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Qian, Guoqing (2023) Nephronectin and the genetics of lung function variability. PhD thesis, University of Nottingham.

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Abstract

Genetic variation at the chromosome 4 locus containing the gene nephronectin (NPNT) is strongly associated with both lung function variability and the risk of developing chronic obstructive pulmonary disease (COPD) in genome wide association studies (GWAS). NPNT is a conserved extracellular matrix protein, which appears to play a crucial role in regulating cell proliferation, differentiation and adhesion. The main aims of the thesis were to i) to investigate the profile of NPNT in different airway structural cell lines and lung tissues; ii) to identify the contribution of targeting NPNT by two approaches (RNA interference, or CRISPR/Cas9) to determine differentially expressed genes and potential pathways involved in human bronchial epithelial cell homeostasis; and iii) to assess the potential for serum NPNT protein levels to be used as a biomarker in either stable COPD stable or patients with exacerbations.

I found that SNP rs17331332 and rs34712979 are eQTLs for NPNT, and the G allele of variant rs34712979 was negatively associated with COPD risk in a phenome wide association study (PheWAS). NPNT mRNA was identified in foetal and adult lung tissues, as well as human bronchial epithelial cells (HBECs), human airway smooth muscle cells (HASMs) and BEAS-2B cells. NPNT protein was detected in foetal, adult healthy controls and COPD lung tissues by immunohistochemistry. NPNT protein is highly expressed in the alveolar and fibro-elastic layers, especially in pneumocytes and fibroblasts. NPNT expression gradually increased with increasing foetal lung development, but was decreased in smokers with COPD.

Two approaches to analyse the differentially expressed genes after knockdown of NPNT by siRNA were used Microarrays and RNA sequencing. There were 24 and 442 differentially expressed genes identified by Microarray and RNA sequencing, respectively but poor agreement between the two platforms. NPNT expression itself was only seen to be significantly reduced using RNA sequencing analysis. Gene set enrichment analysis revealed inflammation pathways were upregulated, and cell proliferation signalling were downregulated by NPNT knockdown. To extend these studies, I successfully established a protocol for fast, robust, cloning-free and high efficiency electroporation-based CRISPR/Cas9 genome editing system for primary HBECs. This will be of value in the future to further assess the downstream genes and pathways regulated by NPNT.

Serum NPNT protein levels in COPD patients were measured by ELISA from a UK (Nottingham) cohort and a China (Ningbo) cohort. Serum NPNT expression levels showed weak positive correlations for age and creatinine blood, but were not associated with lung function. Serum NPNT levels were decreased in patients with COPD exacerbations, and in this group NPNT levels were associated with FEV1 and FEV1/FVC.

In summary, the work described in this thesis has identified the potential casual variant associated with lung function decline and COPD risk, and has defined the expression profiles of NPNT in foetal and adult lung tissue and airway structural cells. NPNT appears to be implicated in HBEC homeostasis. An electroporation based CRISPR/Cas9 genome editing system was established and will be of value for future work. Measuring serum NPNT levels in patients is feasible and may throw further light on the role of NPNT in lung disease.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Hall, Ian Sayers, Ian
Keywords:	Lung function; Chronic obstructive pulmonary disease; Extracellular matrix protein; Airway cell lines; Lung tissue; Bronchial epithelial cell homeostasis; Biomarker
Subjects:	W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	72023
Depositing User:	Qian, Guoqing
Date Deposited:	31 Jul 2023 04:40
Last Modified:	31 Jul 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/72023

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