Investigating the functional roles of microRNA-29b and microRNA-146a in prion diseases
AffiliationBiochemistry and Molecular Biology
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr Wenting Zhao
Neurodegenerative diseases such as Alzheimer’s disease and prion disease are closely related with specific gene and protein dysfunction. Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are characterized by the structural transformation of the cellular prion protein (PrPC) to the disease associated isoform (PrPSc). One hallmark of Alzheimer’s disease is the accumulation of beta amyloid (Aβ) plaques in the brain, resulting from the pathological cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1) and the γ-secretase complex. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b and miR-146a, have been detected in various biological fluids and tissues from prion disease and Alzheimer’s disease patients, as well as in cell and animal models. These miRNAs could be potential diagnostic biomarkers of these diseases, suggesting that investigating the miRNA functional roles and miRNA-target regulation pathways will improve our understanding of the disease regulation networks. The first aspect of the thesis utilized CRISPR/Cas9 gene editing to knockdown miR-29b and miR-146a respectively in a number of cell lines, and cell clones with stable miRNA knockdown were generated. Off-target analysis of the cell clones revealed the high specificity of CRISPR/Cas9 editing of miRNAs. Common and distinct pathways and novel targets of miR-29b were also identified in two cell lines using transcriptome profiling, and potential miR-29b targets in Alzheimer’s and prion diseases were revealed. In the second aspect of the thesis, miR-29b was shown to positively regulate prion protein levels in both miR-29b stable knockdown cell clones and miR-29b overexpressed cells. This regulation is not mediated through miR-29b target SP1 or potential target PPP2CA, which can interact with prion protein or was implicated in prion pathogenesis. miR-29b could further affect PrPSc generation through regulating prion protein levels and potentially affect prion progression. miR-29b was also revealed to regulate APP and BACE1, the two key proteins in Alzheimer’s disease, in in vitro models. Lastly, the dual roles of miR-146a in regulating prion protein and inflammatory pathways were revealed in prion disease. miR-146a can upregulate prion protein levels in both overexpressed and stably downregulated cell models, as well as in miR-146a transgenic mice generated using CRISPR/Cas9 gene editing. miR-146a overexpression also resulted in the decreased formation of PrPSc in prion cell models. The miR-29b/miR-146a-PrP-PrPSc pathways possibly share a similar mechanism involving the interaction of prion protein with Argonaute protein – the key component of miRNA induced silencing complex (miRISC). Prion protein was demonstrated to be a direct target of miR-146a. miR-146a can also target inflammatory regulator TRAF6 in both prion infected cell models and in miR-146a transgenic mice. The findings from this thesis have important implications for the comprehensive understanding of prion disease pathogenesis. The miR-29b/miR-146a-PrP-PrPSc pathways and miR-146a mediated inflammatory pathway are added to the regulation network of prion disease. miRNAs represent novel regulators in prion diseases and other neurodegenerative disorders and hold promise to be future therapeutics to cure prion disease.
KeywordsmicroRNA; miR-29b; miR-146a; CRISPR/Cas9; prion disease; prion protein; Alzheimer's disease; RNA-sequencing
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