Total proteins from cells and tissues were extracted using 500 µL of Radio-Immunoprecipitation Assay lysis buffer (Beyotime, China). Equal amounts of protein (20 µg) were loaded onto 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, Solarbio) and transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen). The membranes were blocked with 5% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies IgG (1:1000, ab181236, Abcam) were added and incubated for 2 h. Signals were visualized using the Enhanced Chemiluminescence (ECL) reagent kit (34080, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Density analysis was performed using ImageJ software. Commercial antibodies used in the experiment included: FHL1, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-caspase-3, mitogen-activated protein kinase kinase 1 (MEK1), phosphorylated-MEK1 (p-MEK1), p38 MAPK, phosphorylated-p38 MAPK (p-p38 MAPK), and GAPDH (GAPDH and FHL1 from Abcam; others from Cell Signaling Technology, dilution for FHL1 was 1:500, and for others 1:1000) [19].

Biochemical marker detection

Levels of factors associated with airway remodeling and inflammatory response, including alpha-smooth muscle actin (α-SMA), collagen I, IL-1β, IL-6 and TNF-α, were measured in patient blood, mouse lung tissue, and cell supernatant using enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific, Waltham, Ma, USA) according to the manufacturer’s instructions. Absorbance at 450 nm was assessed using a Centro LB 960 microplate reader (BERTHOLD, Stuttgart, Germany). Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were detected using standard kits (Nanjing Jiancheng Bioengineering Institute, China) [20].

Cell counting kit-8 (CCK-8)

Post-transfection, 16HBE cells (1 × 106 cells/well) were seeded in 6-well plates and treated with 10 µL of CCK-8 reagent (cat. no. 96992-100TESTS-F; Sigma-Aldrich; Merck KGaA) at different time points (0, 24, 48, and 72 h) and incubated at 37 °C for 2 h. The optical density (OD) at 450 nm was recorded using a Multiskan microplate reader (Thermo Fisher Scientific, Inc.) [21].

5-Ethynyl-2′-deoxyuridine (EdU) assay

Treated 16HBE cells (5 × 104 cells/well) were seeded on a 96-well plate and incubated in EdU medium for 2 h. Cells were washed with PBS and fixed with 4% paraformaldehyde. The EdU assay was conducted using an EdU detection kit (RiboBio, Guangzhou, China). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution, and Edu positive rates were observed under a fluorescence microscope (Nikon-OLYMPUS IX71, Nikon Instruments, Japan) [22].

Flow cytometry

Cell apoptosis was detected using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (Sangon Biotech Co., Ltd). Treated 16HBE cells were resuspended in binding buffer (300 µL). Subsequently, cells were dual-stained with 5 μL Annexin V-FITC and 5 μL PI for 20 min in dark, room temperature conditions. Finally, the percentage of apoptotic cells was evaluated using a FACS Calibur flow cytometer [20]. Quadrant definitions: Q1 (upper left quadrant) is not typically used for traditional Annexin V/PI apoptosis assays; Q2 (upper right quadrant) usually represents late apoptotic or dead cells; Q3 (lower left quadrant) usually indicates live cells; Q4 (lower right quadrant) typically represents early apoptotic cells.

Dual-luciferase reporter assay

16HBE cells were plated in 24-well plates. Constructs containing wild type or mutant binding sites of SNHG4 or FHL1 3′ untranslated region (UTR) (Promega) were inserted into pmirGLO vector (Promega, Madison, WI), named SNHG4/FHL1-WT 3′UTR and SNHG4/FHL1-MUT 3′UTR reporter genes, respectively. The respective luciferase reporter genes were co-transfected with miR-409-3p mimic or miR-NC into 16HBE cells. After 48 h of incubation, luciferase activity was measured using a Dual-Luciferase Reporter Assay Kit (Promega) [23].

RNA immunoprecipitation (RIP) assay

The assay was conducted using anti-Ago2 (ab252812) and anti-IgG (ab109489) antibodies. In brief, 16HBE cells were lysed, and then incubated with protein-G magnetic beads conjugated with anti-Ago2 or IgG antibodies at 40 °C for 6 h. Beads were collected, bound RNA was isolated, and the enrichment levels of SNHG4, miR-409-3p, and FHL1 were detected [13].

