Introduction
Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects preterm infants requiring respiratory support and oxygen therapy after birth [
1]. Oxygen toxicity, inflammatory injury, and genetic predisposition are risk factors associated with this disease [
2‐
4]. Studies have highlighted the involvement of an imbalance between pro-inflammatory and anti-inflammatory mechanisms in the pathogenesis of BPD [
5,
6]. Hyperoxia leads to the excessive production of reactive oxygen species, damages lung epithelial cells, increases lung permeability, and promotes the levels of inflammatory cytokines [
7]. Notably, immature alveolar epithelial cells, particularly type II alveolar epithelial cells, are primary targets of hyperoxia-induced lung injury in premature infants [
8]. Extensive research has demonstrated the role of alveolar epithelial cell apoptosis in hyperoxia-stimulated lungs of neonatal rats [
8‐
10]. Despite considerable progress in the clinical management of BPD, the incidence of this disease remains relatively stable, highlighting the need for novel therapeutic targets [
11].
Hyperoxia-induced methylation has been observed in a rodent model of BPD [
12], and DNA methylation has been detected in autopsy lung samples from preterm infants with BPD [
13]. Among the various internal mRNA modifications, N6-methyladenosine (m6A) is the most common and abundant, recognized by m6A-selective reader proteins of the YTH family [
14]. YTH domain-containing 1 (YTHDC1), a nuclear m6A reader, facilitates the transport of m6A methylated mRNA from the nucleus to the cytoplasm [
15]. The involvement of YTHDC1-mediated m6A RNA methylation has been investigated in numerous diseases, including various human tumors [
16] and osteosarcoma [
17]. Both YTHDF1 mRNA expression and m6A-RNA methylation levels increased in the mouse model of acute respiratory distress syndrome [
18]. However, there have been no previous studies on m6A RNA methylation in BPD nor its reader YTHDC1.
Interleukin-33 (IL-33), a member of the IL-1 cytokine family, is constitutively expressed in healthy mucosal tissues and other organs, with increased expression under inflammatory conditions [
19]. IL-33 plays a role in chronic lung inflammation [
20‐
22], inducing the formation of neutrophil extracellular traps and degrading fibronectin in a mouse model of BPD [
23]. Knockdown of IL-33 has been shown to exert protective effects against hyperoxia-induced lung injury, which is attributed to reduced polarization and proliferation of alveolar macrophages [
24]. Elevated cord blood IL-33 levels have been observed in severe BPD (3.91 ± 1.22 pg/mL) than moderate BPD (2.82 ± 0.74) group in premature infants [
25]. In a neonatal IL-33 transgenic mouse model, enlarged alveolar spaces resembling BPD have been observed [
19]. Moreover, IL-33 is associated with alveolar epithelial cell injury [
26‐
28], underscoring its crucial role in BPD.
Plasmacytoma variant translocation gene 1 (PVT1) is a long non-coding RNA (lncRNA) whose expression is upregulated by inflammatory stimuli [
29‐
31]. PVT1 has been implicated in promoting airway inflammation during asthma [
32] and recently identified as a regulator of the asthmatic phenotype in human airway smooth muscle [
33]. High expression levels of PVT1 have been detected in peripheral blood samples from patients with chronic obstructive pulmonary disease, indicating its potential as a biomarker for this condition [
34]. However, the exact role of PVT1 in BPD remains unclear.
In our study, we performed Arraystar Mouse m6A-mRNA&lncRNA Epitranscriptomic microarray analysis on normal lungs (n = 3, the control group) and hyperoxia-stimulated lungs (n = 3, the BPD group). We identified significantly higher RNA expression levels of IL-33 and increased m6A RNA methylation levels of IL-33 and PVT1 (with the highest significance) in the BPD group compared to the control group. Therefore, we proposed a hypothesis that PVT1 and IL-33 may be involved in the occurrence and development of BPD.Our investigation aimed to elucidate the role of PVT1 in the apoptosis of hyperoxia-stimulated alveolar epithelial cells and a mouse model of BPD. We also examined the molecular interaction between PVT1 and IL-33, along with their relationship with m6A RNA methylation.
