BPD is a chronic lung disease characterized by simplification of the alveolar structure, impaired vascular development, and minor damage to the small airways (17). BPD is a multifactorial disease, and the current treatment effect is poor, focusing on prevention. BPD is now considered to be one of the most difficult problems in the neonatal intensive care unit (18). With continuous improvement in the global critical care neonatal treatment capacity, an increasing number of preterm infants have survived (19).However, the probability of developing BPD, especially severe BPD, has significantly increased (11, 20).Respiratory diseases frequently occur in BPD surviving children, and their quality of life and life expectancy decline, which significantly affects the quality of health services (21). Owing to improvements in the survival rate of very preterm infants and use of pulmonary surfactants, increased incidence of BPD has now been noted in very premature infants with extremely immature lung development (22), and pathophysiological changes, pathogenic factors, pathogenesis, and diagnosis and treatment strategies of BPD have changed significantly. Therefore, to reduce the incidence and complications of BPD, it is necessary to understand the internal mechanisms of BPD, identify its molecular regulatory pathways, and obtain targeted treatment measures.
LncRNAs longer than 200 nucleotides constitute a family of transcripts with no protein-coding function (23). They are involved in various biological processes, including cell proliferation, apoptosis, and genome stability (24). Because lncRNAs show a high degree of tissue specificity (25) and disease specificity, they can be used as disease biomarkers in clinical applications (26). The ceRNA hypothesis states that lncRNAs regulate gene expression by acting as miRNA sponges (13). They act as natural sponges for the competitive adsorption of certain miRNAs and reduce the binding of miRNAs to their corresponding target genes, resulting in changes in the expression of miRNA target genes (27). However, it remains unclear whether abnormal lncRNAs exert ceRNA effects on some miRNAs and indirectly regulate the expression of target mRNAs in the alveoli and pulmonary blood vessels during BPD development.
MiRNAs are short non-coding RNAs, 20–22 nucleotides in length, that help degrade mRNAs or inhibit translation by binding to the 3'-untranslated region of mRNAs (28, 29). A large body of evidence has shown that miRNAs play an important role in the pathogenesis of diseases and are considered potential targets for disease treatment. Dysregulated miRNAs are closely associated with many diseases, including cardiovascular disease (30), diabetes, and autoimmune diseases (31). In addition, miRNAs play crucial roles in lung inflammation and lung cancer (32–35). lncRNAs and miRNAs play important roles in the pathological mechanism of BPD. For example, SNHG6 is highly expressed in BPD models, whereas miR-335 is underexpressed, and knockdown of SNHG6 attenuates hyperoxia-induced lung cell damage. In addition, (36) found that SNHG6 can mediate lung cell damage by regulating the miR-335/KLF5/NF-κB pathway. The mechanism may be that Xist competitively binds miR-101-3p to activate HMGB3, and overexpression of miR-101-3p alleviates lung injury in BPD mice (37). lncRNA MALAT1 inhibits apoptosis and plays a protective role in BPD through the Keap1/Nrf2 signaling pathway (38). Studies have shown that increased lncRNA TUG1 attenuates lung injury in BPD mice and promotes hyperoxia-induced MLE-12 cell proliferation. Meanwhile, TUG1 inhibited the inflammatory response and apoptosis in BPD mouse lung tissue and hyperoxia-induced MLE-12 cells. The inhibitory effect of TUG1 on apoptosis and inflammation in hyperoxia-induced MLE-12 cells could be reversed by decreasing ELN or increasing miR-29a-3p (39). Rian was downregulated and miR-421 was upregulated in lung tissue and hyperoxia-induced MLE-12 cells in a BPD mouse model. Rian-plasmids can inhibit the secretion of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in serum and hyperoxia-induced MLE-12 cells in BPD mice (40). The Rian plasmid also abolished the inhibitory effects of hyperoxia on MLE-12 cell viability and induced apoptosis. Moreover, the effects of Rian were significantly reversed by miR-421 mimics. These results suggest that Rian can attenuate hyperoxic injury in the neonatal lung and may serve as a novel molecular target for BPD therapy (40).
To explore potential interactions between lncRNAs and miRNAs in BPD, we constructed an lncRNA–miRNA–mRNA ceRNA network. A total of 1286 DEGs, 77 DEmiRs, and 104 DElncRs were identified in BPD and normal blood samples. Functional enrichment analysis showed that DEGs were primarily enriched in GO terms, such as B-cell receptor signaling pathways, asthma, FcRI signaling pathways, cell apoptosis, the intestinal immune network that produces IgA, and Th17 cell differentiation signaling pathways. The ceRNA network consisted of 11 miRNAs, 6 lncRNAs, and 61 mRNAs (Fig. 4). lncRNAs indirectly regulated miRNA target genes by competitive binding with miRNAs.
Small nucleolar RNA host gene 16(c) (SNHG16) is an lncRNA located on chromosome 17q25.1. Previous studies have confirmed that lncRNA SNHG16 can act as a promoter of epithelialmesenchymal transition in various cancer types, such as glioma (41), esophageal cancer (42), cervical cancer (43), retinoblastoma development (44), and glioma (45). SNHG16 can sponge miR-146a, and SNHG16 is upregulated and involved in tumor progression in non-small cell lung cancer (46). A recent study showed that SNHG16 promotes TLR4 expression by targeting miR-15a/16, thereby inhibiting the anti-inflammatory effects mediated by miR-15a/16 (47). Previous studies have also reported that SNHG16 is overexpressed in the serum of patients with acute pneumonia, and SNHG16 knockout inhibits the expression of CCL5 by upregulating miR-146a-5p, thereby attenuating lipopolysaccharide (LPS)-induced lung injury which can be used in the diagnosis and treatment of acute pneumonia (48). In Lipopolysaccharide-induced A549 cells, targeting IGF2 via miR-370-3p by SNHG16 inhibits cell viability and promotes apoptosis and inflammatory damage (49). A recent study reported that the lncRNA SNHG16 is an important regulator of pulmonary fibrosis. SNHG16 acts as a miR-455-3p sponge, thereby positively regulating Notch2 expression by binding to miR-455-3p (50). The above studies suggest that SNHG16 substantially contributes to LPS-mediated inflammation, especially in lung injury. Therefore, we conclude that SNHG16 also plays a role in BPD.
