Date of Award

2013

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor N.G. McElvaney

Second Supervisor

Dr Catherine M. Greene

Funder/Sponsor

Malaysian Government Agency MARA

Keywords

a1-antitrypsin; chronic obstructive pulmonary disease;unfolded protein response; miR-199a-5p; promoter hypermethylation

Abstract

The hereditary disorder alpha-1 antirypsin deficiency (AATD) results from mutations in the SERPINA1 gene. The most common form of AATD occurs because of the Z mutation causing the protein to fold aberrantly and accumulate in the endoplasmic reticulum (ER) as opposed to being secreted into the circulation. The loss-of-function of AAT in neutralising neutrophil elastase and other proinflammatory enzymes contribute to the pathogenesis of emphysema. Meanwhile, the gain-of-function effects are thought to derive from the toxicity of the ZAAT forming insoluble polymers within hepatocytes leading to liver disease. The unfolded protein response (EIPR), a component of the ER stress has been shown to occur in monocytes due to ZAAT accumulation intracellularly. In view of both loss-and gain-of-function effects, numerous strategies have been examined for the development of gene therapies to address both aspects of the disease. MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. The regulatory role of miRNAs in regulating both the SERPINA1 gene and the pathophysiology of the UPR and inflammation in AATD has not been investigated.

Very few experimental methods can comprehensively identify multiple miRNAs that target a single mRNA. Elere, an experimental approach to search for miRNAs targeting a specific mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide (miR-CATCH) was developed for capture of AAT, interleukin- 8 and secretory leucoprotease inhibitor mRNAs. AAT mRNA-specific and total miRNAs from monocytic THP-1, bronchial epithelial 16HBE14o- and liver HepG2 cell lines were profiled and validated revealing cell-specific miRNA regulation. Overexpression of miR- 455-3p that was captured as an AAT-targeting miRNA identified in THP-1 monocytic cell lines in primary human monocytes with and without AATD (ie MM and ZZ monocytes respectively) led to significant decreases in both M- and ZAAT mRNA and protein.

To investigate miRNA expression and function in ZZ and MM monocytes, miRNA expression profiling was performed. Forty-three miRNAs were shown to be differentially expressed, with miR-199a-5p most highly upregulated in asymptomatic ZZ (individuals with no evidence of chronic obstructive pulmonary disease or COPD) compared to MM monocytes. MiR-199a-2 promoter hypermethylation inhibits miR-199a- 5p expression and was increased in symptomatic (individuals with COPD) MM and ZZ monocytes compared to their asymptomatic counterparts. The expression of components of the UPR arms, namely GRP 78, ATF6, p50 and p65 was increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or upregulation of these markers was observed after miRNA modulation using precursors and inhibitors of miR-199a-5p. Direct miR-199a-5p targeting of these markers was demonstrated using luciferase reporter systems.

To examine the effects of weekly AAT augmentation therapy (AATAT) on miRNA expression, miRNA profiling was performed. This revealed sets of differentially expressed miRNAs between (i) MM and ZZ monocytes in asymptomatic individuals, and (ii) between symptomatic ZZ monocytes receiving AATAT or not. The canonical dimer of NFkB was downregulated in ZZ monocytes receiving AATAT in vivo and in vitro. NFkB modulation using an NFkB inhibitor or activator altered the expression of miR-199a-5p, - 320a and -528 with potential NFkB binding sites in the regulatory regions of these miRNA genes. Using bioinformatic tools, target genes of these NFicB-regulated miRNAs were identified in a selection of pathways that include inflammation, hypoxia and antioxidant responses.

This study provides the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression in a cell-specific manner in which modulation of AAT using miRNAs in human primary cells was successfully performed. MiR-199a-5p was identified as a key regulator of the UPR in AATD monocytes and epigenetic silencing of its expression regulates this process in emphysema. Another anti-inflammatory property of AATAT in which NFkB was downregulated in ZZ monocytes receiving AATAT altered the expression of miRNAs with potential NFkB binding sites in their regulatory region. These findings in tandem to current knowledge of AATD, could provide potential avenues for future research and new generation gene therapies involving miR-based therapeutics that could tailor organ-specific treatment in AATD.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

10,969

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2013.

Share

COinS