- Email: [email protected]
microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2
Accepted Manuscript microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2 Jing Li, Suhua Zhang PII:
S0006-291X(16)30804-X
DOI:
10.1016/j.bbrc.2016.05.096
Reference:
YBBRC 35846
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 27 April 2016 Accepted Date: 19 May 2016
Please cite this article as: J. Li, S. Zhang, microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.05.096. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2
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Jing Li1 and Suhua Zhang2,* Department of Geratory, Linzi District People’s Hospital of Zibo City, Zibo,
Shandong, China
Department of Health, Qilu Hospital of Shandong University (Qingdao), Qingdao
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2
*
Corresponding author.
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City, Qingdao, China
Qilu Hospital of Shandong University (Qingdao), NO.758, Hefei Road, Qingdao
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266000, China Tel: +86-18561811017 Fax: +86-053296599
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Email: [email protected]
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Abstract
Transformation of macrophages into foam cells plays a critical role in the
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pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 µg/ml oxidized
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low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in
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THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control
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cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small
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interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of
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miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive
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ACCEPTED MANUSCRIPT effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to
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prevent macrophage foam cell formation in atherosclerosis.
Key words: atherosclerosis; cholesterol efflux; foam cell formation; therapeutic
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target.
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Introduction
Atherosclerosis, which is characterized by inflammatory responses and accumulation
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of lipids and cellular and fibrous elements in the arteries, is a leading cause of heart disease and stroke [1,2]. Generation of lipid-laden macrophages, or macrophage foam cells, is a hallmark feature of early stage atherosclerotic lesions [3]. The
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transformation of macrophages into foam cells is causally linked to unbalanced
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cholesterol influx and efflux [4]. Recruited macrophages in the vessel intima readily ingest modified low density lipoproteins (LDL), especially oxidized forms (oxLDL), through scavenger receptors (SRs) such as CD36 and SR-A [5]. Excessive uptake of modified lipoproteins results in the formation of cholesterol-rich foam cells.
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Cholesterol efflux is critical in maintaining lipid homeostasis and preventing macrophage foam cell formation [6]. ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) are known to participate in macrophage cholesterol efflux.
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ABCA1 is implicated in the removal of free cholesterol to lipid-poor apolipoproteins,
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most notably apolipoprotein A1 (apoA1), while ABCG1 is responsible for transporting cholesterol to high-density lipoprotein (HDL) [7,8]. The expression of ABCA1 and ABCG1 is regulated by proliferator-activated receptor gamma (PPARγ)and liver X receptor alpha (LXRα)-dependent pathways [9]. Suppression of macrophage foam cell formation via alteration of cholesterol homeostasis has been suggested as an important strategy for prevention of atherosclerosis [10].
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ACCEPTED MANUSCRIPT microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that play important roles in a wide range of biological processes such as proliferation, differentiation, inflammation, lipid metabolism, and tumorigenesis [11]. miRNAs
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typically repress gene expression through binding to the 3′-untranslated region (3′-UTR) of target mRNAs and causing transcript destabilization or translational inhibition [12]. Several miRNAs such as miR-155 [13] and miR-302a [14] have been
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reported to affect macrophage foam cell formation and atherosclerosis. It has been
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documented that miR-150 is a modulator of pathological inflammatory responses [15,16]. Downregulation of miR-150 has been shown to be correlated with enhanced proinflammatory mediator expression in Krüppel-like factor 2-deficient macrophages
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[17]. These studies suggest a possible implication of miR-150 in atherosclerosis.
The aim of this study was to investigate the potential roles of miR-150 in the formation of macrophage foam cells and cholesterol homeostasis. Additionally, the
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functional target gene(s) of miR-150 was characterized.
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Materials and methods
Cell culture and oxLDL exposure
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THP-1 human monocytic cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml
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streptomycin (Invitrogen, Rockville, MD, USA). To induce differentiation to
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macrophages, THP-1 cells were incubated with 100 nM phorbol 12-myristate 13-acetate (PMA; Calbiochem, San Diego, CA, USA) for 24 h. PMA-differentiated THP-1 macrophages were exposed to 50 µg/ml oxLDL (Biomedical Technologies, Stoughton, MA, USA) for indicated times before gene expression and lipid
Cell transfection
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accumulation analysis.
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miR-150 mimic and negative control miRNA were purchased from RiboBio Company
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(Guangzhou, China). Locked nucleic acid (LNA)-modified anti-miR-150 and negative controls were obtained from Qiagen (Valencia, CA, USA). Small interfering RNA (siRNA) targeting human adiponectin receptor 2 (AdipoR2) and control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
PMA-differentiated THP-1 macrophages at ~70% confluence were transfected with miR-150 mimic, LNA-anti-miR-150, AdipoR2 siRNA, and their corresponding
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ACCEPTED MANUSCRIPT controls (100 nM for each) with FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) per the manufacturer’s protocol. Twenty-four hours later, cells were incubated with oxLDL and examined for gene expression and cholesterol uptake and efflux. In
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some experiments, cells were pretreated with geranylgeranyl pyrophosphate (GGPP; 10 µM) or GW9662 (5 µM; Sigma, St. Louis, MO, USA) for 1 h before transfection of AdipoR2 siRNA. For estimation of transfection efficiency, fluorescent-labeled
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control siRNAs (Santa Cruz Biotechnology) were transfected in parallel.
RNA isolation and real-time quantitative PCR analysis
Total RNA was isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For measurement of mature miR-150, total RNA was
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reverse-transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). miR-150 expression levels were quantified with TaqMan MicroRNA assay kits (Applied Biosystems) on the ABI Prism Sequence
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Detection System 7900HT (Applied Biosystems). Results were normalized to U6
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snRNA using the comparative threshold cycle (Ct) method [18]. For measurement of mRNA levels of interested genes, total RNA was reverse-transcribed using MMLV Reverse Transcriptase (Invitrogen) and random primers. qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems). The sequences of PCR primers used are summarized in Supplementary Table S1. Relative mRNA levels were determined using the comparative Ct method after normalization to β-actin.
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ACCEPTED MANUSCRIPT Oil Red O staining and cholesterol content measurement After incubation with oxLDL, cells were rinsed with ice-cold phosphate-buffered saline and fixed in 4% paraformaldehyde for 1 h. Cells were stained with 0.5% Oil
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Red O (Sigma) in isopropanol for 2 h and visualized under a light microscope. The stained lipids were extracted with isopropanol. Absorbance was measured at 510 nm. After lipid extraction, total cholesterol contents were measured colormetrically with
Cholesterol efflux assay
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the Infinity Total Cholesterol Kit (Thermo Scientific, Waltham, MA, USA).
Assessment of cholesterol efflux from cholesterol-laden macrophages was performed as described previously [19]. In brief, THP-1 macrophages were converted to foam
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cells by incubation with 50 µg/ml oxLDL and [3H]cholesterol (1 µCi/ml; PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) for 48 h. Foam cells were washed and incubated with culture medium supplemented with 0.2% bovine serum albumin
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(Sigma) and apoA-I (10 µg/ml; Sigma) or HDL (100 µg/ml; Biomedical Technologies)
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for an additional 24 h. Radioactivity was determined in centrifuged medium and in cell lysates by a liquid scintillation counter. Cholesterol efflux was expressed as the percentage of [3H]cholesterol counts in the medium relative to the total [3H]cholesterol counts (medium plus cell).
oxLDL uptake assay Fluorescence-labeled oxLDL (Dil-oxLDL) was used to evaluate the uptake ability of
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ACCEPTED MANUSCRIPT macrophages, as described previously [20]. In brief, macrophages were incubated with 10 µg/ml Dil-oxLDL (Biomedical Technologies) for 4 h at 37°C. Fluorescence
Protein extracts and Western blot analysis
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intensity was analyzed with a flow cytometer.
Macrophages were lysed in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.5),
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120 mM NaCl, 1 mM EDTA, 1% NP40, 10 mM pyrophosphate, 10 mM
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β-glycerophosphate , 50 mM sodium fluoride, 1.5 mM sodium orthovanadate, 10 µM okadaic acid, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein (50 µg per lane) were resolved by sodium dodecyl
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sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. The membranes were blocked for 1 h with 5% fat-free milk and incubated overnight with primary antibodies against ABCA1 (1:500), ABCG1 (1:500), AdipoR2
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(1:1000), and β-actin (1:3000; Santa Cruz Biotechnology). After washing, the
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membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunoreactive bands were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). Protein expression was quantitated by densitometry using Quantity-One software (Bio-Rad).
Luciferease reporter assay The entire 3′-UTR of human AdipoR2 was amplified by PCR and inserted into the
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ACCEPTED MANUSCRIPT pMIR-REPORT luciferase reporter vector (Ambion, Austin, TX, USA). Mutant AdipoR2 3′-UTR was obtained by site-directed mutagenesis using the QuikChange Lightning site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and also
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cloned into the pMIR-REPORT plasmid. The insertions were confirmed by DNA sequencing. For luciferase reporter assay, HEK293 cells (obtained from ATCC) were seeded into 24-well tissue plates and co-transfected with 0.1 µg of luciferase reporter
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plasmid, 0.05 µg of pRL-TK control vector (Promega, Madison, WI, USA), and 50
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nM miR-150 mimic or nontargeting control using FuGENE 6. The pRL-TK plasmid, which expresses Renilla luciferase, was used to correct the differences in transfection efficiency. After 24-h incubation, cells were collected and tested for luciferase activities using the Dual-Luciferase Reporter Assay Kit (Promega) according to the
Statistical analysis
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manufacturer’s instructions.
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Data are expressed as the means ± S.E.M. Differences among multiple groups were
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compared with one-way analysis of variance followed by Tukey's multiple comparison test. P < 0.05 was considered to indicate a significant difference.
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Results
miR-150 prevents the transformation of oxLDL-treated macrophages into foam
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cells We first explored whether miR-150 is involved in the formation of macrophage foam cells in atherosclerosis. To this end, we examined the effect of oxLDL on miR-150
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expression in PMA-differentiated THP-1 macrophages. As shown in Fig. 1A,
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exposure to 50 µg/ml oxLDL led to a significant upregulation of miR-150 after treatment for 12 and 24 h, suggesting that miR-150 is implicated in macrophage foam cell generation. To validate this hypothesis, gain- and loss-of-function experiments were
performed.
After
incubation
with
50
µg/ml
oxLDL
for
48
h,
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miR-150-overexpressing THP-1 macrophages displayed less lipid accumulation than control cells, as determined by Oil Red O staining (Fig. 1B). Direct lipid analysis further confirmed about 58% reduction of total cholesterol amounts in
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miR-150-overexpressing THP-1 macropahges (P < 0.05 vs. control cells; Fig. 1C).
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Inhibition of miR-150 was achieved by transfection of LNA-modified anti-miR-150. Notably, the delivery of anti-miR-150 markedly accelerated lipid accumulation in oxLDL-exposed THP-1 macrophages (Fig. 1B and 1C). These results indicate that miR-150 upregulation hampers the formation of macrophage foam cells.
miR-150 facilitates cholesterol efflux from oxLDL-laden macrophages To gain more insight into the suppression of macrophage foam cell formation by
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ACCEPTED MANUSCRIPT miR-150, we examined the effect of miR-150 overexpression or inhibition on cholesterol uptake and efflux in THP-1 macrophages. As shown in Fig. 2A, apoA-Iand HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in
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miR-150-overexpressing macrophages relative to control cells. In contrast, inhibition of miR-150 significantly reduced macrophage cholesterol efflux to apoA-I (47%) and HDL (35%), compared to control cells (Fig. 2A). However, manipulating miR-150
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expression did not alter macrophage lipid uptake, as determined by Dil-oxLDL uptake
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assays (Fig. 2B). At the molecular level, ectopic expression of miR-150 increased the mRNA and protein expression of ABCA1 and ABCG1, either in the absence or presence of oxLDL (Fig. 2C and 2D). However, the expression of SR-A, SR-BI, and CD36 remained unchanged in response to upregulation or downregulation of miR-150
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(data not shown). Taken together, these results suggest that miR-150-mediated suppression of lipid accumulation in macrophages is largely due to the increase of
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cholesterol efflux.
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AdipoR2 is a functional target of miR-150 Bioinformatic analysis using the program Targetscan (http://www.targetscan.org/) demonstrated that AdipoR2 contained a putative binding site for miR-150 in its 3′-UTR (Fig. 3A). To check whether miR-150 negatively regulates the expression of AdipoR2 via binding to the predicated site, we performed luciferase reporter assays using plasmids carrying wild-type or mutant AdipoR2 3′-UTR (Fig. 3A). The results showed that delivery of miR-150 mimic significantly reduced the expression of
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ACCEPTED MANUSCRIPT wild-type AdipoR2 3′-UTR reporter (Fig. 3B). Mutation of the putative miR-150 binding site within the AdipoR2 3′-UTR rendered the reporter resistant to repression by miR-150 (Fig. 3B). In THP-1 macrophages, enforced expression of miR-150
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downregulated the expression of AdipoR2 at both the mRNA and protein levels (Fig. 3C and 3D). Altogether, miR-150 can directly target AdipoR2 in macrophages.
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Downregulation of AdipoR2 phenocopies the effects of miR-150 on macrophage
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foam cell formation
In light of the finding that AdipoR2 deficiency protects against atherosclerosis in a mouse model [21], we hypothesized that this gene may mediate the effects of miR-150 on macrophage foam cell formation. To test this hypothesis, we knocked
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down the expression of AdipoR2 via siRNA technology (data not shown). Silencing of AdipoR2 significantly decreased lipid accumulation (Fig. 4A) and promoted cholesterol efflux (Fig. 4B) in THP-1 macrophages exposed to oxLDL. qRT-PCR
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analysis confirmed that downregulation of AdipoR2 led to a significant increase in the
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mRNA expression of ABCA1 and ABCG1 (Fig. 4C). Additionally, AdipoR2 depletion induced the expression of PPARγ and LXRα in oxLDL-treated THP-1 macrophages, as determined by qRT-PCR analysis (Fig. 4D). Moreover, the addition of LXRα antagonist GGPP or PPARγ antagonist GW9662 blocked the elevation in ABCA1 and ABCG1 expression induced by AdipoR2 silencing (Fig. 4D). Taken together, AdipoR2 downregulation promotes cholesterol efflux through upregulation of ABCA1 and ABCG1 via the PPARγ- and LXRα-dependent pathways.
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Discussion
The transformation of macrophages to foam cells plays a fundamental role in the
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pathogenesis of atherosclerosis [3]. An improved understanding of the mechanisms by which macrophage foam cell formation is regulated should lead to new approaches to atherosclerosis prevention and treatment. In this study, we showed that miR-150 was
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upregulated in THP-1 macrophages in response to oxLDL treatment, suggesting its
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involvement in macrophage foam cell production. Both loss- and gain-of function experiments validated miR-150 as a suppressor of macrophage foam cell formation. Our data demonstrated that miR-150 overexpression antagonized lipid accumulation in oxLDL-exposed THP-1 macrophages. In contrast, inhibition of miR-150 enhanced
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lipid deposition in THP-1 macrophages after oxLDL treatment. A previous study has reported that miR-150 can be packaged into microvesicles (MVs) and secreted from monocytes [22]. Clinical analysis revealed that MVs isolated from the plasma of
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patients with atherosclerosis have greater amounts of miR-150 than those from
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healthy donors [22]. This finding can be explained that during the pathogenesis atherosclerosis, recruited monocytes release via MVs suppressive miRNAs such as miR-150, which prevent lipid accumulation and foam cell formation, thereby leading to an increase in miR-150-containing MVs in the circulation.
Excessive uptake of oxLDL and/or reduced cholesterol efflux contribute to the transformation of macrophages into foam cells. Several miRNAs such as miR-27a/b
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ACCEPTED MANUSCRIPT [23], miR-155 [13], and miR-33 [24] have been found to coordinate cholesterol influx and/or efflux in macrophage foam cell formation. In this study, we demonstrated that miR-150 promoted macrophage cholesterol efflux to apoA-I and HDL and that this
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response was accompanied by increased expression of ABCA1 and ABCG1. However, miR-150 overexpression or inhibition seemed not to affect oxLDL uptake by macrophages. At the molecular level, the key genes involved in cholesterol uptake
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(SR-A and CD36) were not altered by either miR-150 upregulation or downregulation.
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These data highlight an important role for miR-150 in the regulation of cholesterol homeostasis in macrophages. miR-150-mediated inhibition of macrophage foam cell formation is largely ascribed to enhanced cholesterol efflux.
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A number of target genes of miR-150 have been identified, such as Elk1, Etf1 and Myb [15], angiopoetin-2 [25], and FOXO4 [26]. In different biological processes, miR-150 seems to target different transcripts [15,25,26]. Using luciferase reporter
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assays, we showed that miR-150 repressed the expression of AdipoR2 via interaction
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with the putative site within the AdipoR2 3′-UTR. Moreover, overexpression of miR-150 downregulated the endogenous expression of AdipoR2 in macrophages. Our data identified AdipoR2 as a direct target of miR-150 in macrophages. In high fat-induced experimental atherosclerosis, AdipoR2 deficiency yielded beneficial effects, as evidenced by decreased plaque area in the brachiocephalic artery [21]. Moreover, absence of AdipoR2 was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages, suggesting its implication in the regulation
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ACCEPTED MANUSCRIPT of macrophage cholesterol homeostasis. In this study, we provided direct evidence for the regulation of lipid accumulation by AdipoR2. We found that depletion of AdipoR2 antagonized lipid deposition and facilitated cholesterol efflux in oxLDL-treated
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THP-1 macrophages, which was accompanied by upregulation of ABCA1 and ABCG1. Moreover, AdipoR2 silencing upregulated the expression of PPARγ and LXRα. Pharmacological inhibition of PPARγ or LXRα abolished the induction of
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ABCA1 and ABCG1 in AdipoR2-depleted cells. Taken together, AdipoR2 controls
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cholesterol efflux in oxLDL-treated THP-1 macrophages through regulation of PPARγ and LXRα signaling. It has been reported that activation of PPARγ signaling induces the expression of AdipoR2 in macrophages [27] and hepatocytes [28].
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Therefore, there is a complex interaction between PPARγ and AdipoR2.
In conclusion, we demonstrate that miR-150 can hinder lipid accumulation in oxLDL-treated
macrophages
through
promotion
of
cholesterol
efflux.
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miR-150-mediated inhibition of macrophage foam cell formation is causally linked to
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targeting of AdipoR2. These results suggest that delivery of miR-150 may represent a promising strategy for prevention of macrophage foam cell formation in the development of atherosclerosis.
