Abstract
Micro-RNA (miRNA) sequences are short non-coding RNAs which play key roles in epigenetic regulation of gene transcription and translation. Significant changes in miRNA expression occur in macrophage 'foam' cells, laden with cholesterol and cholesteryl ester, which contribute not only to macrophage phenotype and inflammatory status, but also to novel pathways which may influence the development of atherosclerotic lesions, the principle underlying cause of coronary heart disease. An initial microarray identified miRNA sequences miR-150-5p and miR-let-7d-5p as differentially expressed in phorbol ester-induced THP-1 macrophages after cholesterol-loading. Upon validation of this microarray, treatment of THP-1 macrophages with acetylated low-density lipoprotein(AcLDL), induced a 1. 4-fold increase in miR-let-7d-5p expression. Modest increases in miR-let-7d-5p expression, when compared to vehicle control-treated macrophages, were also observed in THP-1 macrophages in response to native LDL (nLDL) and LDL oxidised by hypochlorous acid (HOCI-oxLDL). No change in miR-150-5p expression was found in response to any of the native or modified LDL treatments under investigation.
The role of miR-let-7d-5p expression in macrophage 'foam' cells was examined through delivery of synthetic miR-let-7d-5p mimics and inhibitors. A decrease in expression of the miR-let-7d-5p target, high-mobility AT-hook 2 (HMGA2), and cellular cholesterol mass was observed after delivery of miR-let-7d-5p mimics. Conversely, delivery of miRlet- 7d-5p inhibitors increased expression of HMGA2 and enhanced cholesterol mass. To uncover the underlying mechanism driving these changes, various pathways that regulate cellular cholesterol homeostasis were investigated. Delivery of miR-let-7d-5p inhibitors to foam cells resulted in increased endogenous cholesterol and cholesteryl ester biosynthesis. This pool of cholesterol could readily undergo efflux to apolipoprotein
A-l (ApoA-1). When HMGA2 expression was inhibited directly by short-interfering RNA (siRNA), a decrease in cholesterol mass was observed in macrophage foam cells. Together these results suggest that miR-let-7d-5p regulates cellular cholesterol levels, in part, through regulation of HMGA2 and plays a protective role in the formation of foam cells.
Changes in global miRNA expression were profiled in AcLDL-treated macrophages. Using target prediction and biological pathway analysis software, multiple pathways, such as fatty acid synthesis, poorly described in the context of foam cell formation were found to be regulated by differentially expressed miRNA. Further investigation of each pathway may uncover additional novel miRNA and gene targets which exhibit atheroprotective functions.
| Date of Award | 2023 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Sponsors | Heart Research UK |
| Supervisor | Annette Graham (Supervisor) & Yvonne Dempsie (Supervisor) |