Control of cholesterol metabolism and plasma high-density lipoprotein levels by microRNA-144 - PubMed (original) (raw)

. 2013 Jun 7;112(12):1592-601.

doi: 10.1161/CIRCRESAHA.112.300626. Epub 2013 Mar 21.

Noemi Rotllan, Alexander V Vlassov, Alberto Dávalos, Mu Li, Leigh Goedeke, Juan F Aranda, Daniel Cirera-Salinas, Elisa Araldi, Alessandro Salerno, Amarylis Wanschel, Jiri Zavadil, Antonio Castrillo, Jungsu Kim, Yajaira Suárez, Carlos Fernández-Hernando

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Control of cholesterol metabolism and plasma high-density lipoprotein levels by microRNA-144

Cristina M Ramírez et al. Circ Res. 2013.

Abstract

Rationale: Foam cell formation because of excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including adenosine triphosphate-binding cassette transporter A1 (ABCA1) and adenosine triphosphate-binding cassette transporter G1 (ABCG1). ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters.

Objective: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists.

Methods and results: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed an miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to apolipoproteinA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 seems to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver.

Conclusions: miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression and modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease.

Keywords: ABCA1; cardiovascular research; cholesterol efflux; cholesterol homeostatis; high-density lipoprotein; lipids and lipoprotein metabolism; microRNAs.

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Figures

Figure 1

Figure 1. miR-144 expression is up-regulated in LXR-treated macrophages and has multiple lipid-related predicted target genes

(A) miRNA array analysis of miRNAs up-regulated (red) or down-regulated (green) from peritoneal macrophages treated with T090 (n=4). (B to D) Gene ontology analysis of the miR-144 predicted target genes using Panther software. (E to F) Protein-protein interaction analysis of the miR-144 predicted targets using String software and Navigator 2.2.

Figure 2

Figure 2. LXR induces miR-144 transcriptional expression

(A) qRT-PCR analysis of miR-144 expression in mouse peritoneal macrophages (MØ, left panel), THP-1 cells (middle panel) and Huh-7 cells (right panel) treated with T090. (B) qRT-PCR analysis of pri-miR-144/451 expression in mouse peritoneal macrophages (left panel), THP-1 cells (middle panel) and Huh-7 cells (right panel) treated with T090. (C and D) qRT-PCR analysis of miR-144 (C) and pri-miR-144/451 (D) expression from wild-type (WT) and LXRα,β-/- macrophages treated with T090. (E) miR-144/451 promoter-luciferase reporter activity in Huh-7 cells treated with T090. Data are expressed as relative expression and correspond to mean±SEM of 3 independent experiments. *Significantly different from cells without treatment (Ctrl), _P_≤0.05.

Figure 3

Figure 3. miR-144 levels regulate ABCA1 expression

(A) qRT-PCR analysis of ABCA1, and ABCG1 expression in mouse peritoneal macrophages transfected with miR-144 mimics. Data are expressed as relative expression and correspond to the mean±SEM from 3 independent experiments. *Significantly different from cells transfected with control mimic (Con-miR), *P ≤ 0.05. (B and C) Western blot analysis of ABCA1, ABCG1 and HSP90 in mouse peritoneal macrophages and THP-1 cells transfected with (B) Con-miR or miR-144, or (C) control inhibitor (Con-inh) or anti-miR-144 in the presence or absence of Ac-LDL (Ac) or T090. Data correspond to a representative experiment among three that gave similar results. Values of the band densitometry analysis are shown. (D) Luciferase-reporter activity in COS-7 cells transfected with Con-miR or miR-144 (40 nM) mimic and human Abca1 3’UTR containing the indicated point mutations (PM) in the miR-144 target sites. Data are expressed as mean % of 3’UTR activity of Con-miR ±SEM and are representative of ≥3 experiments. *Significantly different from cells cotransfected with Con-miR and wild-type (WT) 3’UTR. _P_≤ 0.05. (E) Human Abca1 3’UTR containing the indicated point mutations (PM) in the miR-144 target sites.

