Primary Screening And Validation Of Differentially Expressed Genes In Peripheral Blood Of Extremely Preterm Infants With Bronchopulmonary Dysplasia Based On Bioinformatics Analysis

Objective: Bronchopulmonary dysplasia(BPD)is the most common complication of premature infants,which is related to a variety of pathogenic factors.Its pathogenesis is complex and treatment methods are limited.The purpose of this study is to identify differentially expressed genes(DEGs)in the peripheral blood of extremely preterm infants with BPD using bioinformatics methods,conduct immune-infiltrating cell analysis,construct miRNA-mRNA interaction networks,screen and validate key molecules involved in the pathogenesis of BPD,thereby providing new ideas for further elucidating the potential pathogenesis of BPD.Methods: Firstly,the gene expression Omnibus(GEO)database was used to retrieve eligible data sets on peripheral blood of premature infants with BPD,including GSE32472 and GSE108754.GSE32472 was used as the test set and GSE108754 as the validation set.Next,GSE32472 was preprocessed using R software to obtain DEGs from three groups(5,14,and 28 days postnatal)of samples,during which immuno-infiltrating cell analysis was performed.After that,co-expressed differentially expressed genes(co-DEGs)were further screened and subjected to Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis to understand their functions and enrichment pathways.Then,the protein-protein interaction(PPI)network of co-DEGs was obtained using STRING database,based on which Cytoscape software was used as a tool to identify hub genes(highly connected gene,hub gene),followed by exploring the functions and related pathways of hub gene,and constructing the interaction network between hub gene and predicted miRNAs.Subsequently,the expression of hub gene was verified with the validation set GSE108754.Finally,in order to further clarify the expression of related hub genes in the peripheral blood of BPD patients,peripheral blood samples from BPD and non BPD preterm infants with gestational age of 28-32 weeks were collected at 28 days after birth,and the expression level of related molecules validated in the validation set was detected using quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)technology.Results: 1.The immuno-infiltrating cell analysis results of the dataset GSE32472 showed that the expression abundance of neutrophils and M0 type macrophages in the BPD group significantly increased,while the expression abundance of CD4 initial T cells,resting CD4 memory T cells,CD8 T cells,and M2 type macrophages significantly decreased.A total of 117 co DEGs were screened from the GSE32472 dataset,mainly concentrated in biological processes such as "T cell differentiation and activation","positive regulation of epithelial cell apoptosis",and cell components such as "specific particles" and "outer side of the plasma membrane","cytokine-cytokine receptor interactions","NF-kappa B signal transduction pathway","T cell receptor signal transduction pathway" "primary immune deficiency" and other signal pathways.Through PPI network,14 hub genes were identified,mainly concentrated in biological processes such as "cell defense response","regulation of lymphocyte apoptosis process","regulation of leukocyte apoptosis process",and signal pathways such as "hematopoietic cell lineage" and "primary immune deficiency".Predicted 18 target miRNAs for THBS1 and 7 target miRNAs for BCL6,and constructed a miRNA-mRNA interaction network centered on THBS1 and BCL6.2.Using the validation set GSE108754,14 hub genes were validated using R software.The results showed that ICOS and IL1R2 were low expressed in the BPD group and P<0.05.The expression results of ICOS were consistent with the test set results,while the expression results of IL1R2 were opposite to the test set results;The remaining 12 hub genes all showed P>0.05,with no statistically significant difference.In this study,q RT-PCR detection of peripheral blood samples collected from extremely premature infants with BPD and non BPD at a gestational age of 28 to 32 weeks showed that the expression of ICOS in the BPD group was significantly higher than that in the NC group,and there was no significant difference in IL-1R2 between the two groups.Conclusion: In this study,we screened the peripheral blood-related differentially expressed genes in extremely preterm infants with BPD by applying a combined test set and validation set approach through comprehensive bioinformatics analysis,and proposed that the key differentially expressed genes ICOS and IL1R2 may play an important role in the pathogenesis of BPD.And the expression of ICOS and IL1R2 was verified experimentally,which provided new ideas for the study of possible pathogenesis of BPD.