Abstract Rationale Cigarette smoke (CS) induced airway remodelling is a characteristic feature of chronic obstructive pulmonary disease (COPD); however, a clear molecular insight is lacking. In this study, utilizing bioinformatics tools, we aimed to identify crucial hub genes driving COPD pathogenesis, and validate their role in COPD using in-vitro CS-exposed cellular models and COPD patient samples. Methods Bioinformatics tools like String, GEO datasets, CTD, Genecards, Disgenet, Opentargets, and Cytoscape were used of in-silico molecular interactions and identification of hub genes. The hub genes were validated using cigarette smoke extract (CSE)-exposed airway epithelial cells (AECs), followed by assessments of EMT-related parameters and markers using cellular and molecular biology techniques such as the MTT assay, trypan blue assay, AO/EtBr assay, migration and invasion assay, immunoblotting, immunocytochemistry, and RT-qPCR. Furthermore, key hub genes and inflammatory cytokines were assessed in PBMCs from COPD patients and healthy volunteers via RT-qPCR and ELISA. Results Four online databases (CTD, Genecards, Opentargets, and Disgenet) and a clinical dataset from the Gene Expression Omnibus (GEO) were utilised to identify upregulated differentially expressed genes (DEGs). Subsequently, ten hub genes for COPD were identified using MCODE and cytohubba indices of Cytoscape, of which NOTCH3 and matrix metalloprotease (MMP)-2 were selected for further validation owing to their crucial role in COPD. CSE exposure of AECs altered cellular morphology, induced fibrous phenotype, upregulated fibrosis and EMT markers, along with increased expression of NOTCH3 and MMP-2. Chemical inhibition of MMP-2 downregulated NOTCH3, suggesting NOTCH pathway upregulation by CSE-induced MMP-2 activation. Further, Inhibition of either MMP-2 or NOTCH3 reversed CSE-induced EMT-related changes in AECs. COPD patients derived PBMCs showed modulation of NOTCH3 and MMP-2. JAG1, a NOTCH ligand, and many inflammatory markers were also significantly upregulated in COPD patient samples. Conclusion MMP-2 and NOTCH3 could be central drivers of CSE-induced airway epithelial cell remodelling, as identified and validated using in-silico and in-vitro approaches, respectively, and also confirmed through analysis of samples from COPD patients. This abstract is funded by: None
Dubey et al. (Fri,) studied this question.