 ± 0.05. The obtained solution was referred to as 100% CSE.
Cell treatment
16HBECs and HEK293T cells were purchased from PRO-Cell Technology (Wuhan, China). The cells were cultured in 10% fetal bovine serum (Biological Industries, Israel) supplemented RMPI-1640 medium and incubated in 5% CO2 environment at 37°C. 16HBECs were exposed to varying concentrations of CSE for 24 h. After explore the best concentrations (10%) of CSE, the cells were stimulated with 10% CSE for different times. Before exposure to CSE, miR-4458 mimics (sense5'-AGAGGUAGGUGUGGAAGAA-3', antisense5'-CUUCCACACCUACCUCUUU-3'), NC-mimics (sense5'-UUCUCCGAACGUGUCACGUTT-3', antisense5'-ACGUGACACGUUCGGAGAATT-3'), miR-4458 inhibitor (5'-AGAGGUAGGUGUGGAAGAA-3'), NC-inhibitor (5'-CAUUACUUUUGUGUAGUACAA-3') (GenePharma, Shanghai, China), pcDNA3.1(+)-P53 overexpression plasmid (Biosune Biotechnology, Shanghai, China) were transfected into 16HBECs using Lipofectamine 2000(Invitrogen, USA).
Cell Counting Kit 8 (CCK8) assay
In this study, a total of 2 × 103 cells/ml was planted into 96-well plates. Each group had four duplicate wells. Then, 10 µl CCK8 reagents were added into each well. Subsequently, the OD of each well was detected at 450-nmwavelength 2 h later.
Apoptosis assessment
After application of different treatments, 16HBECs were harvested to measure the apoptosis rate using Annexin V-FITC/PI apoptosis detection kit (Elabscience, Wuhan, China). Then, a FACSCalibur flow cytometer (BD Biosciences) was employed to analyze the treated cells.
RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The total RNA of 16HBECs and PBMCs was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Then, cDNA synthesis was conducted with the PrimeScript RT reagent kit (TakaraBio, Japan). RT-qPCR was performed using the TB Green Premix Ex Taq II (TaKaRa) on LightCycler 480II (Roche, Basle, Switzerland). GAPDH and U6 were considered as endogenous controls. The gene specific primers sequences were as follows: GAPDH (F: 5'-GCACCGTCAAGGCTGAGAAC-3', R: 5'-TGGTGAAGACGCCAGTGGA-3'), LC3B (F: 5'-CAGCATCCAACCAAAATCCCG-3', R: 5'-TTGAGCTGTAAGCGCCTTCTAAT-3'), BECN (F: 5'-AACCGCAAGATAGTGGCAGA-3', R: 5'-CTCTCTGATACTGAGCTTCCTCC-3'), P53 (F: 5'-AGTCACAGCACATGACGGAG-3', R: 5'-GCCAGACCATCGCTATCTGA-3'), U6 (F: 5'-CAGCACATATACTAAAATTGGAACG-3', R: 5'-ACGAATTTGCGTGTCATCC-3') and miR-4458 (F: 5'-CAAAACCAACCAGAGGTAGGTG-3', R: 5'-TATGCTTGTTCTCGTCTCTGTGTC-3'). The 2 − ΔCT and 2 − ΔΔCT method were employed to quantified the mRNA levels of the genes.
Western blot assay
Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) and separated by SDS-PAGE gels (Beyotime), then transferred onto PVDF membranes. Thereafter, the membranes underwent blocking and then incubated in specific primary antibody. The primary antibodies used were as follows: LC3B (ab48394, Abcam, Cambridge, UK), BECN (3495, Cell Signaling Technologies, MA, USA), P53 (9282, Cell Signaling Technologies), Bax (89477, Cell Signaling Technologies), Bcl-2 (4223, Cell Signaling Technologies), p-AKT (4060, Cell Signaling Technologies), AKT (4691, Cell Signaling Technologies), p-mTOR (5536, Cell Signaling Technologies), mTOR (2983, Cell Signaling Technologies). Then, the membranes were incubated in secondary antibodies (Beyotime Biotechnology). Finally, the proteins were visualized by ECL solution using a Chemiluminescence Imager (Tanon, China).
Bioinformatics analysis and luciferase reporter assay
The potential target genes of miR-4458 were predicted by miRDB (http://www.mirdb.org/) and Targetscan (http://www.targetscan.org/vert_72/). A total of 1 × 104 cells/well HEK-293T cells were planted into 96-well plates. 24 h later, miR-4458 mimics or its negative control were co-transfected into the cells with pmirGLO-P53-wt or pmirGLO-P53-mut plasmid (Atagenix, Wuhan, China). Then, a Dual-Luciferase Reporter kit (Promega, WI, USA) was employed to detect the luciferase activities.
Statistical analysis
Statistical analysis was performed using GraphPad 7.0 (Graphpad Software, CA, USA). Data are presented as mean ± standard deviation (SD). In this study, Student’s t-test was used for pairwise comparison, and one-way analysis of variance and post-hoc Dunnett t-test were used for multiple comparisons. Spearman correlation analysis was employed for correlation analysis between miR-4458 and FEV1% pred. P < 0.05 was considered to have significant difference.