The regulation of PCSK9 and cholesterol homeostasis by microRNAs

Abstract

Low density lipoprotein (LDL) receptor on the cellular membrane is a receptor responsible for uptake of serum LDL-cholesterol into the cell. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the LDL receptor (LDLR), followed by internalization of the complex and degradation of LDLR, and ultimately regulates cholesterol homeostasis. Transcription of PCSK9 is regulated by SREBP (sterol regulatory element binding protein) but regulation by microRNA (miRNA) is still not known. The purpose of this study is to show the existence of miRNA regulating PCSK9 and involving cholesterol homeostasis. In order to discover the posttranscriptional regulation mechanism of PCSK9, bioinformatic algorithms were utilized to discover miRNAs expected to target PCSK9. Overexpression of these miRNAs reduced both PCSK9 mRNA and protein significantly whereas increased LDLR protein. 3？UTR (untranslated region) luciferase reporter assay showed that these miRNAs downregulated PCSK9 expression by binding 3？UTR of PCSK9. Site-directed mutagenesis of predicted seed region in PCSK9 mRNA revealed specific interaction between miRNAs and PCSK9 mRNA. In Dil-LDL uptake assay, overexpression of these miRNA demonstrated a relative increase in Dil-LDL uptake by 70% or greater. In addition, it was indentified that miR-224 regulated the inducible degrader of the LDL receptor (IDOL) by 3？UTR reporter assay. These indicate that miRNAs may be yet another mechanism involved in cholesterol homeostasis by posttranscriptional regulation of PCSK9 or IDOL and that utilization of miRNA may be a method of treatment of hypercholesterolemia.