Buck, Amy

MacDonald, Andrew

Hoy, Anna Maria

Natural Environment Research Council (NERC)

2016-10-28T14:08:44Z

2016-10-28T14:08:44Z

2015-06-29

Schistosomiasis caused by the parasitic helminth Schistosoma mansoni is a major health problem in tropical and subtropical regions. Its detection is crucial for patient management, evaluation of treatment, and monitoring of disease transmission, thus the development of novel diagnostic assays is of immense importance. The pathology of S.mansoni infection is characterised by formation of granulomatous lesions, hepatic fibrosis and portal hypertension. Liver fibrosis itself, regardless of the causative agent is an important health problem. The fact that there is no treatment for hepatic fibrosis other than transplantation in end stage liver failure emphasizes the importance of investigating the molecular basis of fibrogenesis and development of better therapeutic tools. MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals. Several miRNAs have been implicated in hepatic fibrogenesis; however, the expression profile and the role of miRNAs in S. mansoni infection are yet to be determined. This thesis focuses on the characterization of miRNAs in the liver and serum of mice during S.mansoni infection in order to determine their therapeutic and diagnostic potential in this disease. Profiling of miRNA expression in the liver of mice infected with S.mansoni revealed a set of mouse miRNAs that were differentially expressed in infected compared to naïve mice including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. Further, inhibition of one of the up-regulated miRNAs, miR-199a-3p, in the liver upon S.mansoni infection resulted in reduced levels of collagen and other fibrosis related genes. Our results are consistent with a model where miRNA inhibition influences the clearance or reversion of hepatic stellate cells (HSCs) from a “myofibroblast-like” to an inactivated state. Thus, these results suggest miR-199a-3p inhibitor as potential therapeutic in treatment of liver fibrosis. miRNAs have been shown to be altered in disease process and are present in body fluids in a stable form, indicating that they can be used as novel diagnostic biomarkers. Studies of miRNAs in the circulation revealed that 5 of the mouse miRNAs altered in the liver were also significantly elevated in serum by 12 weeks post-infection. Sequencing of small RNAs from serum confirmed the presence of these miRNAs and further revealed 11 parasite-derived miRNAs that were detectable by 8 weeks post infection. Analysis of host and parasite miRNA abundance by qRTPCR was extended to the serum of patients from low- and high-infection sites in Zimbabwe and Uganda. The host-derived miRNAs failed to distinguish uninfected from infected individuals. However, analysis of three of the parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect infected individuals from low- and high-infection intensity sites with specificity/sensitivity values of 89%/80% and 80%/90%, respectively. Moreover, sequencing of small RNAs from serum revealed that specific tRNA fragments of 29-33 nt exist in mouse serum and occur at a >10 fold higher copy number than known microRNAs. The tRNA fragments appear to be the product of a specific cleavage event near the anti-codon loop, which has previously been associated with oxidative stress inside cells. We detected a clear bias in the abundance of 5’ versus 3’ products of tRNAGly(GCC), indicating specificity in the mechanism of stabilization of these products following cleavage. Our findings suggest that these specific tRNA cleavage products are either generated in, or exported to, serum and are protected against serum RNase activity by protein rather than encapsulation within vesicles. Further work is necessary to investigate the role and potential involvement of tRNA halves in cell-to-cell communication in oxidative stress induced by S.mansoni infection. In summary, this thesis reveals three major findings, which provide an important base for further studies of the role of miRNAs in S.mansoni infection and liver fibrosis. Firstly, it characterises host miRNAs dysregulated during S.mansoni infection in the liver and identifies a miR-199a-3p inhibitor as a potential anti-fibrotic agent. Secondly, it identifies parasite-derived miRNAs as novel markers of S. mansoni infection in the serum of both mice and humans, with the potential to be used with existing techniques to improve S.mansoni diagnosis. Lastly, it identifies and characterises novel tRNA-derived fragments in the serum, whose properties as biomarkers awaits further characterisation.

http://hdl.handle.net/1842/17078

en

The University of Edinburgh

Hoy A.M., Lundie JR, Ivens A, Quintana JF, Nausch N, Forster T, Jones F, Kabatereine NB, Dunne DW, Mutapi F, MacDonald AS, Buck AH (2014). Parasite-derived microRNAs in host serum as novel biomarkers of helminth infection. PLOS Neglected Tropical Diseases. 8(8(2): e2701

Hoy, A.M. & Buck, A.H. (2012). Extracellular small RNAs: what, where, why? Biochem Soc Trans. 40(4); 886-90

S.mansoni

microRNAs

fibrosis

Functional and diagnostic role of microRNAs in Schistosoma mansoni infection

The functional and diagnostic role of microRNAs in Schistosoma mansoni infection

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy