Over the last several decades, numerous treatments have been incrementally improved and developed, including increased prenatal corticosteroids, surfactant replacement, and less intrusive breathing procedures. Unfortunately, none of these techniques significantly reduced the incidence of bronchopulmonary dysplasia (BPD) 33. Most fatalities were caused by abrupt respiratory distress, with computational and postmortem evidence of airway diseases including distal inflammatory responses, extensive fibroproliferative alterations, and hypertensive pulmonary vascular remodelling. Notably, Dr Northway and colleagues uncovered additional characteristics of BPD in their investigation, including prematurity-related retinopathy and brain injury 3. Bronchopulmonary dysplasia, the newborn form of chronic lung disease, has contributed to substantial increases in morbidity and mortality in recent years. However, the failure to unravel the molecular mechanisms behind the pathogenesis is a primary reason for the lack of specific therapies. With the recent discovery of several non-coding RNAs, including microRNAs, long non-coding RNAs (lncRNAs), and circular RNAs, more spotlights have been focused on their gene regulation. Previous research has indicated the probable relationship between lung injury and non-coding RNAs 34, revealing the essential roles in lung injury. Numerous studies have shown a link between miRNAs and the advancement of lung injury and fibrosis, cell proliferation, and cell apoptosis, including miR-29a-3p 34, miR-34a 35, and miR-184 36 in BPD mice models. Besides, lncRNAs are not only involved in lung maturation 37 but are also regarded as crucial regulators of biological activities and BPD progression 10, 34, 38. According to research, lncRNA Rian rescued lung cells from hyperoxia-induced damage by targeting miR-421 39. Along with the tangible expression of lncRNA H19 in the lung tissues of BPD model mice 40, it was shown that lncRNA CASC2 protects against lung damage caused by hyperoxia exposure, with CASC2 decreasing aberrant cell apoptosis in both animal and cell models 41. However, owing to the challenges of computational prediction in the context of poor sequence conservation and low homology, lncRNAs remain relatively unexplored and need more additional studies to elucidate. This study examined the abnormal patterns of circRNAs expression in the peripheral blood of severe BPD patients throughout their early life (7–10 days after birth). Microarray profiling detected 252 differentially expressed genes, including 171 upregulated and 81 downregulated genes in the BPD group compared to the control group, with a fold change of more than 1.5, a P-value less than 0.05, and an FDR less than 0.05. Following that, we began screening potential genes for additional investigation.
CircRNAs were initially discovered in 1979 via electron microscopy in human Hela cells. Mounting evidence demonstrated the widespread of circRNAs in various species 42. CircRNA could be categorised into three groups, exonic (ecircRNA), exon-intron (elcircRNA), and intronic (ciRNA) 43, with lengths varying from 100 to several kilobases, depending on the amount of back-spliced exons and introns. The median size is typically between 500 and 700 nucleotides and consists of 2–3 exons 44. Most circRNAs are ecircRNAs, which reside mainly in the cytoplasm but may also be expelled into the extracellular environment through exosomes. Additionally, certain exonic circRNAs are found in the nucleus, enhancing protein retention or attracting proteins to chromatin 45. In contrast, elcircRNAs and ciRNAs are most often detected inside of nucleus 24.
Until recently, circular RNAs (circRNAs) were not expected to participate in various biological activities. Researchers have found evidence to support the hypothesis that the miRNA-binding protein (RBP) circRNAs regulate biological and pathological processes by sponging miRNA, modulating the expression of parental genes, and even monitoring protein translation or being translated into peptides 28, 46. In the cell nucleus, circRNAs serve as decoys for miRNAs, preventing them from binding and inhibiting their target mRNAs 47, 48. CircABCC4 was uncovered by sponging miR-663a and upregulating PLA2G6 expression to promote BPD evolution 49. However, on the other, certain circRNAs have the potential to sponge proteins due to their propensity to bind proteins. Circular RNAs are crucial in transcription, alternative splicing, and chromatin looping when locked in the nucleus 50. For instance, the binding of circPABPN1 to HuR inhibits HuR from binding to PABPN1 mRNA and hinders PABPN1 translation 51. It has been established that circMBl, for instance, dominates the expression of its parent gene, MBL, by interfacing with RBPs and creating a negative feedback loop between MBL and the creation of circMBl 52. CircHuR blocked the binding of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) binding to the HuR promoter, reducing HuR production and tumour development 53.