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X.Y. Liu, J. Peng, K. Chen, P.P. He, Y.C. Lv, X.P. Ouyang, F. Yao, D.P. Tang, F.S. Cayabyab, D.W. Zhang, X.L. Zheng, G.P. Tian, C.K. Tang, MicroRNA-27a/b regulates cellular cholesterol efflux, influx and esterification/hydrolysis in THP-1 macrophages, Atherosclerosis. 234(2014):54-64. 24. D. Karunakaran, A.B. Thrush, M.A. Nguyen, L. Richards, M. Geoffrion, R. Singaravelu, E. Ramphos, P. Shangari, M. Ouimet, J.P. Pezacki, K.J. Moore, L. Perisic, L. Maegdefessel, U. Hedin, M.E. Harper, K.J. Rayner, Macrophage Mitochondrial
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ACCEPTED MANUSCRIPT Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis, Circ. Res. 117(2015):266-278. 25. C. Rajput, M. Tauseef, M. Farazuddin, P. Yazbeck, M.R. Amin, V. Avin Br, T.
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Sharma, D. Mehta, MicroRNA-150 Suppression of Angiopoetin-2 Generation and Signaling Is Crucial for Resolving Vascular Injury, Arterioscler. Thromb. Vasc. Biol. 36(2016):380-388.
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26. J. Li, L. Hu, C. Tian, F. Lu, J. Wu, L. Liu, microRNA-150 promotes cervical
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cancer cell growth and survival by targeting FOXO4, B.M.C. Mol. Biol. 16(2015):24. 27. Chinetti G, Zawadski C, Fruchart JC, Staels B. Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha,
PPARgamma,
and
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Biophys.
Res.
Commun.
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314(2004):151-158.
LXR..
28. X. Sun, R. Han, Z. Wang, Y. Chen, Regulation of adiponectin receptors in hepatocytes by the peroxisome proliferator-activated receptor-gamma agonist
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rosiglitazone, Diabetologia. 49(2006):1303-1310.
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Figure legends
Fig. 1. miR-150 prevents the transformation of oxLDL-treated macrophages into foam
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cells. (A) qRT-PCR analysis of miR-150 expression in THP-1 macrophages after treatment with 50 µg/ml oxLDL for indicated times. *P < 0.05, compared to cells without oxLDL treatment. (B) THP-1 macrophages transfected with indicated
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constructs were treated with 50 µg/ml oxLDL and tested for lipid deposition by Oil
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Red O staining. Stained lipids were extracted with isopropanol and absorbance was measured at 510 nm. (C) Measurement of cholesterol contents in THP-1 macrophages transfected with indicated constructs. Bar graphs represent the results of three
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independent experiments. *P < 0.05.
Fig. 2. miR-150 facilitates cholesterol efflux from oxLDL-laden macrophages. (A) THP-1 macrophages transfected with miR-150, anti-miR-150, or their controls were
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loaded with [3H]cholesterol. After incubation with apoA-I or HDL, cholesterol efflux
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was measured. (B) THP-1 macrophages were transfected with indicated constructs and then incubated with Dil-oxLDL. Mean fluorescence intensity was determined by flow cytometry. (C and D) THP-1 macrophages were transfected with negative control miRNA or miR-150 and treated with 50 µg/ml oxLDL or medium. The mRNA (C) and protein (D) levels of ABCA1 and ABCG1 were measured. Bar graphs represent the results of three independent experiments. *P < 0.05.
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ACCEPTED MANUSCRIPT Fig. 3. AdipoR2 is a functional target of miR-150. (A) A potential target site for miR-150 in the 3′-UTR of human AdipoR2 mRNA is shown. To confirm miR-150 binding to the putative site, the predicted site was mutated. (B) HEK293 cells were
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co-transfected with miR-150 or control miRNA together with wild-type or mutant AdipoR2 3′-UTR constructs and subjected to luciferase reporter assays. (C and D) THP-1 macrophages were transfected with miR-150 or control miRNA and tested for
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the protein (C) and mRNA (D) levels of AdipoR2. *P < 0.05.
Fig. 4. Downregulation of AdipoR2 phenocopies the effects of miR-150 on macrophage foam cell formation. (A) THP-1 macrophages transfected with AdipoR2 siRNA or control siRNA were treated with 50 µg/ml oxLDL and tested for lipid
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deposition by Oil Red O staining. (B) Measurement of cholesterol efflux in THP-1 macrophages with indicated treatments and incubated with apoA-I or HDL. (C) THP-1 macrophages were transfected with AdipoR2 siRNA or control siRNA and
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examined for the mRNA levels of ABCA1 and ABCG1. (D) THP-1 macrophages
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were pretreated with GGPP (10 µM) or GW9662 (5 µM) for 1 h before transfection of AdipoR2 or control siRNA and examined for the mRNA expression of ABCA1 and ABCG1. Bar graphs represent the results of three independent experiments. *P < 0.05 vs. control siRNA; #P < 0.05 vs. AdipoR2 siRNA alone.
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Highlights
miR-150 inhibits macrophage foam cell formation.