Materials and Methods
Cell Culture
Mouse alveolar epithelial cell line MLE12 (#IM-M015) and human alveolar epithelial cell line A549 (#IM-H113) were purchased from IMMOCELL (Xiamen, China). MLE12 cells were cultured in DMEM/F12 (94%) + fetal bovine serum (2%) + GlutaMAX-1 glutamine (1%) + HEPES 1 M Buffer solution (1%) + penicillin–streptomycin (1%) + ITS (Insulin + transferrin + Selenium) (1%) + hydrocortisone (10 nM) + 10 nM (estradiol). A549 cells were cultured in 90% DMEM + 10% fetal bovine serum + penicillin–streptomycin. To construct the
in vitro model of BPD, MLE12, and A549 cells were exposed to 100% oxygen for 16 h [
35].
Vector Construction and Transfection
Short hairpin-RNAS (sh-RNAs) targeting PVT1 (sh-PVT1), YTHDC1 (sh-YTHDC1), IL-33 (sh-IL-33), and scrambled shRNAs as negative control (sh-NC) were synthesized by GenePharma (Shanghai, China). The coding region sequences of YTHDC1 were inserted into pcDNA3.1 vector (GenePharma) to construct the overexpression vector, and the empty vector was used as negative control (NC). All vectors were transfected into MLE12 and A549 cells using lipofectamine 2000 (ThermoFisher Scientific) at room temperature based on the manufacturer’s instructions.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from MLE12 and A549 cells, as well as hyperoxia-stimulated lungs, using TRIzol Reagent (#15596026), followed by reverse transcription using a RevertAid RT kit (#K1691). Subsequently, q-PCR was performed using SYBR™ Green PCR Master Mix (#4309155). All the reagents were purchased from ThermoFisher Scientific, and all procedures were performed according to the instructions of supplier. The results were calculated by the 2−ΔΔCt method, and each sample was run in triplicate.
Western Blotting
Proteins were extracted from MLE12 and A549 cells, as well as hyperoxia-stimulated lungs. The protein samples were mixed with the loading buffer, separated by 10% SDS-PAGE at 100 V for 3 h, and transferred to PVDF membranes at 100 V for 50 min. After blocking in 5% skimmed milk for 3 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies, including rabbit monoclonal anti-Bax (ab32503; 1:2000), rabbit monoclonal anti-Bcl-2 (ab182858; 1:2000), or rabbit monoclonal anti-GAPDH (ab181602; 1:10000). The membranes were then washed with TBST three times and incubated with goat anti-rabbit secondary antibody anti-IgG (ab6721; 1:10000) for 2 h. Protein bands were visualized using enhanced chemiluminescence reagents (ThermoFisher Scientific). All antibodies were purchased from Abcam (Shanghai, China). The Image Lab 5.0 software (Bio-Rad) was used to analyze the densitometry values of Bax and Bcl-2, which were standardized relative to GAPDH.
Cell Counting Kit-8
Cells were seeded in 96-well plates at a density of 5 × 103 cells/well and cultured for 24 h. CCK8 solution (Beyotime) was added to each well, and the plates were further incubated at 37 °C for 3 h. Cell viability, indicated by the optical density of each well, was measured using a microplate reader (Bio-Rad) at 450 nm.
Flow Cytometry Analysis
An Annexin V-FITC/PI Apoptosis Detection Kit (#A211-01, Nanjing, China) was used according to the manufacturer’s instructions. Transfected MLE-12 and A549 cells were seeded into 6-well plates at a concentration of 1 × 105 cells/well, washed with cold PBS, and then resuspended in annexin-binding buffer. Next, cells were incubated with 5 µL of Annexin V-FITC and 10 µL of PI for 10 min at room temperature in the dark. Apoptotic cells were detected with the CytoFLEX flow cytometer and analyzed using the CytExpert software (Beckman). Cells in Q3 were defined as apoptotic cells.