We observed that the low expression of lncRNA SNHG16 and DCAF8, EFHD2, EPHA4, GATM, GNG5, KPNA5, KREMEN1, MAPK6, PBX2, PPP2R2A, and ZC3HAV1 in children with BPD led to the upregulation of Lhasa-let-7g-5p, as well as that SNHG16 can regulate CCNE1, CDK1, CEP55, FURIN, JARID2, PHF19, PSAT1, RUNX1T1, and SNCG by competitively binding to hsa-miR-15b-5p; SNHG16 can also competitively bind to hsa-miR-30b-5p to regulate the expression of CDCA7, GLCE, KREMEN1, NACC2, PRDM1, SNAI1, and SOCS3. Thus lncRNA SNHG16 can regulate the occurrence and development of BPD through multiple pathways in the peripheral blood of children with BPD.
Studies have shown that downregulation of hsa-circ-0107593 promotes the osteogenic differentiation of hADSCs through miR-20a-5p/SMAD6 signaling (51). Atorvastatin may be involved in lipid-lowering therapy by downregulating microRNA-20a-5p (52). hsa-miR-20a-5p attenuates allergic inflammation in HMC-1 cells by targeting HDAC4 (53). hsa-circ-0107593 inhibits cervical cancer progression by sponging hsa-miR-20a-5p/93-5p/106b-5p (54). Bioinformatics analysis revealed that SNHG16 acted as a sponge for hsa-miR-20a-5p. Plasma hsa-miR-20a-5p/SNHG16 levels were significantly associated with more severe disease and IPI/FLIPI scores. Furthermore, patients with an hsa-miR-20a-5p/SNHG16 risk expression profile had a higher risk of positive bone marrow involvement and plasma levels of the hsa-miR-20a-5p/SNHG16 pair were associated with overall and progression-free survival in patients with Non-Hodgkin lymphoma and were independent prognostic factors in multivariate Cox analysis (55). However, hsa-miR-20a-5p/SNHG16 has not yet been reported in BPD studies. In this study, bioinformatics analysis showed that hsa-miR-20a-5P was upregulated to bind SNHG16 and downregulate the expression level of MAP3K5 in the blood samples from children with BPD, which is involved in the occurrence of BPD.
MAP3K5 (apoptosis signal-regulated kinase 1, ASK1) is a member of the MAPK group and is required for reactive oxygen species–induced cell death and inflammation (56, 57). ASK1 is subject to multiple stimuli, including oxidative stress, calcium influx, endoplasmic reticulum stress, DNA damage inducers, and signaling mediated by the tumor necrosis factor receptor (56). Hyperoxia injury induces ASK1 expression, which is a key event in hyperoxia-induced apoptosis (57). ASK1 plays a key role in mediating the various mechanisms of oxidative stress–induced cellular dysfunction and death, and ASK1 deficiency has protective effects (58, 59). Studies have confirmed that ASK deletion prevents hyperoxia-induced apoptosis of lung epithelial cells (57, 60) and that deletion of ASK1 can prevent acute lung injury caused by hyperoxemia (60).
In this study we found thatMBNL1-AS1 is highly expressed in children with BPD and competes with CHD9, E2F5, FBXO11, GOLGA1, IPO5, MOB3B, and TIAL1 to bind to hsa-miR-181a-5p, resulting in its downregulation and the formation of a ceRNA control network. Similarly, UBL7-AS1 downregulated hsa by competitively binding with ADPGK, ANXA2, DDX5, E2F5, EPB41L2, FOXP1, HNRNPU, MON2, PRKCE, SEC62, STK35, TNKS2, UST, ZNF280C. Moreover, hsa-miR-1-3p, constituting the ceRNA regulatory network in children with BPD.
LINC01915 constitutes a ceRNA regulatory network by competitively binding to hsa-miR-32-5p with ARRDC4, DDIT4, HIVEP1, IKZF4, and KIF1B. However, MBNL1-AS1 and UBL7-AS1 have been mainly studied in cancer and tumors (61–66) and less in lung diseases; therefore, more cell and animal experiments are needed to verify and explore the related mechanisms.
To understand the pathophysiological and biological mechanisms related to the occurrence and development of BPD, abnormally expressed circRNAs, miRNAs, and lncRNAs should be identified and verified in relevant human BPD samples or relevant animal models. Extracting total RNA from key cell types, such as lung epithelial cells and macrophages, can provide insight into cell-type-specific changes during BPD progression. When differential expression is validated, it will be necessary to test whether lncRNAs regulate BPD progression at the molecular level in vitro and in vivo. Predictive models incorporating ceRNA network expression analysis showed improved predictive power compared with models that only considered clinicopathological variables. Few studies have used ceRNA networks as potential prognostic biomarkers, especially in BPD, which may allow for a more personalized management of these patients. Nonetheless, our findings were only obtained from bioinformatic analyses, predicted modes of action based on measured RNA networks, and partially validated in animal models. However, it has not been demonstrated using dual-luciferase reporter assay, gene overexpression, or gene knockout. Therefore, although several related genes were screened for the first time in this study, further in vitro clinical studies and in vivo experiments are required to confirm their expression and functional mechanisms in BPD.