Human sample collection

Human sample collection was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Approval No: 2023-326). Written informed consent was obtained from all subjects included in this study. Human lung tissue samples were obtained from patients undergoing lung resection surgery for diagnostic and/or therapeutic purposes due to isolated lung nodules or lung tumors at the First Affiliated Hospital of Chongqing Medical University. All subjects were classified into obese (BMI ≥ 30.0 kg/m2) and normal weight (BMI 18.5–24.9 kg/m2) groups according to the World Health Organization (WHO) BMI standards (Additional file 1: Table S1). All lung tissue samples were meticulously collected at a safe distance of 5 cm from the nodule, strictly ensuring the exclusion of tumor-affected areas. Exclusion criteria included any underlying lung disease, previous chemotherapy or radiotherapy, significant health complications such as cancer or heart disease, pregnancy, or the presence of any other known acute or chronic inflammation.

Animal studies

Male C57BL/6 mice, aged 6–8 weeks old, provided by the Experimental Animal Center of Chongqing Medical University were used in this study. The animal experiment protocol was approved by the Animal Ethics Committee of Chongqing Medical University (IACUC-CQMU-2023-0062). During the study, mice (5 per cage) were housed in a dedicated pathogen-free facility with temperature control (24 ± 1 °C) and a 12-h light/dark cycle. All mice were randomly assigned to either the normal chow group (Lean group) or a high-fat diet with 60% kcal fat (TP2330055A, Trophic Animal Feed High-tech Co., Ltd., China) group [diet-induced obese (DIO) group]. After 24 weeks of feeding, the mouse ALI model was established according to the literature [22]. Briefly, after anesthesia with pentobarbital (50 mg/kg) (Sigma-Aldrich), 50 μL LPS (Sigma-Aldrich, 5 mg/kg) or saline was instilled intratracheally. To deplete pulmonary macrophages, 50ul of clodronate liposomes (YEASEN, Shanghai, China) or its control liposomes were instilled intratracheally in obese mice 24 h before LPS modeling [23]. To overexpress lung miR-192 levels in mice, miR-192 agomir (10 nmol in a 50 µl volume per mouse) or its control was administered 24 h before LPS injection. 24 h after LPS administration, bronchoalveolar lavage fluid (BALF) or lung tissue was carefully collected for subsequent analysis.

Cell culture and treatment

The mouse alveolar macrophage cell line (MH-S) was purchased from ATCC and maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, and cultured in a humidified incubator at 37 °C with 5% CO2. To mimic the in vivo characteristics of HFD exposure, cells were treated with 100 μM sodium palmitate (PA) or solvent control (Kunchuang, Xian, China) [24, 25]. After 24 h of PA treatment, MH-S cells were stimulated with 100 ng/ml LPS (Sigma, USA) for an additional 24 h.

Cell transfection and construction of stable cell lines

According to the manufacturer’s instructions, MH-S cells were transfected with a final concentration of 30 nM miR-192 agomir or antagomir and their respective controls using GP-transfect-Mate reagent (GenePharma). Cells were harvested 24 h after transfection for subsequent studies. Lentiviral constructs for BIG1 or FTO overexpression were purchased from Genechem (Shanghai, China). MH-S cells were seeded in a 6-well plate and infected with lentiviral vectors (MOI = 50) in the presence of HitransG A infection enhancer (GeneChem, Shanghai, China) for 12 h. After replacement with complete medium, the cells were further cultured for 60 h. At 72 h post-transfection, stable transductions were selected with puromycin (3 μg/ml) (Meilunbio, MA0318). Agomir and antagomir sequences are listed in Additional file 1: Table S2.

Collection of bronchoalveolar lavage fluid and extraction of alveolar macrophages

Mice were euthanized, and the trachea was exposed. An 18-gauge catheter was inserted into the trachea through a midline incision extending from the upper abdomen to the mid-neck region. The lungs were lavaged with 1.2 ml of cold PBS containing 0.5 mM EDTA. After 1 min, the lavage fluid was gently aspirated and collected in a 15 ml centrifuge tube. This procedure was repeated 10 times to obtain bronchoalveolar lavage fluid (BALF). The centrifuge tube was then spun at 400 × g at 4 °C for 10 min. The supernatant was used for total protein assay, and the cell pellet was resuspended in RPMI-1640 medium supplemented with 10% FBS and seeded in a 6-well plate. Alveolar macrophages were allowed to adhere for 2.5 h, after which non-adherent cells were washed away with PBS [26]. Lung tissue after alveolar lavage was not used for follow-up studies.