RNA-Binding Protein Immunoprecipitation (RIP)
A RNA Immunoprecipitation Kit (#P0102, Geneseed, Guangzhou, China) was used following the manufacturer’s instructions. A/G magnetic beads were incubated with anti-IgG (#ab133470, Abcam), anti-YTHDC1 (#ab264375, Abcam), and anti-m6A (#ab208577, Abcam) and then added to the cell lysates. After elution, RNA was isolated from the complexes and analyzed by q-PCR, as mentioned above, to detect the enrichment of PVT1 or IL-33.
RNA Pull-Down Assay
Biotinylated RNA was treated with structure buffer to induce secondary structure formation, denatured by heating and ice-bath for ion, and then incubated with BeyoMag™ Streptavidin Magnetic Beads (#P2151, Beyotime) for 2 h at 4 °C. Cell lysates of MLE12 and A549 cells were divided into four groups (Input, Bio-NC, Bio-IL-33-wt, and Bio-IL-33-m6A mut) and incubated with the bead-probe complex at overnight 4 °C. After extracting total proteins from pull-down products, Western blot was conducted to detect the enrichment of YTHDC1 as mentioned above.
Luciferase Reporter Assay
A Dual Luciferase Reporter Gene Assay Kit (RG027, Beyotime, Shanghai, China) was used according to the manufacturer’s instructions. The full sequence of IL-33 was subcloned into the pGL3 vector to construct the pGL3-IL-33 (m6A-Wt) vector. IL-33 with mutated m6A binding site was inserted into the pGL3 vector to create the pGL3-IL-33 (m6A-Mut) vector. The empty pGL3vector served as NC. After co-transfection of the reporter vectors mentioned above with pcDNA3.1-YTHDC1 or pcDNA3.1 into MLE12 and A549 cells, the luciferase reporter activity was determined using the Dual-Luciferase® Reporter Assay System (E1910, Promega), with Renilla luciferase used as an internal reference.
Establishment of Hyperoxia-Induced BPD Mouse Model
All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health, under the approval of the Ethics Committee of The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University. Neonatal mice were placed in a sealed polypropylene cage and exposed to continuous oxygen at a flow rate of 5 L/min with an inspired fraction of O
2 > 95% [
36,
37], as measured by an oxygen analyzer, from postnatal day 1 to 7. The temperature was maintained between 22–27 °C, with humidity levels between 50 and 70%. The cage contained soda lime to absorb CO
2 and color-changing silica gel to absorb water. To maintain lung inflation, the trachea of the mice was intubated, exsanguination was performed, the chest was opened, and the lungs were subsequently extracted.
Intervention of the Hyperoxia-Treated Mice
Before hyperoxia treatment, mice in the BPD/PVT1 KO group were intratracheally instilled with 5 μL adenovirus vector expressing sh-PVT1 at a titer of 1 × 109 pfu/100 μL. Mice in the BPD/PVT1 KO + IL-33 group were intratracheally instilled with 5 μL adenovirus vector expressing sh-PVT1 and 5 μL adenovirus vector expressing IL-33. The adenovirus vectors were synthesized by GenePharma Co. Ltd. (Shanghai, China).
Hematoxylin-Eosin (HE) Staining
Hyperoxia-stimulated lung tissues were fixed with 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, and sectioned into 5 μm slices. Subsequently, the sections were deparaffinized, hydrated, and stained with HE. A light microscope (Nikon, Tokyo, Japan) was used to observe the pathological changes of lung tissues under five randomly selected fields. The radial alveolar count was calculated along a vertical line from the center of the respiratory bronchiole to the distal pleura [
36]. The alveolar chord length was measured using Image-Pro Plus 6.0 software.