We carefully filtered critical circRNAs for the following criteria: differential circRNAs with P values of less than 0.05 and a fold change of more than 2, acceptable lengths between 200 and 1000 bp, and exonic type. Then Real-time Q-PCR validated candidate genes picked by microarray analysis. The hypergeometric distribution test was used to perform differential RNA GO (http://www.geneontology.org/) and KEGG (https://www.genome.jp) analyses.
In our study, four of five upregulated circRNAs (hsa_circ_0007054, hsa_circ_0057950, hsa_circ_0050386, hsa_circ_0120151) were confirmed by qRT-PCR, which was consistent with microarray assays. Hsa_circ_0007054 is derived from the TMEM50A/SMP1 gene. This gene is situated between the RHD and RHCE genes in the RH gene locus 54, with a sequence of potential transmembrane domains, yet its protein product's function is elusive. The gene hsa_circ_0057950 is generated from parental genes that encode multiple functional proteins — CREB1. This gene encodes several transcription factors through alternation splicing, acting as homodimer binding to the cAMP-responsive element or stimulating hormone-induced gene transcription through the cAMP pathway. CREB1 is one of the pivotal transcription factors (TFs) in human malignancies, believed to function effectively as oncogenes or tumour suppressors like miRNAs, as shown by the broader range of cancer-related papers 55–57. Furthermore, CREB1 could modify the VE-cadherin expression of endothelial cells in mediating sepsis-induced inflammatory lung injury 56. Another circRNA was hsa_circ_0050386, derived from VARP, also known as ANKRD27, which codes VPS9-ankyrin-repeat protein. Numerous molecules interacting with VARP have been identified during the past years, and considerable research focuses on VARP's multiple activities in endosomal trafficking, including positive regulators of neurite outgrowth 58 and retromer-mediated sorting transmembrane proteins from endosomes to plasma membranes 59. Calcium and the second messenger diacylglycerol could activate the protein kinase C (PKC) family of serine and threonine-specific kinases encoded by the PRKCE parent gene has_circ_0120151 60. This kinase might influence cellular energy metabolism and, consequently, cell function, from phosphorylating many protein targets, participating in diverse cellular signalling pathways, neuron channel activation 61, apoptosis, and cardioprotection from ischemia 60, as well as insulin exocytosis. Additionally, a group of tumour promoters called phorbol esters are crucial receptors for PKCs 62–64. Furthermore, preeclampsia's aetiology is linked to the process of apoptosis in the placental syncytiotrophoblast, as shown in the most recent publication on PRKCE 65.
Depending on bioinformatics techniques, the circRNA-miRNA-mRNA network of the four differently expressed circRNAs was ascertained. Has-miR-57887, hsa-miR-6842-3p, hsa-miR4505, hsa-miR-4638-5p, and hsa-miR-6071 were anticipated to be adversely regulated by the upregulated has_circ_0057950.
The hsa-miR-5787 has previously been shown to limit fibroblast cell proliferation by binding to the eIF5 gene. However, no direct evidence indicates their function in BPD. Yoo H et al. have previously validated this 66. Similarly, Bao Z et al.67 revealed that hsa-miR-5787 might attenuate the inflammatory response mediated by LPS/TLR4 in macrophages through the NF-κB signalling pathway downregulating its expression and producing pro-inflammatory cytokines such as IL-6 and TNF-α. Due to the increased expression of hsa_circ_0057950 under this study, we hypothesised that hsa-miR-5787 is downregulated, which stimulates fibroblast cell proliferation while boosting LPS/TLR4-mediated inflammatory response via NF-κB in macrophages, leading to increased release of inflammatory factors such as IL-6 and TNF-α. These actions may further exacerbate the progression in bronchopulmonary dysplasia, but more investigation is required.
To get a clear grasp of the interaction of the ceRNA network's target genes, we conducted function and pathway analysis to provide more underlying mechanisms of BPD. Gene Ontology (GO) terminology from three categories (GO: biological process, GO: cellular component, and GO: molecular function) was employed for route enrichment analysis and biological interpretation. According to GO analysis, these target genes were most enriched in the categories "negative control of transcription from the RNA polymerase II promoter" (GO:0000122) and "transcription DNA-templated" (GO:00006351). Non-coding RNAs are so numerous in eukaryotes as a significant regulator of the transcriptional process at multiple levels, function through controlling transcription factor occupancy and epigenetic modifications, to direct interference with RNAP II function 68, 69. Although transcription of a lncRNA may indirectly regulate nearby mRNA genes, either positively or negatively 69, these circRNAs were novel ones and different from previous results in this field. Nevertheless, the exact role and underlying mechanism are still in progress.