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miR-150 accelerates cholesterol efflux from oxLDL-laden macrophages.
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miR-150 suppresses macrophage foam cell formation by targeting AdipoR2.
S0006-291X(16)30804-X
DOI:
10.1016/j.bbrc.2016.05.096
Reference:
YBBRC 35846
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 27 April 2016 Accepted Date: 19 May 2016
Please cite this article as: J. Li, S. Zhang, microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.05.096. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2
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Jing Li1 and Suhua Zhang2,* Department of Geratory, Linzi District People’s Hospital of Zibo City, Zibo,
Shandong, China
Department of Health, Qilu Hospital of Shandong University (Qingdao), Qingdao
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2
*
Corresponding author.
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City, Qingdao, China
Qilu Hospital of Shandong University (Qingdao), NO.758, Hefei Road, Qingdao
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266000, China Tel: +86-18561811017 Fax: +86-053296599
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Email: [email protected]
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Abstract
Transformation of macrophages into foam cells plays a critical role in the
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pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 µg/ml oxidized
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low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in
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THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control
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cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small
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interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of
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miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive
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ACCEPTED MANUSCRIPT effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to
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prevent macrophage foam cell formation in atherosclerosis.
Key words: atherosclerosis; cholesterol efflux; foam cell formation; therapeutic
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target.
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Introduction
Atherosclerosis, which is characterized by inflammatory responses and accumulation
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of lipids and cellular and fibrous elements in the arteries, is a leading cause of heart disease and stroke [1,2]. Generation of lipid-laden macrophages, or macrophage foam cells, is a hallmark feature of early stage atherosclerotic lesions [3]. The
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transformation of macrophages into foam cells is causally linked to unbalanced
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cholesterol influx and efflux [4]. Recruited macrophages in the vessel intima readily ingest modified low density lipoproteins (LDL), especially oxidized forms (oxLDL), through scavenger receptors (SRs) such as CD36 and SR-A [5]. Excessive uptake of modified lipoproteins results in the formation of cholesterol-rich foam cells.
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Cholesterol efflux is critical in maintaining lipid homeostasis and preventing macrophage foam cell formation [6]. ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) are known to participate in macrophage cholesterol efflux.
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ABCA1 is implicated in the removal of free cholesterol to lipid-poor apolipoproteins,
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most notably apolipoprotein A1 (apoA1), while ABCG1 is responsible for transporting cholesterol to high-density lipoprotein (HDL) [7,8]. The expression of ABCA1 and ABCG1 is regulated by proliferator-activated receptor gamma (PPARγ)and liver X receptor alpha (LXRα)-dependent pathways [9]. Suppression of macrophage foam cell formation via alteration of cholesterol homeostasis has been suggested as an important strategy for prevention of atherosclerosis [10].
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ACCEPTED MANUSCRIPT microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that play important roles in a wide range of biological processes such as proliferation, differentiation, inflammation, lipid metabolism, and tumorigenesis [11]. miRNAs
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typically repress gene expression through binding to the 3′-untranslated region (3′-UTR) of target mRNAs and causing transcript destabilization or translational inhibition [12]. Several miRNAs such as miR-155 [13] and miR-302a [14] have been
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reported to affect macrophage foam cell formation and atherosclerosis. It has been
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documented that miR-150 is a modulator of pathological inflammatory responses [15,16]. Downregulation of miR-150 has been shown to be correlated with enhanced proinflammatory mediator expression in Krüppel-like factor 2-deficient macrophages
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[17]. These studies suggest a possible implication of miR-150 in atherosclerosis.
The aim of this study was to investigate the potential roles of miR-150 in the formation of macrophage foam cells and cholesterol homeostasis. Additionally, the
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functional target gene(s) of miR-150 was characterized.
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Materials and methods
Cell culture and oxLDL exposure
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THP-1 human monocytic cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml
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streptomycin (Invitrogen, Rockville, MD, USA). To induce differentiation to
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macrophages, THP-1 cells were incubated with 100 nM phorbol 12-myristate 13-acetate (PMA; Calbiochem, San Diego, CA, USA) for 24 h. PMA-differentiated THP-1 macrophages were exposed to 50 µg/ml oxLDL (Biomedical Technologies, Stoughton, MA, USA) for indicated times before gene expression and lipid
Cell transfection
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accumulation analysis.
