Abstract 63: Post-transcriptional Control of the LDLR Pathway by Cholesterol-responsive microRNAs.

Abstract

Despite the success of statins to reduce levels of LDL-cholesterol (LDL-C), cardiovascular disease remains the leading cause of mortality in westernized countries. PCSK9 inhibition is emerging as an exciting new therapy to further lower LDL and holds tremendous promise to reduce the significant residual cardiovascular risk in many individuals. Recent advances from our group and others have shown that microRNAs (miRNAs) have emerged as critical components in the regulation of cholesterol metabolism. In addition to miR-33, which we discovered as a key regulator of metabolic programs including cholesterol and fatty acid homeostasis, we identified two new cholesterol responsive miRNAs, miR-520d-5p (miR-520d) and miR-224. Here we show that miR-520d and miR-224 target the 3’UTR of both PCSK9 and Idol, “accessory” proteins that regulate cell surface expression of the LDL receptor (LDLR). Moreover, miR-520d and miR-224 simultaneously target the 3’UTR of HMGCR, which codes for the limiting enzyme in cholesterol synthesis and is the target of statins. In mouse and human cells, the overexpression of miR-520d inhibits PCSK9, Idol and HMGCR mRNA levels by 30-50% compared to a control miRNA, while overexpression of miR-224 results in an even more potent 75% decrease in PCSK9, Idol and HMGCR mRNA compared to controls. In human hepatocytes (HepG2/Huh7) and mouse peritoneal macrophages, miR-520d and miR-224 overexpression caused a repression of PCSK9, Idol and HMGCR protein levels, and a decrease in PCSK9 protein secretion. Importantly, these miR-224 and miR-520d-induced changes in PCSK9, Idol and HMGCR expression were associated with a concomitant increase in LDLR protein expression, increased cell surface expression of an LDLR-GFP fusion protein and enhanced LDL binding to the cell surface. Notably, these increases in LDLR were above and beyond those achieved with statin treatment. Together, these findings suggest a role of miR-224 and miR-520d in contributing to the post-transcriptional regulation of LDLR expression, and represent the first evidence of miRNA control of the LDLR pathway. This highlights the therapeutic promise of miR-224/miR-520d for reducing LDL cholesterol beyond statin therapy alone.