Additionally, we employed KEGG analysis to elucidate the function of the ceRNA network's target genes and acquired notable signalling pathways, some of which were congruent with the present understanding of BPD and others that were entirely novel. BPD formation and progression are modulated by diverse signalling pathways, including the p53 signalling pathways 31, TGF-β signalling pathway 70, NF- κb signalling 71 and MAPK signalling pathways 40. Both human and animal-derived tissues or cells are suitable for microarray and high-throughput sequencing of non-coding RNA expression patterns. More in-depth bioinformatics research has uncovered a complex network of molecular connections. For example, researchers once used deep Illumina sequencing to reveal variable expression of long non-coding RNAs in hyperoxia-induced bronchopulmonary dysplasia rat models. The result indicated that enriched KEGG terms are tied directly to ECM-receptor interaction, the cell cycle, and cytokine-cytokine receptor interaction 38. Moreover, significant pathways enriched in VEGF signalling, vascular smooth muscle contraction, thyroid hormone signalling, platelet activation, and oxytocin signalling were identified in another hyperoxia-induced bronchopulmonary dysplasia rat model 72, 73. The authors of another study applied RNA deep sequencing and bioinformatics analysis to identify circRNAs that were substantially changed by HCMV productive infection in human embryonic lung fibroblasts (HELF). According to their investigations, the host genes of different circRNAs were linked to focal adhesion, ECM-receptor interaction, amoebiasis, and the PI3K-Akt signalling pathway 74. The circRNA expression patterns from the peripheral blood of BPD patients and normal preterms were obtained through bioinformatics, conforming critical metabolic processes including ECM-receptor interactions, protein digestion and absorption, RNA transport, and bacterial invasion of epithelial cells 49.
In our study, the KEGG pathway analysis revealed several lung developments, aberrant injury, and repair-associated pathways that have recently been confirmed 75, 76. Hippo, bacterial invasion of epithelial cells, cGMP-PKG, and MAPK are all inextricably linked with the pathogenesis of BPD 77. For instance, the Hippo signalling pathway, crucial for embryonic lung maturation and postnatal airway homeostasis, could lose function due to lung injury 78. Also, in complicated pregnancies, including preeclampsia (PE) and chorioamnionitis (CA) 79, a postal bacterial infection of the lungs may increase the chance of preterm newborns developing bronchopulmonary dysplasia (BPD) 80, 81.
Furthermore, we observed another signalling pathway that may play an essential role in lung agenesis, namely the Wnt signalling pathway. The Wnt gene family contains 19 glycoproteins interacting with cell surface receptors 82, 83. Canonical Wnt/β-catenin signalling pathway, which initially appears in the anterior forgetting region, forms the trachea and two lung buds (E9.5). As a result, mice lacking endoderm-specific catenin expression or Wnt2/2b−/− mutants have an increased risk of lung agenesis 84, 85. Inactivation of β-catenin at the epithelial level leads to decreased branching and anomalous proximal-distal patterning 86, 87. As an additional bonus, non-canonical Wnt pathways govern stem cell maintenance and modulate activating protein kinase C and CaMKII signalling pathways 88. Conversely, the inflammatory response plays another vital role in lung injury, and activation of the Wnt/catenin pathway in AEC2 leads to increased production of IL-1, which promotes the inflammatory pro-fibrotic response 89. These findings reveal that circRNAs may modulate molecular mechanisms of BPD, and in the future, more specific circRNAs could come into light function as prospective diagnostic and therapeutic targets.
The current research has certain limitations. To begin, differentially expressed circRNAs were found in a limited number of patients and should be confirmed in a larger cohort of patients with severe BPD. Second, using bioinformatics methods, the roles of differentially expressed circRNAs were hypothesised. We validated two circular RNAs using human embryonic lung epithelial cells and animal BPD models while collecting further clinical samples. Moreover, we received municipal funds (Item ID: SKJYD2021112) to support our future projects. In short, our future research project will focus on the functional impact of their interplay.