Animal experiments

Animal studies were authorized by the Animal Care and Use Committee of the Sixth Medical Center of PLA General Hospital (No. 201803CH6) and strictly followed the animal research charter. Twenty-four male C57BL/6 mice (6–8 weeks old) were acquired from Hunan SJA Laboratory Animal Co., Ltd and housed in standard laboratory conditions (12 h light/dark cycle, temperature 24 ± 2 °C, humidity 50%). After a week of acclimatization, mice were randomly divided into four groups: control, CS, sh-SNHG4, and sh-NC. Except for the Control group, remaining mice were exposed to CS from 10 cigarettes using a smoking machine (TE-10, Teague Enterprises) twice daily [24], 5 days a week, for 12 weeks. The total particulate matter concentration measured indoors was 160–180 mg/m3. Control group mice were maintained in ambient air. To knock down SNHG4, after the first CS exposure, mice were injected intravenously with a lentiviral vector targeting SNHG4 shRNA (GenePharma, Shanghai, China). After 12 weeks, mice were euthanized, and a portion of lung tissue was fixed in 4% paraformaldehyde for histopathological analysis, while the remainder was preserved at − 80 °C for subsequent gene extraction. Twelve weeks post-treatment, mice were euthanized, and the upper lobe of the left lung was fixed in 4% paraformaldehyde for histopathological analysis. The upper lobe of the right lung was preserved at − 80 °C for subsequent genetic material extraction.

Pulmonary function testing

Pulmonary function was assessed in mice using a whole-body plethysmograph (Buxco Electronics, Ltd., USA). Briefly, mice were placed randomly in a chamber connected to a sensitive pressure transducer, which measures slight pressure changes inside the chamber. The expiration time (Te), relaxation time (Tr), peak inspiratory flow (PIF), and peak expiratory flow (PEF) are all parameters reflecting restricted airflow. Enhanced pause (Penh, Penh = (Te/Tr − 1) × (PEF/PIF)) was recorded using FinePoint software (Buxco Electronics, Ltd., USA) when mice were quiet to evaluate pulmonary resistance. Values were averaged and reported as absolute Penh values.

Hematoxylin and eosin (H&E) staining

Lung tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, then cut into consecutive 4-μm-thick sections and stained with H&E using widely adopted standard procedures. Sections were observed under a microscope (Nikon, Japan) [25].

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

Apoptotic cells in lung tissues were detected using previous methodologies. Briefly, lung tissue sections were deparaffinized and rehydrated. They were then treated with proteinase K (20 ng/μL). Sections were incubated with bromodeoxyuridine (BrdU) solution at 37 °C for 1 h, then incubated with BrdU antibody, followed by three 5-min washes in PBS. All sections were counterstained with DAPI (20 mmol/L) at room temperature in the dark. Images were captured using a fluorescence microscope (Leica, Germany), and TUNEL-positive cells were manually counted in each microscopic image [26].

Immunohistochemistry (IHC) analysis

FHL1 expression was assessed through IHC [27]. Briefly, deparaffinized and rehydrated sections were blocked with 3% hydrogen peroxide. Tissue sections were incubated with FHL1 antibody (ab23937, Abcam) overnight at 4 °C. Subsequently, sections were incubated with biotinylated anti-rabbit secondary antibody (1:250) at room temperature for 1 h. Finally, sections were incubated with ABC reagent kit provided peroxidase substrate solution and developed with diaminobenzidine. Sections were counterstained with hematoxylin. Images were taken using an optical microscope (Olympus, Japan).

Statistical analysis

Data were analyzed using SPSS 21.0 (SPSS, Inc, Chicago, IL, USA) statistical software. The data were confirmed to be normally distributed by the Kolmogorov–Smirnov test. Results are expressed as mean ± standard deviation (SD). Comparisons between two groups were made using t-tests, and comparisons among multiple groups were made using one-way analysis of variance (ANOVA). Two-way analysis of ANOVA was employed to evaluate the variations in inflammatory markers, SNHG4, and FEV1% among patients with COPD, non-smokers, and smokers. P-value of < 0.05 was considered statistically significant. All experiments were performed with at least three biological replicates.