Hematoxylin and Eosin (H&E) staining

The lower lobe of the right lung was fixed in 4% paraformaldehyde (Servicebio, G1101) for 24 h, followed by paraffin embedding, and sectioning at 5 μm. Tissue sections were stained according to the standard H&E staining protocol. The pathological scoring was determined as previously described [27].

Biochemical analysis

Lung LDH activity was measured using a commercial kit (Solarbio, Beijing, China). Levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in lung tissue and cell culture medium were determined using commercial ELISA kits (MEIKE Jiangsu Sumeike Biological Technology Co., Ltd, Shanghai, China).

Lung wet/dry (W/D) weight ratio

The right apical lung tissue was weighed to record its wet weight (W). The tissue was then dehydrated at 80 °C for 48 h and weighed again to determine its dry weight (D).

Immunofluorescence

After dewaxing and rehydration, paraffin sections of lung tissue were subjected to antigen retrieval using citrate sodium antigen retrieval solution (Beyotime, P0081). After fixation in 4% paraformaldehyde, MH-S cells were permeabilized with 0.1% Triton X-100 (Beyotime, ST797). Tissues / cells were incubated with 10% fetal bovine serum (Beyotime, C0234) for 1 h at room temperature to block non-specific antigens. The slides were incubated with the primary antibody overnight at 4 °C, followed by incubation with fluorescence-conjugated secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature. Sections were mounted with anti-fade mounting medium containing DAPI (Beyotime, P0131). Tissue fluorescence was captured using an upright fluorescence microscope (Nikon, Tokyo, Japan), while cell fluorescence was captured under a confocal microscope (ZEISS, Oberkochen, Germany). Immunofluorescence intensity and the number of positive cells were determined using FlowJo software (version 10, FlowJo, LLC). Primary antibodies used included anti-F4/80 (Thermo Fisher Scientific, Waltham, MA, USA), anti-FTO (Abcam, Cambridge, UK) and anti- phospho-NF-κB p65 (Cell Signaling Technology, MA, USA).

RT-PCR

Total RNA was extracted from lung tissue and cells using the TRIzol reagent (Takara Biotechnology) according to the manufacturer’s instructions, and then reverse transcribed into cDNA using the Evo M-MLV RT Mix Kit (Accurate Biology, AG11728) or miRNA 1st strand cDNA synthesis kit (Accurate Biology, AG11716). Subsequently, qPCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, AG11702). The relative expression levels of miRNAs or mRNAs were normalized to U6 small nuclear RNA (snRNA) or β-actin using the 2-ΔΔCt comparison method. Primers used in this study are listed in Additional file 1: Table S3.

Dual-luciferase reporter assay

Potential target genes and their binding sites for miR-192 were predicted using the TargetScan, miRDB and miRWalk databases. The ARFGEF1-3 ‘UTR wild-type (WT) and mutant (MUT) reporter vectors were constructed by Jima Company, Shanghai, China. MH-S cells were cultured in 12-well plates and co-transfected with luciferase vectors and miR-192 agomir or its control (NC) using the GP-transfect-Mate reagent (GenePharma). Cells were lysed 48 h post-transfection and luciferase activity was measured using the dual-luciferase reporter assay system (Promega, San Luis Obispo, CA, USA). Results were expressed as the ratio of firefly luciferase to Renilla luciferase luminescence intensity.

