Investigation of the miRNA-mRNA Regulatory Circuits and Immune Signatures Associated with Bronchopulmonary Dysplasia

Abstract

Background: Bronchopulmonary dysplasia (BPD) has become a major cause of morbidity and mortality in preterm infants worldwide, yet its pathogenesis and underlying mechanisms remain poorly understood. The present study sought to explore microRNA-mRNA regulatory networks and immune cells involvement in BPD through a combination of bioinformatic analysis and experimental validation.

Methods: MicroRNA and mRNA microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed microRNAs (DEMs) were identified in BPD patients compared to control subjects, and their target genes were predicted using miRWalk, miRNet, miRDB, and TargetScan databases. Subsequently, protein-protein interaction (PPI) and functional enrichment analyses were conducted on the target genes. 30 hub genes were screened using the Cytohubba plugin of the Cytoscape software. Additionally, mRNA microarray data was utilized to validate the expression of hub genes and to perform immune infiltration analysis. Finally, real-time PCR (RT-PCR), immunohistochemistry (IHC), and flow cytometry were conducted using a mouse model of BPD to confirm the bioinformatics findings.

Results: Two DEMs (miR-15b-5p and miR-20a-5p) targeting genes primarily involved in the regulation of cell cycle phase transition, ubiquitin ligase complex, protein serine/threonine kinase activity, and MAPK signaling pathway were identified. APP and four autophagy-related genes (DLC1, PARP1, NLRC4, and NRG1) were differentially expressed in the mRNA microarray dataset. Analysis of immune infiltration revealed significant differences in levels of neutrophils and naive B cells between BPD patients and control subjects. RT-PCR and IHC confirmed reduced expression of APP in a mouse model of BPD. Although the proportion of total neutrophils did not change appreciably, the activation of neutrophils, marked by loss of CD62L, was significantly increased in BPD mice.

Conclusion: Downregulation of APP mediated by miR-15b-5p and miR-20a-5p may be associated with the development of BPD. Additionally, increased CD62L^- neutrophil subset might be important for the immune-mediated injury in BPD.

Keywords: CD62L− neutrophil; bronchopulmonary dysplasia; immune infiltration analysis; miRNA-mRNA regulatory circuits.

Figure 1

Flow chart of the whole study.

Figure 2

Identification of DEMs and their target genes. (A) Heatmap of miRNAs in GSE108755. (B) Venn diagram of potential target genes of DEMs predicted by miRNet, miRWalk, miRDB and TargetScan. (C) DEMs-target genes network.

Figure 3

GO and KEGG pathway analysis of the target genes of DEMs. (A) Enriched GO terms. (B) KEGG pathway analysis. Terms with p.adjust < 0.05 and q value < 0.2 were identified to be significant. The x-axis showed the gene count (A) and gene ratio (B) of each GO or KEGG term, and the y-axis showed names of enriched terms. Dot color and size represented p.adjust and gene counts, respectively.

Figure 4

Identification of hub genes and potential upstream transcription factors. (A) PPI network of the top 30 hub genes for DEMs. The redder the node, the larger the MCC score. (B) Predicted upstream transcription factors of hub genes using ChEA3.

Figure 5

Validation of hub genes in the GSE108754 dataset. Student’s t-test. *p < 0.05. ns not significant.

Figure 6

Relative proportions of 22 immune cell types in BPD and normal groups. (A) Compositions of immune cells in each sample. (B) Correlation heatmap of immune cells. Spearman’s method. *p < 0.05, **p < 0.01. (C) Differences in immune cell compositions between BPD and normal groups. Student’s t-test. **p < 0.01.

Figure 7

Experimental verification of bioinformatics findings in BPD mice. (A) The morphology of lung tissues in the RA and hyperoxia groups under the microscope at magnifications of 100 x and 200 x. (B) Quantitation of lung histopathological changes via RAC and MLI. Student’s t-test. **p < 0.01. (C) RT-PCR analysis of App, Nlrc4, Parp1 and Dlc1. Student’s t-test. *p < 0.05. ns, not significant. (D) Representative IHC staining images of APP under the microscope at magnifications of 200 x. The black arrows point to APP-positive areas. (E) Proportion of total neutrophils in the RA and BPD groups. Student’s t-test. ns, not significant. (F) Proportions of CD62L⁺ and CD62L⁻ neutrophil subsets in the RA and BPD groups. Student’s t-test. **p < 0.01. (G) Mean fluorescence intensity of CD62L on neutrophils in the RA and BPD groups. Student’s t-test. **p < 0.01.