An agomir of miR-144-3p accelerates plaque formation through impairing reverse cholesterol transport and promoting pro-inflammatory cytokine production

Abstract

Aims: ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to lipid-poor apolipoproteins, which then form nascent HDL, a key step in the mechanism of reverse cholesterol transport (RCT). While a series of microRNAs (miRNAs) have been identified as potent post-transcriptional regulators of lipid metabolism, their effects on ABCA1 function and associated mechanisms remain unclear.

Methods and results: ABCA1 was identified as a potential target of miR-144-3p, based on the results of bioinformatic analysis and the luciferase reporter assay, and downregulated after transfection of cells with miR-144-3p mimics, as observed with real-time PCR and western blot. Moreover, miR-144-3p mimics (agomir) enhanced the expression of inflammatory factors, including IL-1β, IL-6 and TNF-α, in vivo and in vitro, inhibited cholesterol efflux in THP-1 macrophage-derived foam cells, decreased HDL-C circulation and impaired RCT in vivo, resulting in accelerated pathological progression of atherosclerosis in apoE-/- mice. Clinical studies additionally revealed a positive correlation of circulating miR-144-3p with serum CK, CK-MB, LDH and AST in subjects with AMI.

Conclusions: Our findings clearly indicate that miR-144-3p is essential for the regulation of cholesterol homeostasis and inflammatory reactions, supporting its utility as a potential therapeutic target of atherosclerosis and a promising diagnostic biomarker of AMI.

Figure 1. Effects of miR-144-3p on ABCA1 expression, cholesterol homeostasis and inflammation.

(A) hsa-miR-144-3p directly targeted the 3′-untranslated region (3′UTR) of ABCA1. (a) Sequence alignment of the human hsa-miR-144-3p mature sequence with the binding sites of human ABCA1 3′UTR. (b) Changes in luciferase activity induced by the hsa-miR-144-3p mimic in binding to ABCA1 3′-UTR (n = 3; *p<0.01 vs. 293T cells transfected with negative control miRNAs). (B) miR-144-3p expression levels in various cells were analyzed by qRT-PCR. (C) THP-1 macrophages and primary macrophages were exposed to 50 µg/mL of oxidized LDL for 48 h and then treated with 50 nM miRNA mimics, as indicated, for 48 h. ABCA1 mRNA and protein levels were measured using qRT-PCR and western blot analysis, respectively. (D) THP-1 cells were differentiated for 72 h with 100 nM PMA and then macrophages were treated with 50 nM miRNA mimics for 48 h. Then, the cells were labeled with 0.2 µCi/mL [³H] cholesterol and cholesterol-loaded using 50 µg/ml oxidized LDL. The percentage of cholesterol efflux to (a) apoAI and (b) HDL was analyzed with the liquid scintillation counting assay. (E) THP-1 cells were differentiated for 72 h with 100 nM PMA and then macrophages were transformed into foam cells by incubation in the presence of 50 µg/mL of oxidized LDL for 48 h. THP-1 macrophage-derived foam cells were treated with 50 nM miRNA mimics for 48 h, as indicated, and the cholesterol content measured using HPLC. (F) THP-1 cells were differentiated for 72 h with 100 nM PMA and then macrophages were transformed into foam cells by incubation in the presence of 50 µg/mL of oxidized LDL for 48 h. THP-1 macrophage-derived foam cells were treated with 50 nM miRNA mimics, as indicated, for 48 h, and inflammatory cytokines in the medium measured with ELISA. All results are expressed as mean values ± S.D. of three independent experiments, each performed in triplicate. *p<0.05 vs. control group.

Figure 2. Effects of miR-144-3p on RCT and hepatic lipid deposition.

(A) After 12 weeks of the indicated treatment, apoE^−/− mice were injected subcutaneously with ³H-cholesterol-labeled, Ac-LDL-loaded bone marrow-derived macrophages. Data are expressed as a percentage of the ³H-cholesterol tracer relative to that of total cpm tracer injected ± S.D.; n = 5. * p<0.05 vs. control group. (a) Time-course of ³H-cholesterol distribution in plasma. (b) Hepatic ³H-cholesterol tracer levels after 48 h. (c) Fecal ³H-cholesterol tracer levels. Feces were collected continuously from 0 to 48 h post-injection. (B) Liver cryosections were stained with Oil Red O and hematoxylin. Data are presented as mean values ± S.D.; n = 10. * p<0.05 vs. control group.

Figure 3. Effects of miR-144-3p on atherosclerosis initiation and development in apoE^−/− mice.

(A) (a) Representative staining of aortic sinus with Oil Red O. (b) Representative staining of en face aorta with Oil Red O. (c) Lesions in aortic valves were analyzed in apoE^−/− mice. (d) En face lesions were analyzed in apoE^−/− mice. Data are presented as mean values ± S.D.; n = 10. * p<0.05 vs. control group. (B) Protein levels in tissues of apoE^−/− mice were analyzed using western blot analysis. Data are presented as mean values ± S.D.; n = 5. * p<0.05 vs. control group. (C) (a) Cryo-sections of aortic valves from apoE^−/− mice were immunohistochemically stained for the macrophage marker, CD68. (b) The integral optical density of CD68 in aortic valve cryo-sections from apoE^−/− mice was analyzed. Data are presented as mean values ± S.D.; n = 10. * p<0.05 vs. control group.