Statistical Analysis
All data were presented as mean ± standard deviation and analyzed using GraphPad Prism9. Difference comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey's post hoc test for pairwise comparisons. The t-test was used for comparing differences between two groups. P < 0.05 indicated statistical significance. For in vitro assays, three biological repeats and three technical repeats were performed. For in vivo assays, either three or six biological repeats and three technical repeats were conducted.
Discussion
Previous studies have highlighted the crucial roles of lncRNAs in the progression and treatment of BPD [
38‐
41]. In this study, we have made an innovative discovery by demonstrating the significant upregulation of PVT1 in hyperoxia-stimulated lung alveolar epithelial cells through PCR analysis. Additionally, using epitranscriptomic microarray analysis, we have identified high levels of m6A RNA methylation in PVT1 in BPD mice. Our findings indicate that PVT1 knockdown reduces apoptosis and enhances viability in hyperoxia-stimulated lung alveolar epithelial cells. Furthermore, silencing PVT1 reduces alveolar size, increases radial alveolar count, and decreases mean chord length in hyperoxia-stimulated mice. These findings suggest that targeting PVT1 and suppressing its expression could have a beneficial effect in the treatment of BPD.
IL-33 exhibits dual functions depending on its cellular location. While the full-length IL-33 is found within the nucleus of cells, the mature IL-33 acts as an extracellular cytokine and is released when cells detect inflammatory signals or undergo necrosis [
42‐
44]. The conversion from full-length IL-33 to mature IL-33 is facilitated by neutrophil-derived proteases [
42]. Previous research by Larouche
et al. revealed lower DNA methylation levels of IL33 in bronchial epithelial cells of asthmatic individuals compared to controls [
45]. In our study, we observed higher levels of m6A RNA methylation and mRNA expression of IL33 in BPD mice using epitranscriptomic microarray analysis. We also observed upregulation of IL33 mRNA expression in hyperoxia-stimulated lung alveolar epithelial cells. Considering that hyperoxia can induce methylation in BPD [
12], it can be inferred that the upregulation of IL-33 mRNA in hyperoxia-induced BPD is dependent on its m6A RNA methylation. Furthermore, our study demonstrated that silencing IL33 has a negative effect on the apoptosis of hyperoxia-stimulated lung alveolar epithelial cells, consistent with previous studies highlighting the pro-apoptotic role of IL-33 [
46,
47]. The results of our rescue assays further indicated that IL-33 partially reverses the effects of PVT1 silencing in both
in vivo and
in vitro models of BPD.
Moreover, our study suggests that the interaction between PVT1 and IL-33 is mediated by YTHDC1-mediated m6A methylation. We confirmed that YTHDC1 is a RBP that binds to both PVT1 and IL-33 in lung alveolar epithelial cells. Both PVT1 and YTHDC1 positively regulate IL-33 mRNA expression. Upon silencing YTHDC1, the binding of m6A to IL-33 is inhibited. PVT1 positively regulates IL-33 expression by recruiting YTHDC1 to mediate m6A modification on IL-33, without affecting YTHDC1 expression. As an m6A reader, YTHDC1 can stabilize or destabilize mRNAs by functioning as a RBP [
48,
49], and it has been implicated as a risk factor in lung cancer [
50] and acute respiratory distress syndrome [
18]. However, the direct role of YTHDC1 in BPD remains unclear, which is a limitation of our study.
Moving forward, our research will focus on elucidating the source of the m6A modification in PVT1 in BPD. Building upon the studies conducted by Shen
et al. [
51] and Chen
et al. [
52], which indicates that ALKBH5 promotes the stability of PVT1 through m6A modification, our future investigations will validate the interaction between ALKBH5 and PVT1 and explore the potential roles of ALKBH5 in BPD. Moreover, hyperoxia-induced oxidative stress in lung alveolar epithelial cells leads to apoptosis [
7]. PVT1 has been identified as a regulator of oxidative stress [
53], and there is a bi-directional cause-and-effect relationship between oxidative stress and IL-33 [
54,
55]. Therefore, our upcoming studies will also investigate the relationship between PVT1/IL-33 and oxidative stress in BPD.
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