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miR-150 mimic and negative control miRNA were purchased from RiboBio Company
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(Guangzhou, China). Locked nucleic acid (LNA)-modified anti-miR-150 and negative controls were obtained from Qiagen (Valencia, CA, USA). Small interfering RNA (siRNA) targeting human adiponectin receptor 2 (AdipoR2) and control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
PMA-differentiated THP-1 macrophages at ~70% confluence were transfected with miR-150 mimic, LNA-anti-miR-150, AdipoR2 siRNA, and their corresponding
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ACCEPTED MANUSCRIPT controls (100 nM for each) with FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) per the manufacturer’s protocol. Twenty-four hours later, cells were incubated with oxLDL and examined for gene expression and cholesterol uptake and efflux. In
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some experiments, cells were pretreated with geranylgeranyl pyrophosphate (GGPP; 10 µM) or GW9662 (5 µM; Sigma, St. Louis, MO, USA) for 1 h before transfection of AdipoR2 siRNA. For estimation of transfection efficiency, fluorescent-labeled
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control siRNAs (Santa Cruz Biotechnology) were transfected in parallel.
RNA isolation and real-time quantitative PCR analysis
Total RNA was isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For measurement of mature miR-150, total RNA was
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reverse-transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). miR-150 expression levels were quantified with TaqMan MicroRNA assay kits (Applied Biosystems) on the ABI Prism Sequence
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Detection System 7900HT (Applied Biosystems). Results were normalized to U6
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snRNA using the comparative threshold cycle (Ct) method [18]. For measurement of mRNA levels of interested genes, total RNA was reverse-transcribed using MMLV Reverse Transcriptase (Invitrogen) and random primers. qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems). The sequences of PCR primers used are summarized in Supplementary Table S1. Relative mRNA levels were determined using the comparative Ct method after normalization to β-actin.
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ACCEPTED MANUSCRIPT Oil Red O staining and cholesterol content measurement After incubation with oxLDL, cells were rinsed with ice-cold phosphate-buffered saline and fixed in 4% paraformaldehyde for 1 h. Cells were stained with 0.5% Oil
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Red O (Sigma) in isopropanol for 2 h and visualized under a light microscope. The stained lipids were extracted with isopropanol. Absorbance was measured at 510 nm. After lipid extraction, total cholesterol contents were measured colormetrically with
Cholesterol efflux assay
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the Infinity Total Cholesterol Kit (Thermo Scientific, Waltham, MA, USA).
Assessment of cholesterol efflux from cholesterol-laden macrophages was performed as described previously [19]. In brief, THP-1 macrophages were converted to foam
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cells by incubation with 50 µg/ml oxLDL and [3H]cholesterol (1 µCi/ml; PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) for 48 h. Foam cells were washed and incubated with culture medium supplemented with 0.2% bovine serum albumin
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(Sigma) and apoA-I (10 µg/ml; Sigma) or HDL (100 µg/ml; Biomedical Technologies)
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for an additional 24 h. Radioactivity was determined in centrifuged medium and in cell lysates by a liquid scintillation counter. Cholesterol efflux was expressed as the percentage of [3H]cholesterol counts in the medium relative to the total [3H]cholesterol counts (medium plus cell).
oxLDL uptake assay Fluorescence-labeled oxLDL (Dil-oxLDL) was used to evaluate the uptake ability of
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Protein extracts and Western blot analysis
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intensity was analyzed with a flow cytometer.
Macrophages were lysed in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.5),
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120 mM NaCl, 1 mM EDTA, 1% NP40, 10 mM pyrophosphate, 10 mM
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β-glycerophosphate , 50 mM sodium fluoride, 1.5 mM sodium orthovanadate, 10 µM okadaic acid, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein (50 µg per lane) were resolved by sodium dodecyl
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sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. The membranes were blocked for 1 h with 5% fat-free milk and incubated overnight with primary antibodies against ABCA1 (1:500), ABCG1 (1:500), AdipoR2
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(1:1000), and β-actin (1:3000; Santa Cruz Biotechnology). After washing, the
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membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunoreactive bands were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). Protein expression was quantitated by densitometry using Quantity-One software (Bio-Rad).
Luciferease reporter assay The entire 3′-UTR of human AdipoR2 was amplified by PCR and inserted into the
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ACCEPTED MANUSCRIPT pMIR-REPORT luciferase reporter vector (Ambion, Austin, TX, USA). Mutant AdipoR2 3′-UTR was obtained by site-directed mutagenesis using the QuikChange Lightning site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and also
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cloned into the pMIR-REPORT plasmid. The insertions were confirmed by DNA sequencing. For luciferase reporter assay, HEK293 cells (obtained from ATCC) were seeded into 24-well tissue plates and co-transfected with 0.1 µg of luciferase reporter
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plasmid, 0.05 µg of pRL-TK control vector (Promega, Madison, WI, USA), and 50
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nM miR-150 mimic or nontargeting control using FuGENE 6. The pRL-TK plasmid, which expresses Renilla luciferase, was used to correct the differences in transfection efficiency. After 24-h incubation, cells were collected and tested for luciferase activities using the Dual-Luciferase Reporter Assay Kit (Promega) according to the
Statistical analysis
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manufacturer’s instructions.
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Data are expressed as the means ± S.E.M. Differences among multiple groups were
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compared with one-way analysis of variance followed by Tukey's multiple comparison test. P < 0.05 was considered to indicate a significant difference.
