Induction of experimental COPD

Eight to ten-week-old male C57BL/6 mice were procured from Vital River Laboratory Animal Technology (Beijing, China) and housed under sterile conditions with a 12-hour light/dark cycle, allowing for a one-week acclimatization period. To induce experimental COPD, mice were subjected to chronic exposure to cigarette smoke from Chinese Lion cigarettes or normal air through nasal inhalation for 75 min, twice daily, five days a week for 16 weeks. For miRNA agomir intervention, mice were intranasally administered with MiR-1307-5p specific or scrambled antagomir once a week (2.5 mg/kg) under isoflurane anesthesia. For FBXL16 overexpression in lung tissues, mice were intranasally administered with control or FBXL16 expressing AAV6 twice, at the 4th and 8th weeks, under isoflurane anesthesia. All animal experiments were conducted following protocols approved by the Laboratory Animal Management and Ethics Committee of Hangzhou Medical College in Zhejiang Province (license number of animal use permit: SYXK 2023-0011, license number of approval of animal ethical and welfare: ZJCLA-IACUC-202387).

Pulmonary function assessment

Pulmonary function tests were conducted using a forced pulmonary maneuver system (Shanghai Yuyan Instrument Co., Ltd.), following the manufacturer’s instructions and established protocols [16]. Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by tracheal cannulation. Each maneuver was performed three times, and the average results were calculated.

Human subjects

Normal and COPD lung tissue specimens were obtained from patients who underwent lobectomy or pneumonectomy for lung cancer in the Quzhou Affiliated Hospital of Wenzhou Medical University. COPD was diagnosed based on a combination of medical history and physical examination including pulmonary function test using spirometry. Informed written consent was obtained from all participants. All studies have been approved by the Institutional Review Board at the Quzhou Affiliated Hospital of Wenzhou Medical University (permission No.2021-03-005). The basic medical information of the volunteers was listed in Table 1. Each patient signed an informed consent form for sample collection.

Table 1 Patient characteristics for mir-1307-5p analysisCell culture

Human lung fibroblast MRC-5 and HEK293T cells was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and was cultured in DMEM containing 10% FBS and antibiotics in 5% CO2 at 37℃ in a humidified atmosphere.

Isolation of primary lung fibroblasts and epithelial cells

Primary human lung fibroblasts and lung epithelial cells were isolated as described previously [17]. Lung tissues were cut into small pieces and incubated with digestion buffer (0.1% collagenase, 0.05% trypsin, and 100 mg/mL DNase in Hanks’ balanced salt solution) for 1 h at 37℃. The digested tissue suspensions were filtered through a 40 μm cell strainer, centrifuged at 500×g for 5 min and pellets were collected. After the lysis of red blood cells, the cells were resuspended and incubated for 1 h with biotin-conjugated anti-CD16/32, anti-CD45, and anti-CD31 antibodies. The cells were then rinsed, resuspended, and incubated for 30 min with streptavidin magnetic beads. Tubes containing the incubated cells were then applied to a magnet to deplete endothelial cells, lymphocytes, monocytes/macrophages, natural killer (NK) cells, neutrophils, and other haematopoietic cells. Supernatants were collected and plated into tissue culture plates. After 1 h incubation at 37 ℃, the suspended lung epithelial cells were harvested for experiments. The adherent lung fibroblasts were grown in MEM media supplemented with 10% FBS. Fibroblasts at passage 3–5 were used for experiments.

Plasmids, transfection and lentiviral infection

miR-1307-5p agomir (HY-R00240A) and antagomir (HY-RI00240A) were purchased from MedChemExpress LLC (Shanghai, China). Compared with common mimics/inhibitors, miRNA agomir/antagomir has higher stability and inhibitory effect in mammalian cells, and is more likely to pass through cell membranes to be enriched in target cells. Cells were transfected in 6-well plates using RNAiMAX transfection reagent (Life Technologies, Thermo Fisher Scientific) and OptiMEM media according to manufacturer’s instruction.

For FBXL16 expression in MRC-5 cells, the cDNA ORF clone was purchased from Sino Biological (Cat# HG24168-UT). Then the FBXL16 cDNA ORF fragment was subcloned into pCDH-CMV-puro lentiviral expression vector. The method for lentivirus production and transduction was introduced previously [18]. FBXL16 miRNA 3’-UTR clone (#MiUTR1R-01841) in pMirTarget 3’-UTR Assay Vector was ordered from Creative Biogene (NY, USA). Mutated 3’-UTR of FBXL16 was generated using the Hieff Mut™ Site-Directed Mutagenesis Kit (YEASEN, Shanghai, China). Lipofectamine 3000 reagents were performed for transfection according to the manufacturer’s instruction (Invitrogen). HA-Clover-HIF-1α wild-type (#163365) and HA-Clover-HIF-1ɑ S31D (#163370) were ordered from Addgene.

