Abstract
Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3′-UTR) of human microRNA library screen, we identified microRNAs targeting the 3′ untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3′-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.
Highlights
Cholesterol homeostasis is essential for human health, and its dysregulation results in cardiometabolic diseases, including atherosclerosis
To identify putative miRNA candidates controlling the expression of human proprotein convertase subtilisin/kexin type 9 (PCSK9), we conducted an unbiased screen for human miRNAs, which could modulate the activity of a firefly luciferase reporter vector harboring the 3′-UTR of PCSK9
100 microRNAs with composite scores of
Summary
Cholesterol homeostasis is essential for human health, and its dysregulation results in cardiometabolic diseases, including atherosclerosis. Hepatocytes play a major role in the regulation of systemic cholesterol homeostasis through the assembly and secretion of plasma lipoproteins, as well as their eventual clearance through the low-density lipoprotein (LDL) receptor. This highly synchronized process is achieved through a complex network of regulatory and counter-regulatory mechanisms that function at both the transcriptional and post-transcriptional levels. Imbalances in hepatic cholesterol synthesis and uptake can result in elevated levels of LDL cholesterol (LDL-C), a strong, independent risk factor for atherosclerotic cardiovascular disease (ASCVD). The regulatory networks that maintain cholesterol homeostasis have been the subject of intense research efforts for over 30 years, and discoveries in this area have had major impacts on the management of cardiovascular disease
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