Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740796
Title: Investigation of extracellular microRNAs and Serum Protein Biomarkers in dystrophic Muscle Disease
Author: Coenen-Stass, Anna
ISNI:       0000 0004 7229 0035
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Abstract:
Extracellular microRNAs (ex-miRNAs) and serum proteins are promising biomarkers for a variety of pathological conditions, including muscular dystrophies and, Duchenne Muscular Dystrophy (DMD) in particular. miR-1, miR-133 and miR-206 are muscle-specific miRNAs (myomiRs) that regulate myoblast proliferation and differentiation. These myomiRs are highly abundant in serum of DMD patients and dystrophic mdx mice and have therefore been investigated as biomarkers of dystrophic disease. The biological significance of miRNAs present in the extracellular space is currently not well understood. The work presented here demonstrates that ex-myomiRs are selectively released during periods of myogenic differentiation in cell culture and in vivo. Consequently, their release can be independent of dystrophic pathology, thus indicating that the presence of myomiRs in the serum may serve a physiological function in some contexts. In summary, serum myomiR abundance appears to be a function of the regenerative/degenerative status of the muscle, overall muscle mass, tissue expression levels and myomiR stability in serum. These findings have implications both for miRNA biology in normal muscle physiology as well as for the use of ex-myomiRs as biomarkers for DMD. Secondly, size-exclusion chromatography was identified as a useful methodology to fractionate biofluids and thereby separate extracellular vesicles (EV) from protein complexes and lipoproteins. Using this tool, it was demonstrated that ex-myomiR carriers in cell culture supernatant and murine serum are predominantly non-vesicular, and furthermore, that their release is independent of ceramide-mediated vesicle secretion. In addition, it was found that EVs isolated from dystrophic mice are smaller and more numerous compared to EVs derived from wild-type mice, thereby indicating a role for dystrophic pathology in the regulation of vesicle biogenesis and/or secretion. There is currently an urgent need for minimally invasive, therapy-monitoring DMD biomarkers for use in clinical trials. Serum protein biomarkers are desirable since they can typically be quantified by clinical biochemistry assays, which are technically facile and allow for rapid screening of large numbers of patients. Previously, identification of such biomarkers by mass spectrometry has been limited due to analytical challenges resulting from the massive complexity of the serum proteome. Here, an aptamer-based proteomics approach was utilised to profile 1,129 proteins in the serum of wild-type, mdx mice and, mdx mice treated with exon skipping therapy to restore dystrophin expression. A number of novel, therapy-responsive biomarkers were identified, and the leading candidate (ADAMTS5) was also found to be significantly elevated in DMD patient serum. In conclusion, this work demonstrates that myomiR release accompanies myogenic differentiation and shows that serum myomiR levels are influenced by a number of physiological and pathological factors. Furthermore, multiple novel, therapy-responsive protein biomarkers were identified in the serum of the mdx mouse with potential utility in clinical trials for DMD.
Supervisor: Wood, Matthew ; Roberts, Thomas Sponsor: St John's College ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.740796  DOI: Not available
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