MiR-192 activity assay

To assess changes in miR-192 activity after a high-fat diet (HFD), we used the R software (version 4.3.1, R Foundation for Statistical Computing, Vienna, Austria) and its ggplot2 and dplyr packages for our analyses. First, we performed microarray analysis of mouse lung lobes on normal diet or HFD diet for 12 weeks. RNA labelling and microarray hybridisation were performed by Kangchen Biotechnology Ltd. according to Agilent Monochrome Microarray Gene Expression Analysis protocol. Samples were then amplified and transcribed into fluorescent cRNA using a random priming method, followed by purification, hybridisation and scanning on an Agilent DNA microarray scanner. Microarray probe signals were acquired using Agilent Feature Extraction software and data were normalised using Agilent GeneSpring GX v12.1 software. Using these data, we calculated the log2 fold change (log2FC) for each gene between HFD and control. Based on the list of target genes of miR-192 predicted from the three databases above, we further categorised the genes as either target or non-target genes of miR-192. the Kolmogorov–Smirnov (KS) test was used to compare the log2FC distributions of the genes in these two groups. Distribution differences were visualised by cumulative distribution function plots. The microarray data have been uploaded to the NCBI Gene Expression Omnibus (GEO) under accession number GSE 229262.

m6A quantification

Total RNA was extracted from lung tissues using the TRIzol reagent. The relative m6A content was measured using the EpiQuik m6A RNA Methylation Quantification Kit (colorimetric) (Epigentek, P-9005) according to the manufacturer’s instructions. The percentage of m6A in total RNA can be calculated using the formula: m6A% = [(sample OD − NC OD)/S]/ [(PC OD − NC OD)/P] × 100%, where NC (negative control) represents RNA without m6A, PC (positive control) represents m6A oligonucleotides, normalized to 100% m6A, S is the amount of sample RNA input, and P is the amount of positive control input.

RNA-binding protein immunoprecipitation (RIP)

RIP assays were performed according to the manufacturer’s instructions using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Cat. No. 17-700). Briefly, single cell suspensions of lung tissue and MH-S cells were prepared in ice-cold PBS and lysed in RIP lysis buffer on ice for 5 min. After centrifugation, the supernatant was incubated overnight at 4 °C with magnetic beads conjugated to either anti-m6A antibody (Abcam, ab151230) or control immunoglobulin G (IgG, Millipore, USA). RNA from the immunoprecipitates was then extracted with TRIzol and subjected to qRT-PCR using primers specific for pri-miR-192.

Flow cytometry analysis

Cells were scraped from the culture dish and then washed three times with cold PBS. Staining was performed according to the manufacturer’s recommended protocol using PE anti-mouse CD86 (12-0862-82, Thermo Fisher, USA) and APC anti-mouse CD206 (17–2061-82, Thermo Fisher, USA) antibodies. Data acquisition and further analysis was conducted using the CytExpert software (version 10.0.7; Tree Star, Ashland, OR, USA).

Western blot

Lung tissues and cells were collected, and proteins were extracted using RIPA lysis buffer (Beyotime, China). Protein concentration was determined using the BCA method (Beyotime, China). Proteins were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked with 5% nonfat milk for 1 h at room temperature. Immunoblotting was carried out at 4 °C overnight using primary antibodies directed against BIG1 (1:1000, Abcam, ab183747), FTO (1:1000, Abcam, ab126605), METTL3 (1:1000, Abcam, ab195352), ALKBH5 (1:1000, Abcam, ab195377), YTHDC2 (1:1000, Abcam, ab220160), phospho-AKT (1:1000, Cell Signaling Technology, 4060T), AKT1 (1:1000, Cell Signaling Technology, 2938T), phospho-NF-κB p65 (1:1000, Cell Signaling Technology, 3033T), NF-κB (1:1000, Cell Signaling Technology, 8242), and β-actin (1:2000, Proteintech, 20536-1-AP). After washing three times, the membranes were incubated with secondary antibodies (SA00001-2, Proteintech) for 1 h at room temperature. The relative intensities of the protein bands were analyzed using the Bio-Rad Quantity One software. Results for phospho-AKT and phospho-NF-κB p65 were normalized to their respective total protein levels (AKT1 and NF-κB), while other protein levels were normalized to β-actin.

Statistical analysis

All data are expressed as the mean ± SD. Statistical analyses were conducted using the GraphPad Prism 9.5 software (USA). The Student’s t-test was used for two group comparisons, and one-way ANOVA was employed for multiple group comparisons. In all analyses, statistical significance was set at P < 0.05.