Cigarette smoke extract (CSE)
CSE preparation was done based on the method below. Briefly, three cigarettes without filters (0.9mg flue gas nicotine, 11mg coke, 14mg flue gas carbon monoxide) were collected using a negative pressure suction device and poured into a flask with 3 mL PBS. After shaking well, the suspension was filtered using 0.22 m filter membrane to filter bacteria. To ensure the same CSE concentration, the absorbance value was determined at the optimal absorption wavelength (270-280nm), and the absorbance value of the prepared CSE was the same in each preparation. The CSE concentrate was diluted using 10% FBS containing PRMI-1640 culture medium to obtain 2%, 5%, and 10% CSE. Notably, the CSE were prepared 30 min before use to ensure the effectiveness of CSE ingredients.
Cell culture and treatment
The rat pulmonary alveolar macrophage cell line NR8383 was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd, and cultured in F12K medium supplemented with 15% fetal bovine serum at 37°C in a humidified mixture of air (95%) and CO2 (5%). Cells were placed in 6-well plates with cell density of 1*106 cells per well and cultured in serum-free F12K medium overnight. Cells were treated with CSE for 24 h and 48 h.
Quantitative real-time PCR
Total RNA extraction was performed utilizing Trizol Reagent in light of the instructions of supplier. After determining the RNA concentration and quality, complementary DNA (cDNA) was synthesized using a First Strand cDNA Synthesis Kit (Sangon biotech, China). Real-time qPCR was performed to validate gene expression using 2× SYBR Green PCR Master Mix (Sangon biotech, China) on a Mx3000P QPCR System (Stratagene, USA) with the following thermal cycling conditions: 95°C for 3 min, followed by 40 cycles at 95°C for 12 s and 62°C for 40 s. The specific primer pairs used were shown in supplemental table 1.
Total protein extraction was conducted according to the M-PER Mammalian Protein Extraction Reagent instructions before separating proteins using a 10% SDS-polyacrylamide gel. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) using Semi-Dry Cell at 30 mA for 60 min. 1× Ponseau S solution was used to dye and mark the protein marker spots. Blocking Buffer (1×TBS, 0.1% Tween-20, and 5% w/v nonfat dry milk) was used to block nonspecific binding for 2 h at 37°C. After washing three times with 1× TBST, the membranes were incubated with anti-HAT1 (1:1000, SAB) and anti-TLR4 (1:1000, SAB) primary antibodies at 4°C overnight. The membrane was washed with 1× TBST five times before incubating with HRP-secondary antibodies (1:2000, Jackson) for 2 h at 37°C. After washing five times with 1× TBST, the membrane was exposed with SuperSignal West Pico Chemiluminent Substrates, and gray scanning was preformed using a Gel-Pro Analyzer.
Luciferase reporter assays
The NR8383 cells were placed into 96-well plates for 15-18 h culture. Next, 5pmol miR-486-5p mimic/negative control (NC) and 0.16ug HAT1 3’ UTR/HAT1 3’ UTR-muta (Sangon biotech, China) were transfected to cells using Lipofectamine 2000 (Invitrogen) based on the supplier’s instructions. The medium was replaced with fresh medium after transfection for 6h. Luciferase activity of reporter vectors was determined after 48 h using the Promega Dual-Luciferase system according to manufacturer's instructions.
After treating with CSE, the cell supernatants were collected and analyzed with ELISA kits (Sangon biotech, China) based on the supplier’s instructions.
MiRNA mimics/inhibitors and small interfering RNA
miR-486-5p mimics, inhibitors, and corresponding NC were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and transfected into cells using Lipofectamine 2000 (Invitrogen) to over-express and inhibit miR-486-5p in cells. The specific sequences of miR-486-5p-mimics, inhibitors, and HAT-1 specific siRNA sequences were shown in supplemental table 2.
Fluorescence in situ hybridization
Cells were seeded in a confocal petri dish and cultured for 24 h. After fixing with 4% paraformaldehyde, the cells were impregnated with 50%, 80%, and 98% (mass ratio) ethanol for 3 min before dehydrating. The fixed cells were prehybridized in hybridization buffer (formamide, 50 mM Tris-HCl, 5 mol NaCl, and 0.05% SDS) based on the method previously described. The 5' oligonucleotide-labeled miR-486-5p probe sequence was 5’-Cy3-CUCGGGGCAGCUCAGUACAGGA, and the 5' oligonucleotide-labeled HAT1 probe sequence was 5-FAM-UUCUCCACCGCACUCUUAUAUU. Next, DAPI was added in the dark at 4°C for 5 min before washing using 4°C PBS buffer. Cells were observed under a fluorescence microscope with a 360 nm excitation wavelength and a 460nm emission wavelength filter.
Clinical sample collection
The bronchoalveolar lavage fluid was collected from 14 normal individuals and 36 COPD patients. Next, alveolar macrophages (PAM) were isolated. In brief, the bronchoalveolar lavage fluid was centrifuged at 4°C at 1500 rpm for 10 min. After discarding the supernatant, the cells were washed twice with Hanks solution. The cells were cultured in serum-free RPMI1640 at 37°C in an incubator with 5% CO2 and 100% humidity for 2-3h. Non-adherent cells were removed, and the cells adhering to the culture dish wall were PAM. Peripheral blood was collected from 128 participants (No-smoker: n=33; smoker: n=42; COPD I-II: n=30; COPD III-IV: n=23). Monocytes were isolated from peripheral blood, and monocyte-derived macrophages were induced. All participants were recruited in January 2019, 1 solstice, June 30, 2019 in the respiratory department and physical examination center at the Affiliated Huai’an Hospital of Xuzhou Medical University. Written informed consent was obtained from participants before collecting samples. This study was conducted under the approval of the Ethics Committee of the Affiliated Huai’an Hospital of Xuzhou Medical University.
Microarray and data analysis
The microarray datasets GSE38974 and GSE53519 were downloaded from the GEO database. The miRNA expression data of lung tissue from 19 subjects with COPD and eight normal smokers without COPD in GSE38974 and the miRNAs expression data of small airway epithelium from nine non-smokers and ten smokers in GSE53519 were used in this study. Differential expression analysis was performed using the Bayesian method in the Limma package. The mRNA targets of miRNAs were predicted using miRTarBase  (http://mirtarbase.mbc.nctu.edu.tw/php/ index.php), TargetScan  (http://www.targetscan.org/vert_72/), miRWalk 2.0  (http://mirwalk.umm.uni‑heidelberg. de/), and miRPathDB  (https://mpd.bioinf.uni-sb.de/overview.html). The overlapped mRNAs were selected using Venny analysis.
Data analyses were conducted using SPSS19.0 and GraphPad Prism 7. Mean ± standard deviation (SD) was selected to present all the results. Student’s t-test was used to compare the data between the two groups. Variance analysis was performed to compare multiple groups. One-way ANOVA was used for homogeneity of variances, and the Lsd-q test was selected for pairwise comparison between groups. The Welch method was used when the variances were uneven, and Dunnett's T3 method was used for pair-wise comparison among multiple groups. Differences were statistically significant if P < 0.05.