COPD-related molecular role of FBXO38 gene expression in epithelial airway cells

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a chronic and progressive inflammatory pathway disease which is, the third leading cause of death globally. It is mainly associated with smoking, however there are also genetic and environmental factors at play. Symptoms of the disease include breathing difficulties, excessive phlegm production, chesty coughing and, wheezing and airway structural changes. COPD is known to be a multifactorial and polygenic disease. When the right environmental and genetic factors interact, the risk of the disease increases significantly. FBXO38 has been identified as an expression quantitative trait locus (eQTL) and splicing quantitative trait locus (sQTL) target of SNV rs6889822. This SNV is a marker for COPD, which was found through GWAS studies. The FBXO38 gene is also a co-activator of KLF7, a transcription factor known to be involved in airway inflammation, however no studies have looked into the possible contribution of FBXO38 to COPD molecular pathways. Aim: The aim of this research is to study the effects of FBXO38 gene expression on COPDrelevant molecular pathways in human airway cells. Methods: H520, a high FBXO38 expressing epithelial airway cell line, was selected for the study, and the FBXO38 gene expression was knocked down using siRNA technology. Transfection parameters were first optimised using siGLO and after the ideal parameters were selected, cell samples were then transfected with FBXO38 mRNA-targeting siRNA, together with scrambled, non-transfected and untreated controls. RNA extraction was then carried out, and RNA-Sequencing (RNA-Seq) analysis was carried out on FBXO38 knockdowns and scrambled controls. Differential gene expression was carried out on the RNA-Seq data, followed by pathway analysis, in order to identify COPD-related molecular pathways that are modified by FBXO38 downregulation. Results: The results of the study showed that the knockdown of the FBXO38 gene resulted in the up regulation and down regulation of 25 genes with a significant q-value. KEGG pathway analysis identified 20 pathways which were enriched following FBXO38 knockdown of which nearly half are well known to be associated with COPD. GO analysis found two DEGs to be highly correlated with Transcription Factor AP-1 complex. Discussion: Four pathways from the KEGG analysis were shortlisted as being the most important due to being known to be highly related to COPD. These were the cAMP signalling pathway, interleukin-17 (IL-17) signalling pathway, Th1 and Th2 cell differentiation and finally, Th17 cell differentiation. Transcription factor complex AP-1 was identified from the GO cellular component analysis, being driven by two statistically significant DEGs. Conclusion: This project has determined that FBXO38 knockdown enriched several major COPDrelated pathways. This suggests that the gene relevance to COPD may be related to increased activity of these pathways. The results of this project add to current knowledge of molecular events in COPD, however more research is required to further enhance and solidify the data obtained.