MiR-33 contributes to the regulation of cholesterol homeostasis

Abstract

Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element-binding factor-2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate-binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux.

Fig. 1

Regulation of miR-33 is inversely correlated with cellular cholesterol levels. (A) Schematic representation of the SREBF2 gene locus, demonstrating the miR-33 coding sequence within intron 16 and its conservation among species (reference: the mouse genome). (B) Quantitative real-time fluorescence polymerase chain reaction (QRT-PCR) analysis of miR-33, SREBF2, SREBF1, and ABCA1 expression in control macrophages or macrophages loaded with cholesterol by AcLDL treatment or depleted of cholesterol by statin treatment. *P ≤ 0.05. (C) QRT-PCR analysis of miR-33 in liver of C57BL6 mice (n = 5 per group) fed a chow diet, high-fat diet (HFD), or rosuvastatin-supplemented diet (statin) (D) QRT-PCR analysis of miR-33 in liver and peritoneal macrophages (PMø) from Ldlr^−/− mice fed a chow or HFD for 12 weeks (n = 6 per group). **P ≤ 0.01. (E) QRT-PCR analysis of miR-33 tissue expression in C57BL6 mice (n = 3). In (B) to (E), data are the mean ± SEM and are representative of ≥3 experiments.

Fig. 2

Posttranscriptional regulation of ABCA1, ABCG1, and NPC1 by miR-33. (A to C) Western blot analysis of ABCA1, ABCG1, NPC1, and HSP90 in primary mouse macrophages transfected with (A) a control (Con) miR or miR-33, (B) increasing concentrations of control miR or miR-33, or (C) control miR or miR-33 in the presence or absence of a control inhibitor or anti-miR-33. Ac, AcLDL; T, T0901317. (D) Expression of ABCA1, ABCG1, and NPC1 expression in human (THP-1 and HepG2) and mouse (HEPA) cells of the indicated origin transfected with control miR or miR-33. (E) Expression of ABCA1, ABCG1, and NPC1 in human (THP-1 and HepG2) and mouse (peritoneal Mø and HEPA) macrophages and hepatic cells transfected with control inhibitor or anti-miR-33. Data are the mean ± SEM and are representative of ≥3 experiments.

Fig. 3

miR-33 specifically targets the 3′UTR of ABCA1, NPC1, and mouse, but not human, ABCG1. (A) Activity of luciferase reporter constructs fused to the 3′UTR of human ABCA1, human NPC1, and mouse or human ABCG1 in HEK293 cells transfected with increasing concentrations (0, 5, 50, or 500 ng) of control miR or miR-33. (B to D) Luciferase reporter activity in COS-7 cells transfected with control miR or miR-33 of the (B) hABCA1, (C) hNPC1, and (D) mABCG1 3′UTRs containing the indicated point mutations (PM) in the miR-33 target sites. Data are expressed as mean % of 3′UTR activity of control miR ± SEM and are representative of ≥3 experiments. *P ≤ 0.05.

Fig. 4

Modulation of miR-33 regulates cellular cholesterol efflux and serum HDL levels. (A and B) Cholesterol efflux to (A) apoA1 and (B) HDL in mouse J774 and human THP-1 macrophages stimulated with AcLDL (Ac) or T0901317 (T) and expressing a control miR or miR-33. (C) Cholesterol efflux to apoA1 in mouse J774 and human THP-1 macrophages stimulated with AcLDL or T0901317 and expressing a control inhibitor or anti-miR-33. (D) Analysis of hepatic gene expression 6 days after infection with the control, miR-33, or anti-miR-33 lentiviruses. Western blots are of liver tissue from three representative mice per treatment. (E) Plasma HDL levels in mice infected with the control, miR-33, or anti-miR-33 lentiviruses. (n = 6). (F) Percentage change in serum HDL 6 days after lentiviral delivery of miR-33 or anti-miR-33 (relative to control). Data are the mean ± SEM. *P ≤ 0.05