MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis

Abstract

Proper coordination of cholesterol biosynthesis and trafficking is essential to human health. The sterol regulatory element-binding proteins (SREBPs) are key transcription regulators of genes involved in cholesterol biosynthesis and uptake. We show here that microRNAs (miR-33a/b) embedded within introns of the SREBP genes target the adenosine triphosphate-binding cassette transporter A1 (ABCA1), an important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport, for posttranscriptional repression. Antisense inhibition of miR-33 in mouse and human cell lines causes up-regulation of ABCA1 expression and increased cholesterol efflux, and injection of mice on a western-type diet with locked nucleic acid-antisense oligonucleotides results in elevated plasma HDL. Our findings indicate that miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis and suggest that miR-33 may represent a therapeutic target for ameliorating cardiometabolic diseases.

Figure 1

SREBPs are host genes to conserved intronic miRNAs, miR-33a/b, which are co-expressed with SREBPs. Human SREBP-1 (A) and SREBP-2 (B) genes harbor related intronic miRNAs (miR-33b and miR-33a, respectively). The sequences encoding the pri-miRNAs are shown, with the mature miRNA sequences highlighted in red. C-E Expression profile of miR-33a/b and SREBP host genes in selected human tissues. Error bars represent experimental S.D.

Figure 2

MiR-33 regulates the cholesterol transporter ABCA1. A. Transfection of siRNA against Drosha and Dicer results in increased protein levels of ABCA1 in human HepG2 hepatoma cells (top row), human IMR-90 fibroblasts (middle row), and the mouse macrophage cell line J774 (bottom row). B. Introduction of miR-33a or miR-33b precursors into the three cell lines represses ABCA1 protein levels. C. MiR-33a/b antisense oligonucleotides (A33a/b) increase ABCA1 levels. E. Insertion of the ABCA1 3’UTR fragment into a Luciferase reporter results in decreased Luciferase expression in HEK293 cells. F. Co-transfection with wild-type, but not mutated, miR-33a/b precursors causes further repression of the Luciferase-ABCA1 3’UTR reporter. A scrambled sequence was used as precursor control (PC). G. MiR-33a/b antisense oligonucleotides result in de-repression of the Luciferase-ABCA1 3’UTR reporter. A scrambled sequence was used as antisense control (AC). Error bars represent S.D. * denotes p<0.05.

Figure 3

Reciprocal regulation by cholesterol and SREBP/miR-33. Lovastatin/β-cyclodextrin-mediated depletion of cholesterol in J774 mouse macrophages results in increased expression of (A) SREBP-2 and (B) miR-33a, and (C) in decreased ABCA1 protein levels. D. Cholesterol-depleted J774 mouse macrophages exhibit upregulation of ABCA1 protein in response to miR-33a antisense inhibition. E. Transfection of J774 macrophages cultured in the presence of serum/cholesterol with miR-33a precursor results in decreased levels of ABCA1. F. Introduction of miR-33a antisense oligonucleotides into J774 macrophages loaded with radio-labeled cholesterol results in increased cholesterol efflux, whereas miR-33a precursors inhibit cholesterol efflux. Error bars represent S.D. ** denotes p<0.01.

Figure 4

Regulation of HDL by miR-33a in vivo. A. Tail vein injection of mice fed a western-type diet with Locked Nucleic Acid (LNA)-antisense oligonucleotides directed against mouse miR-33a results in elevated total serum cholesterol. B. FPLC analysis of pooled serum from 5 mice on a western-type diet treated revealed an increase in the magnitude of the HDL-cholesterol peak in the LNA-antimiR-treated animals. Elution of lipoprotein standards is indicated by the labels. Plasma triglycerides (C), glucose (D), AST (E), and ALT (F) were unaffected by the treatments. Error bars represent s.e.m. ** denotes p<0.01.