Real-time PCR

The extraction of total RNA was performed utilizing Trizol reagent in conjunction with the conventional liquid phase method incorporating chloroform. The RNA sample underwent reverse transcription to generate complementary DNA (cDNA) utilizing the RT-Mix kit (Takara, RR036A) with either random primer or gene specific RT primer. The real time PCR experiment was conducted using the SGExcel FastSYBR Master premix (Sangon Biotech, B532955-0005) following standard reaction conditions. The primers employed in this experiment are documented in Table 2. The relative expression level of the target gene or miRNA was determined utilising the 2−ΔΔCt methodology, with β-actin or U6 serving as the internal reference gene.

Table 2 Sequence of primers used for miRNA agomir, antagomir and qRT-PCRWestern blotting

Cells were washed with 1× PBS and lysed in 1× SDS buffer containing protease inhibitors. The lysate was collected, boiled at 100 °C for 10 min, and centrifuged to remove cell debris. Equal amounts of protein were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 0.1% casein at room temperature for 60 min and then incubated overnight at 4 °C with the appropriate primary antibodies. Subsequently, the membrane was incubated with an HRP-conjugated secondary antibody at room temperature for about 60 min. Finally, the protein bands were detected using an ECL protein blotting substrate. The antibodies used for western blotting were listed in Table 3. The intensity of bands was quantified using the Image J software.

Table 3 The reagents and antibodies used in this studyELISA

The levels of proteins in lung tissue homogenates were quantified using ELISA kits listed in Table 3, following the manufacturer’s instructions. Lung tissue homogenates were prepared by rapidly freezing and homogenizing on ice in RIPA buffer (Beyotime, #R0010) with phenylmethylsulfonyl fluoride (PMSF). The homogenates were sonicated three times for 5 s each, centrifuged at 500× g for 10 min at 4℃, and the central layer was collected. Total protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific Pierce). Lung tissue homogenates were then diluted with RIPA buffer to a final protein concentration of 500 mg/mL. For consistency, the obtained values were normalized to the total protein content.

Bronchoalveolar lavage

Airway inflammation was assessed by differential enumeration of inflammatory cells in bronchoalveolar lavage fluid (BALF) as previously described [16]. Briefly, mice were anesthetized, after which the trachea was carefully exposed. A 20 G intravenous catheter was delicately inserted. 1 mL of Sterile PBS was gently infused into the lungs, followed by a series of gentle aspirations to retrieve the fluid. Transfer the fluid into a 15 mL tube placed on ice. Repeat these steps three times. After centrifugation at 1,000 g for 3 min, the pellets were resuspended in 1 mL PBS. Total and differential cell counts were assessed using a hemocytometer and a Diff-Quick stain kit (Sysmex, Kobe, Japan).

Immunofluorescence staining

For ɑ-SMA visualization and quantification, MRC5 cells were treated as indicated and fixed in a 4% paraformaldehyde. Then, they were permeabilized using 0.5% PBS-Triton X-100 for 10 min and blocked with 5% goat serum for 1 h. Next, they were incubated with anti-ɑ-SMA (1:200) antibody at room temperature for 4 h. Remove primary antibody solution and wash the cells three times with 1× PBS. Then the cells were stained by Alexa Fluor 488 secondary antibody for 1 h at room temperature. DAPI staining was used to determine the number of nuclei. The fluorescence was visualized by a SUNNY RX50 fluorescence microscope. An average score of the immunofluorescence intensity was normalized by the number of nuclei.

Luciferase reporter assay

The recombinant vector containing either wild-type (WT) or mutated (MT) 3’-UTR of FBXL16 was transfected into HEK293T cells or primary fibroblasts using Lipofectamine 3000. miRNA agomirs were subsequently transfected into cells depends upon the performance of experiments. The luciferase activity was measured 24 h after transfection.

Statistical analysis

Data is presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla California USA). Differences between the groups were analyzed using Student’s t-test or one-way ANOVA followed by Dunnett’s multiple comparisons test. p < 0.05 was considered as significance.