Clinical samples

Lung tissues were obtained from 33 IPF patients (male, 25; female, 8; mean age, 40.14 ± 13.63 years) who underwent surgical resection at the third Affiliated Hospital of Guangzhou Medical University. Prior to the procedure, none of the patients had received any therapy. In addition, normal lung tissues (tumor-adjacent tissues) were obtained and used as controls. Informed consent was obtained from all participants.

Isolation and culture of primary pericytes

All experiments have been approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University, and pericytes were purified as previously described (Hung et al. 2017). Fresh lung tissues were collected from mice and cut into small pieces. After digestion and filtration through nylon meshes, single cells were resuspended in culture media. Pericytes with negative CD45, CD31, and CD326 expressions and positive PDGFRβ expression were selected using MACS (Miltenyi Biotec, USA). The isolated pericytes were seeded into gelatin-coated culture plates and maintained using DMEM/F-12 (11330032, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% FBS (10100, Thermo Fisher).

Cell transfection

For the induction of fibrogenesis, mouse pulmonary microvascular pericytes were treated with TGF-β1 (HY-P7117, MCE, NJ, USA) for 30 min at 37 °C. The shRNA targeting GAS5 (shGAS5), shPDGFRα, shPDGFRβ, shKDM5B, and negative control (shNC) were synthesized by GenePharma (Shanghai, China). The specific sequences for shRNAs are presented in Table 1. For the overexpression of GAS5 and PDGFRα/β, GAS5 and PDGFRα/β sequences amplified by RT-qPCR were ligated with the pcDNA3.0 plasmids (#13031, Addgene, Watertown, MA, USA) to establish the pcDNA3.0-GAS5 and pcDNA3.0-PDGFRα/β recombinant plasmids, respectively. The primers used for establishing the overexpression constructs are presented in Table 2. The aforementioned plasmids and sequences were transfected into cells using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific) following the manufacturer’s protocols.

Table 1 Sequences for shRNAsTable 2 The primers used for establishing the overexpression constructsMouse model of pulmonary fibrosis (PF)

As previously described (Pan et al. 2020), 7–8-week-old male C57BL/6 mice weighing 20–22 g were obtained from Hunan Slac Jingda Laboratory Animal Co., Ltd. (Changsha, China). The mice were randomly assigned into six groups (n = 7 per group): control, bleomycin, bleomycin + NC, bleomycin + GAS5, bleomycin + shNC, and bleomycin + shGAS5. Intraperitoneal injection with 30-mg/kg sodium pentobarbital was adopted to anesthetize the mice. To inducePF, 2.5-mg/kg bleomycin (HY-108345, MCE) was intratracheally administered to the mice. The control group was administered with the same volume of PBS (10010001, Thermo Fisher Scientific). Five days before bleomycin administration, the mice were intratracheally injected with lentiviruses containing overexpression of GAS5, shGAS5, or NC (1 × 107 TU, GenePharma). Then, 2 weeks after bleomycin administration, the mice were sacrificed, and lung tissues were collected for further analysis. All animal protocols were approved by the Animal Care and Use Committee of Guangzhou Medical University.

Hematoxylin and eosin staining

Before being paraffin-embedded, the lung samples of mice were fixed in 4% paraformaldehyde (P1110, Solarbio, Beijing, China). Subsequently, the lungs were cut into 4-µm slices and stained with the H&E staining kit (G1120, Solarbio) and examined under a microscope (Olympus, Japan) for pathological and fibrosis assessment.

Masson staining

Collagen deposition was evaluated via Masson staining on lung slices. In brief, the 4-μm slices of lungs were stained using the Masson trichrome staining kit (G1340, Solarbio). The images were photographed under a microscope.

Immunohistochemical staining

The 4-µm paraffin-embedded lung slices were dewaxed and rehydrated in ethanol gradient. After blocking endogenous peroxidases with 0.3% hydrogen peroxide (7722-84-1, Sigma–Aldrich, Saint Louis, MO, USA), the slices were probed overnight at 4 °C with primary antibodies against α-SMA (ab124964, 1:50, Abcam, Cambridge, MA, UK), collagen I (ab88147, 1:50, Abcam), and desmin (ab227651, 1:2000, Abcam). Then, the slices were rinsed with PBS and reacted with the secondary antibody. Subsequently, 3,3′-diaminobenzidine was used to visualize the slices. A light microscope was used to obtain the photographs.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Using the TRIzol reagent (T9424, Sigma–Aldrich), total RNA was isolated from the pericytes and tissues, followed by reverse transcription into cDNA using the PrimeScript RT reagent kit (RR047AA, Takara, Tokyo, Japan). Then, qPCR was performed using the SYBR Premix Ex Taq II kit (RR820A, Takara). GAPDH was used as the housekeeping gene to calculate the relative gene expression using the 2−ΔΔCT method as previously described (Zuo et al. 2022). The primers used in RT-qPCR are listed in Table 3.

