Overexpression of FBXO38 in airway epithelial cells

Abstract

Background: Chronic Obstructive Pulmonary Disease (COPD) is a multifactorial disease caused by genetic variants and environmental factors like smoking and air pollution, leading to airway inflammation, emphysema, and chronic bronchitis, with 22 genome-wide significant loci identified, including FBXO38, and ranking as the fourth leading global cause of death. FBXO38, an F-box protein, regulates protein homeostasis by targeting proteins for ubiquitination and modulating apoptosis via KLF7, with dysregulation potentially contributing to COPD and cancer; located on chromosome 5, it spans 58,879 base pairs, with 47,371 in the coding region containing 21 exons. Current treatments focus on symptom management and slowing the disease progression. Aim: To study the effects of FBXO38 gene overexpression on COPD-relevant molecular pathways in airway cells. Methods: The H520 airway epithelial cell line was cultured in RPMI at 37°C and 5% CO2 as a model for this study. Initial optimisation involved seeding cells in ninety six-well plates and magnetofecting them with a GFP plasmid at varying magnetofection/DNA ratios, with fluorescence assessed after 24 hours to determine optimal transfection conditions. For the main experiments, cells in 6-well plates were magnetofected with either an FBXO38 expression vector or a scrambled-sequence control plasmid using the optimised parameters. After 48 hours, RNA was extracted, and FBXO38 overexpression was confirmed via qPCR. Samples were then submitted for RNA-Seq analysis. Differential gene expression between FBXO38-overexpressing and scrambled control cells was analysed using pathway analysis software to identify affected molecular pathways. These pathways were evaluated based on their statistical significance and relevance to COPD. Results: The study findings revealed that FBXO38 gene overexpression significantly altered the expression of 113 genes (q<0.05 [multiple comparison corrected p]), inducing both upregulation and downregulation. KEGG pathway analysis detected 20 enriched pathways, including 8 with established links to COPD. Additionally, GO analysis identified 19 DEGs showing strong associations with COPD-related mechanisms. Discussion: KEGG pathway analysis revealed that FBXO38 overexpression affected multiple cellular pathways, with GO enrichment analysis highlighting significant involvement in immune system processes and transcription regulation, primarily driven by four DEGs (IFNAR2-IL10RB, TRIB3, CD47, CD74). Five key pathways, IL-17, RIG-I-like receptor, NF-κB, TNF, and NOD-like receptor signalling, were selected for detailed discussion due to their established links to COPD. Conclusion: This study demonstrated that FBXO38 overexpression activates several key pathways implicated in COPD pathogenesis, indicating the gene's potential role in disease progression through modulation of these pathways. While these findings contribute valuable insights to our understanding of COPD's molecular mechanisms, additional research remains necessary to validate and expand upon these results which may eventually offer insights to novel druggable targets.