The Role And Mechanism Of Transcription Factor JunD In Cigarette Smoke-Induced Airway Inflammation And Oxidative Stress Of COPD

Background Cigarette smoke(CS)-induced oxidative stress and airway inflammation are important pathologic features of chronic obstructive pulmonary disease(COPD).Nevertheless,the hub genes and mechanisms underlying CS-induced airway inflammation and oxidative stress in COPD remains unclear.Transcriptome and bioinformatics analyses are helpful to investigate the molecular mechanisms and key targets in pathophysiological processes or diseases,with integration and re-analysis of transcriptome data in public databases,such as the Gene Expression Omnibus(GEO)database,providing valuable new insights.Recently,weighted gene co-expression network analysis(WGCNA)has been used to take into account correlations among transcripts and identify potential disease-related gene co-expression modules by considering associations between the gene co-expression modules and disease traits as well as intramodular associations.Key driver analysis(KDA)can be used to identify key regulatory components of sub-networks in various biological contexts.These techniques have been widely used in biomedical research.In this study,we used WGCNA to conduct data mining based on GEO data,and we used KDA to identify the key genes,such as JunD,involved in lung function(which is the most import ant index of COPD).Increasing studies indicate a potential role of JunD,a member of the Jun family of transcription factors,in the regulation of various pathophysiological processes such as inflammation and oxidative stress through activation or inhibition the expression of a variety of target genes.But its roles in CS-induced oxidative stress and airway inflammation have not been reported.We then investigated the role of JunD in CS-induced airway inflammation and oxidative stress response both in vitro and in vivo,and we explored the potential taregets and mechanisms of JunD using chromatin immunoprecipitation(Ch IP)-seq and mRNA-seq.Methods Data from the Gene Expression Omnibus(GEO)database(GSE37147)were analyzed using weighted gene co-expression network analysis(WGCNA)and key driver analysis(KDA),and these analyses were validated using the GSE47460 dataset.The expression of JunD was examined in lung tissues of COPD patients and CS-exposed mice and in CS extract(CSE)-exposed BEAS-2B and HSAE cells by western blot and RT-q PCR.The effects of cigarette on oxidative stress and airway epithelial inflammatory damage after JunD knockdown or overexpression were investigated.mRNA-seq and chromatin immunoprecipitation(Ch IP)-seq were used to explore the targets and mechanisms of JunD-mediated CS-induced oxidative stress and airway inflammation,then target genes and signaling pathways were verified via JunD overexpression models both in vitro and in vivo.Results WGCNA and KDA of the lung function-related gene modules in GSE47460 and GSE37147 revealed a potential role of JunD in inflammation and oxidative stress response of COPD.JunD was downregulated in lung tissues of COPD patients and CS-exposed mice as well as in BEAS-2B and HSAE cells.JunD knockdown aggravated CSE-induced tumor necrosis factor(TNF)-α and interleukin(IL)-1β in BEAS-2B cells,while JunD overexpression attenuated these effects and increased superoxide dismutase activity and glutathione levels and decreased malondialdehyde levels.Ch IP-seq and mRNA-seq identified JunD-regulated genes(NR1D1,RELB and PALM3),which are involved in NF-κB and MAPK signaling pathways and are commonly dysregulated in cell models of oxidative stress and airway inflammation.The overexpression of JunD cells and mice samples showed that relative mRNA expression NR1D1 and RELB was significantly up-regulated respectively,PALM3 was significantly down-regulated,the phosphorylation level of p65 was significantly decreased.Conclusion Both in vitro and in vivo,JunD is down-regulated in COPD patients and CS-stimulated mice and cells.JunD overexpression attenuated the CS-induced inflammatory cytokine release and oxidative stress damage,as well as p65 phosphorylation level.In conclusion,JunD may participate in the regulation of cigarette-induced airway inflammation and oxidative stress response through NF-κB signaling pathway,suggesting that JunD-based therapy may be useful in CS-induced airway disorders.