Table 3 Primers used for RT-qPCR analysisWestern blotting

The cells or tissues were lysed in RIPA solution (P0013B, Beyotime, Haimen, China) and quantified using BCA protein assay (P0012, Beyotime). The protein (50 µg) was separated on SDS-PAGE and then transferred to PVDF membranes (3010040001, Roche, Basel, Switzerland). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: PDGFR-α (ab203491, 1:1000, Abcam), PDGFR-β (ab69506, 1:1000, Abcam), α-SMA (ab124964, 1:2000, Abcam), NG-2 (ab275024, 1:1000, Abcam), collagen I (ab260043, 1:1000, Abcam), desmin (ab227651, 1:5000, Abcam), KDM5B (ab181089, 1:1000, Abcam), H3K4me2 (#9725, 1:1000, CST, USA), H3K4me3 (#9751, 1:1000, CST, Danvers, MA, USA), and β-actin (ab8226, 1:1000, Abcam). After incubation with a secondary antibody, the membranes were treated with the ECL-chemiluminescent kit (34580, Thermo Fisher Scientific) to visualize the protein bands.

Immunofluorescence staining

Primary pericytes were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (T8787, Sigma–Aldrich), and blocked with 1% BSA (A1595, Sigma–Aldrich). The antibodies, including α-SMA (ab124964, 1:200, Abcam) and desmin (ab227651, 1:500, Abcam), were applied overnight at 4 °C. The pericytes were probed with goat anti-rabbit IgG (H + L) secondary antibody (ab150077, 1:200, Abcam) for 1 h. A fluorescence microscope (Zeiss, Germany) was used to observe and photograph the outcomes of the immunofluorescence assays.

Fluorescence in situ hybridization (FISH)

FISH was employed to determine GAS5 localization in pericytes. Briefly, the pericytes were seeded in a 24-well plate. After fixation and permeabilization, the cells were probed with GAS5 probe. Subsequently, the cells were rinsed in a hybridization solution and stained with DAPI (MBD0015, Sigma–Aldrich) in the dark. Images were obtained using a laser scanning confocal microscope (Leica, Germany).

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was conducted using the Magna ChIP and EZ-Magna ChIP kit (17-10461, Millipore, Billerica, MA, USA). Sequences for the PDGFRα/β promoter containing the wild-type (WT) or mutant (MUT) binding sites in KDM5B are presented in Table 4. Generally, pericytes were extracted and cross-linked with 1% formaldehyde before being treated with 125-mM glycine (67419, Sigma–Aldrich). The cell suspension was then sonicated and precipitated with anti-KDM5B (#15327, CST), anti-H3K4me2 (#9725, CST), anti-H3K4me3 (#9751, CST), or anti-control rabbit IgG (ab37415, Abcam). PDGFRα/β enrichment in the immunoprecipitated complex was assessed via quantitative PCR.

Table 4 Sequences for PDGFRα/β promoter containing the wild type (MT) or mutant (MUT) binding sites in KDM5BDual-luciferase reporter assay

A luciferase reporter assay was conducted to confirm the target relationship between KDM5B and PDGFRα/β. The sequences of the PDGFRα/β promoter containing the WT or MUT binding sites in KDM5B were synthesized and introduced into the pmirGLO vector (E1330, Promega, Madison, WI, USA). In 96-well plates, 293 T cells were co-transfected with the aforementioned reporter vectors and shKDM5B or shNC using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific). After 48 h, the luciferase activity was measured using a dual-luciferase reporter system (E1960, Promega).

RNA pull-down assay

The biotin-labeled GAS5 probe was provided by RiBobio (Guangzhou, China) and transfected into pericytes. Then, cell lysate was prepared using lysis buffer and incubated with streptavidin–agarose beads (16-126, Millipore) for 3 h at 4 °C. The RNA-binding protein complexes in agarose beads were washed and boiled at 95 °C–100 °C. Finally, the eluted protein was assessed via Western blotting.

RNA immunoprecipitation assay

Pericytes were lysed using the lysis buffer from the Magna RIP kit (17-700, Millipore). The cell lysate was treated overnight at 4 °C with RIP buffer containing magnetic beads pre-coated with anti-IgG (#3900, CST) or anti-KDM5B (#15327, CST). To remove the protein, proteinase K (70663, Sigma–Aldrich) was added to the eluted samples at 55 °C for 30 min. Subsequently, the immunoprecipitated RNA was isolated, and GAS5 enrichment was determined via RT-qPCR.

Statistical analysis

The data are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism. Student’s t-test was employed to determine the difference between two groups. One-way analysis of variance was employed to compare multiple groups, followed by Tukey’s post hoc test. P < 0.05 was considered to indicate statistical significance.