Abstract

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3′-untranslated region (3′-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3′-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr+/− mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

Highlights

  • Elevated levels of apoB-containing lipoproteins, low-density lipoprotein (LDL), are a major risk factor for atherosclerotic cardiovascular diseases (CVDs)

  • We demonstrate that miR-224 and miR-520d target both PCSK9 and inducible degrader of the LDLR (IDOL), chaperone proteins that promote the degradation of LDL receptor (LDLR), as well as targeting HMG-CoA reductase (HMGCR), an enzyme whose inhibition increases LDLR expression

  • Using miRNA mimics, we found that miR-224 and miR-520d dose-dependently reduced the activity of the 3′-untranslated region (3′-UTR) of PCSK9, IDOL, and HMGCR by 50–80% compared to a control miRNA mimic (Figures 1B–D)

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Summary

INTRODUCTION

Elevated levels of apoB-containing lipoproteins, low-density lipoprotein (LDL), are a major risk factor for atherosclerotic cardiovascular diseases (CVDs). PCSK9 and IDOL have been shown to function in non-redundant complementary pathways and could be targeted in concert to prevent LDLR degradation [10] Together, these discoveries highlight how expanding our understanding of how the LDLR is regulated at the posttranscriptional level can enable the further discovery of novel classes of cholesterol-lowering agents that go beyond transcriptional activation of LDLR. Functional studies in hepatocytes revealed that miR-224 and miR-520d reduce mRNA and proteins of PCSK9, IDOL, and HMGCR, resulting in increased LDLR cell surface expression and binding of LDL. These data demonstrate roles for miR-224 and miR-520d in controlling LDLR cell surface expression and LDL homeostasis

EXPERIMENTAL PROCEDURES
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DATA AVAILABILITY STATEMENT
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