microRNA, Dental Pulp Cell Mineralisation and Therapeutic Epigenetic Modification
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Kearney, Michaela, microRNA, Dental Pulp Cell Mineralisation and Therapeutic Epigenetic Modification, Trinity College Dublin, School of Dental Sciences, Dental Science, 2022Download Item:
Abstract:
Current clinical approaches to the management of deep caries and symptomatic pulpitis are destructive and costly, generally involving removal of the entire pulp as part of a root canal treatment (RCT) procedure. Contemporary restorative materials aimed at preserving the pulp and stimulating its natural reparative abilities have demonstrated improved success rates; however, there remains limitations to their use. In order to address these shortcomings and improve on current treatments, a deeper understanding of the molecular mechanisms regulating reparative processes in the pulp is critical. Notably, the role of epigenetic modifications such as histone modifications, DNA methylation and, more recently, miRNAs in dental pulp cell (DPC) proliferation and differentiation has been the subject of intense research within the field of regenerative endodontics. It is possible that these mechanisms can be targeted pharmacologically to induce dental pulp repair and regeneration, thus avoiding the need for RCT. This thesis aimed to investigate the expression of miRNAs during epigenetically-mediated DPC mineralisation, in order to identify potential therapeutic targets for use in regenerative endodontics.
In Chapter 3, the relationship between epigenetic modifications and miRNAs in mineralising DPCs was investigated by exposing mineralising DPCs to a pan-HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-Aza-2?-deoxycytidine (5-AZA-CdR). The effects on the expression profiles of three candidate miRNAs was investigated using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). These miRNAs - miR-135b-5p, miR-320-3p and mir-29b-1 - were selected based on previous literature suggesting links to mineralisation or stem cell differentiation processes, and indeed were shown to be differentially regulated during epigenetically-enhanced mineralisation. Furthermore, both SAHA and 5-AZA-CdR were determined to have a positive effect on rodent DPC mineralisation and a negative effect on proliferation.
Chapter 4 expanded on these findings to include RNA sequencing (RNAseq) analysis of mineralising rodent DPCs, with and without supplementary SAHA and 5-AZA-CdR, to characterise the global miRNA expression profile after 4 days in culture. This study is the first to use RNAseq to not only investigate miRNA expression in mineralising DPCs, but also to characterise the relationship between miRNAs and epigenetic-modifying agents during DPC mineralisation. Gene Ontology (GO) enrichment and pathway analysis were carried out to identify miRNAs which may have a role in epigenetically-mediated DPC mineralisation. Four miRNAs - miR-182, miR-200b-3p, miR-221-5p and miR-205 - were selected for further investigation using qRT-PCR, which established differential expression both during mineralisation and upon exposure to SAHA and 5-AZA-CdR.
As the ultimate goal of this study was to identify potential therapeutic targets for use in regenerative endodontics, translating the experimental work to human DPCs (hDPCs) represented a fundamental stage of experimentation. In Chapter 5, the effects of SAHA and 5-AZA-CdR on the morphology, proliferation and mineralisation potential of hDPCs was established. Subsequently, qRT-PCR analysis of miR-182, miR-200b-3p, miR-221-5p and miR-205 was carried out, which revealed significantly altered expression in the experimental groups. One of these - miR-205 - was selected for further investigation using a miRNA Power Inhibitor. Ultimately, the extent of miR-205 knockdown was unclear, as was the effect on mineralisation; however, it was apparent that there was a relationship between histone acetylation and miR-205 during DPC mineralisation.
Overall, the findings presented in this thesis describe a clear interaction between miRNAs and epigenetic modifications in mineralising DPCs and have identified a number of potentially important miRNAs which represent attractive targets for further investigation in epigenetically-mediated mineralisation, highlighting exciting opportunities for the development of novel therapeutic and diagnostic tools.
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https://tcdlocalportal.tcd.ie/pls/EnterApex/f?p=800:71:0::::P71_USERNAME:MIKEARNEDescription:
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Author: Kearney, Michaela
Advisor:
Duncan, HalPublisher:
Trinity College Dublin. School of Dental Sciences. Discipline of Dental ScienceType of material:
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