Figure 4

Figure 4. miR-33 and miR-144 cooperate to regulate ABCA1 expression

(A) Western blot analysis of Huh-7 cells transfected with 5nM non-targeting control mimic (Con-miR), miR-33, miR-144 and miR-33/miR-144 (upper panel). Data correspond to a representative experiment among three that gave similar results. Quantification of specific bands was conducted by densitometric analysis and is shown as the bottom of each band. (B) Activity of the luciferase reporter construct fused to the 3’UTR of Abca1 in COS-7 cells. Cells were transfected with Con-miR or miR-33, or miR-144, or miR-33 and miR-144 together. All microRNA constructs were transfected at 5νM final concentration. Relative luciferase activity is presented and data are the mean±SEM of 3 independent experiments in triplicate. *Significantly different from cells cotransfected with Con-miR and Abca1 3’UTR. _P_≤ 0.05. # Significantly different from cells cotransfected with miR-33 or miR-144 and Abca1 3’UTR. _P_≤ 0.05. (C) Total cholesterol efflux to ApoA1 in Huh-7 cells stimulated with T090 and expressing control miR (Con-miR), miR-33, miR-144 or miR-33 and miR-144. Data are the mean±SEM of 2 independent experiments performed in triplicate.

Figure 5

Figure 5. Modulation of miR-144 regulates macrophage cholesterol efflux

(A) Total cholesterol efflux to ApoA1 in J774 mouse macrophages stimulated with Ac-LDL (Ac) or T090 and expressing control miR (Con-miR) or miR-144. Upper panel shows the ABCA1 protein expression assessed by western blotting in the same conditions. (B) Total cholesterol efflux to ApoA1 in J774 mouse macrophages stimulated with Ac-LDL (Ac) or T090 and expressing control inhibitor (Con-inh) or Inh-miR-144. Upper panel shows ABCA1 protein expression assessed by western blotting in the same conditions. Data are the mean±SEM of 3 independent experiments performed in triplicate. *Significantly different from cells transfected with Con-miR (A) or Con-inh (B). _P_≤ 0.05.

Figure 6

Figure 6. Delivery of miR-144 oligonucleotides significantly reduces liver ABCA1 expression and HDL levels in mice

(A) qRT-PCR analysis of ABCA1 and ABCG1 expression from mouse livers treated with miR-144 mimics. Data are the mean±SEM from 6 mice per group. *Significantly different from mice injected with non-targeting control mimic (Con-miR) conjugated particles. _P_≤ 0.05. (B) Analysis of hepatic gene expression 6 days after injection of Con-miR or miR-144. Western blot analysis of liver tissue from three representative mice per group. Quantification analysis is shown in the right panel. Data are the mean±SEM from 6 mice per group. *Significantly different from mice injected with Con-miR conjugated particles. _P_≤ 0.05. (C) Total cholesterol (left panel), HDL cholesterol (middle panel) and TG (right panel) levels in mice treated with Con-miR or miR-144 mimics (n=6). *Significantly different from mice injected with non-targeting Con-miR conjugated particles. _P_≤ 0.05. (D) Lipoprotein profile from mice treated with Con-miR or miR-144 mimics (n=6). Cholesterol distribution across plasma fractions was analyzed by FPLC.

Figure 7

Figure 7. Inhibition of miR-144 increases hepatic ABCA1 expression and raises HDL plasma levels

(A) qRT-PCR analysis of miR-144 levels from mouse livers treated with miR-144 inhibitor (Inh-miR-144) conjugated particles. Data are expressed as relative expression and correspond to the mean±SEM from 6 mice per group. *Significantly different from mice injected with control inhibitor (Con-inh), #significantly different from mice untreated and injected with Con-Inh or treated with T090 and injected with Inh-miR-144 compared with mice treated with T090 and injected with Con-Inh, _P_≤0.05. (B) qRT-PCR analysis of hepatic ABCA1 gene expression mouse liver treated or untreated with T090 and control inhibitor (Con-inh) or Inh-miR-144. Significantly different from mice injected with Con-inh, _P_≤0.05. (C) Western blot analysis of ABCA1 expression from liver tissue of mice injected with Con-inh or Inh-miR-144 and treated with vehicle (upper panel) or T090 (bottom panel). Quantification of specific bands was conducted by densitometric analysis and is shown at the bottom of each band. (D) Total cholesterol, HDL cholesterol and TG plasma levels in mice injected with Con-miR or miR-144 mimics and treated or not with T090 (n=6). Data are the mean ± SEM from 6 mice per group. Significantly different from mice injected with Con-inh, *P ≤ 0.05. (E) Lipoprotein profile analysis from mice treated with Con-inh or Inh-miR-144 (n=6). Cholesterol distribution across plasma fractions was analyzed by FPLC.

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