Study On Pathogenic Pathways And Biomarkers Of Obstructive Bronchiolitis Based On Bioinformatics Analysis

BackgroundBronchiolitis Obliterans(BO)is a irreversible respiratory disease with a difficult diagnosis,no specific treatment and a poor prognosis.Bioinformatics is an integrated discipline of biology,computer science,information engineering,mathematics and statistics,which is frequently used to explore the biological significance of transcriptome sequencing results,such as the discovery of potential signal pathways and biomarkers.In the current era of big digital data,the GEO database provides multispecies,multidisease and multisample transcriptome sequencing data for researchers,so it is feasible and necessary to combine GEO database with bioinformatics to explore potential signalling pathways and biomarkers of BO.In this study,we identified potential signal pathways and biomarkers for occlusive bronchitis based on bioinformatics and performed molecular experiments in lung tissue from BO model mice to validate the aberrant expression of critical molecules and biomarkers of signal pathways in BO.ObjectivesThe aim of this study was to explore potential signalling pathways and biomarkers of BO based on bioinformatics.Methods1.Predicting potential signal pathways for BO:In this study,we first obtained BO-related transcriptomic datasets from the GEO database and then performed differential gene expression analysis on each dataset in order to get critical differential genes.Gene Ontolog(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,and Gene Set Enrichment Analysis(GSEA)were used to explore potential signal pathways of BO.Screening and validation of potential BO signaling pathways2.Predicting potential biomarkers for BO:Key differentially expressed genes were imported into the Cytohubba plugin in Cytoscape software to compute hubgenes applying 12 topological algorithms.3.Construction of BO mouse models and experimental validation of predicted potential BO-related signalling pathways and biomarkers:In this study,BO mouse model was constructed using the tracheal 2,3-butanedione drip method.In this study,Western Blot(WB)was used to detect the protein levels of critical molecules of the signal pathway in the lung tissue of BO mice and RT-q PCR was used to detect the m RNA expression of hub genes in the lung tissue of BO mice.Results1.This study identified 57 key differential genes of BO,including 39 up-regulated genes and 18 down-regulated genes.2.The KEGG result suggested that the lysosomal pathway and apoptotic pathway might be important signal pathways in BO.3.The GO result suggested that transmembrane transport of cations might be an important biological process in BO.4.GSEA result suggested that 33 pathways including NETs formation pathway might be up-regulated in BO.5.CC Motif Chemokine Receptor 2(CCR2),CD1 D Molecule(CD1D),GM2 Ganglioside GM2 Activator(GM2A),EC Transcription Factor EC(TFEC)Transcription Factor EC(TFEC),Macrophage Expressed Gene 1(MPEG1),Cathepsin S (CTSS),Glycoprotein(GPNMB),Baculoviral IAP Repeat Containing 2(BIRC2)and Cathepsin Z(CTSZ)genes were predicted as potential biomarkers for BO.6.TFEC,IRF8 and SPI1 were predicted to be important transcription factors for BO.7.BO pathology was found in the lungs of mice after day 14 of tracheal drip administration of 30% 2,3-butanedione.8.Compared with the control group,the lung tissues of mice with BO induced by 2,3-butanedione showed increased expression levels of NE and MPO with statistically significant differences(P<0.01;P<0.01).9.The m RNA expression levels of Ccr2,Mpeg1,Ctsz,Ctss and Tfec genes in lung tissues of 2,3-butanedione-induced BO mice were significantly higher than control group,and the differences were statistically significant(P<0.05;P<0.0001;P<0.0001;P<0.0001;P<0.01).ConclusionsIn this study,we find that the NETs formation pathway is dysregulated in BO,and the protein levels of NE and MPO,the key molecules of NETs,are significantly increased in lung tissues of BO mice,as verified by WB experiments results.In this study,CCR2,CD1 D,GM2A,TFEC,MPEG1,CTSS,GPNMB,BIRC2,and CTSZ were predicted as potential biomarkers of BO,and the levels of Ccr2,Mpeg1,Ctsz,Ctss,and Tfec m RNA were up-regulated in lung tissues of BO mice,as verified by RT-PCR assay.This study provides new options for exploring the molecular mechanisms,diagnostic indicators,and target therapies of BO.