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Results
miR-150 prevents the transformation of oxLDL-treated macrophages into foam
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cells We first explored whether miR-150 is involved in the formation of macrophage foam cells in atherosclerosis. To this end, we examined the effect of oxLDL on miR-150
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expression in PMA-differentiated THP-1 macrophages. As shown in Fig. 1A,
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exposure to 50 µg/ml oxLDL led to a significant upregulation of miR-150 after treatment for 12 and 24 h, suggesting that miR-150 is implicated in macrophage foam cell generation. To validate this hypothesis, gain- and loss-of-function experiments were
performed.
After
incubation
with
50
µg/ml
oxLDL
for
48
h,
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miR-150-overexpressing THP-1 macrophages displayed less lipid accumulation than control cells, as determined by Oil Red O staining (Fig. 1B). Direct lipid analysis further confirmed about 58% reduction of total cholesterol amounts in
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miR-150-overexpressing THP-1 macropahges (P < 0.05 vs. control cells; Fig. 1C).
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Inhibition of miR-150 was achieved by transfection of LNA-modified anti-miR-150. Notably, the delivery of anti-miR-150 markedly accelerated lipid accumulation in oxLDL-exposed THP-1 macrophages (Fig. 1B and 1C). These results indicate that miR-150 upregulation hampers the formation of macrophage foam cells.
miR-150 facilitates cholesterol efflux from oxLDL-laden macrophages To gain more insight into the suppression of macrophage foam cell formation by
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ACCEPTED MANUSCRIPT miR-150, we examined the effect of miR-150 overexpression or inhibition on cholesterol uptake and efflux in THP-1 macrophages. As shown in Fig. 2A, apoA-Iand HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in
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miR-150-overexpressing macrophages relative to control cells. In contrast, inhibition of miR-150 significantly reduced macrophage cholesterol efflux to apoA-I (47%) and HDL (35%), compared to control cells (Fig. 2A). However, manipulating miR-150
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expression did not alter macrophage lipid uptake, as determined by Dil-oxLDL uptake
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assays (Fig. 2B). At the molecular level, ectopic expression of miR-150 increased the mRNA and protein expression of ABCA1 and ABCG1, either in the absence or presence of oxLDL (Fig. 2C and 2D). However, the expression of SR-A, SR-BI, and CD36 remained unchanged in response to upregulation or downregulation of miR-150
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(data not shown). Taken together, these results suggest that miR-150-mediated suppression of lipid accumulation in macrophages is largely due to the increase of
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cholesterol efflux.
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AdipoR2 is a functional target of miR-150 Bioinformatic analysis using the program Targetscan (http://www.targetscan.org/) demonstrated that AdipoR2 contained a putative binding site for miR-150 in its 3′-UTR (Fig. 3A). To check whether miR-150 negatively regulates the expression of AdipoR2 via binding to the predicated site, we performed luciferase reporter assays using plasmids carrying wild-type or mutant AdipoR2 3′-UTR (Fig. 3A). The results showed that delivery of miR-150 mimic significantly reduced the expression of
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ACCEPTED MANUSCRIPT wild-type AdipoR2 3′-UTR reporter (Fig. 3B). Mutation of the putative miR-150 binding site within the AdipoR2 3′-UTR rendered the reporter resistant to repression by miR-150 (Fig. 3B). In THP-1 macrophages, enforced expression of miR-150
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downregulated the expression of AdipoR2 at both the mRNA and protein levels (Fig. 3C and 3D). Altogether, miR-150 can directly target AdipoR2 in macrophages.
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Downregulation of AdipoR2 phenocopies the effects of miR-150 on macrophage
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foam cell formation
In light of the finding that AdipoR2 deficiency protects against atherosclerosis in a mouse model [21], we hypothesized that this gene may mediate the effects of miR-150 on macrophage foam cell formation. To test this hypothesis, we knocked
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down the expression of AdipoR2 via siRNA technology (data not shown). Silencing of AdipoR2 significantly decreased lipid accumulation (Fig. 4A) and promoted cholesterol efflux (Fig. 4B) in THP-1 macrophages exposed to oxLDL. qRT-PCR
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analysis confirmed that downregulation of AdipoR2 led to a significant increase in the
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mRNA expression of ABCA1 and ABCG1 (Fig. 4C). Additionally, AdipoR2 depletion induced the expression of PPARγ and LXRα in oxLDL-treated THP-1 macrophages, as determined by qRT-PCR analysis (Fig. 4D). Moreover, the addition of LXRα antagonist GGPP or PPARγ antagonist GW9662 blocked the elevation in ABCA1 and ABCG1 expression induced by AdipoR2 silencing (Fig. 4D). Taken together, AdipoR2 downregulation promotes cholesterol efflux through upregulation of ABCA1 and ABCG1 via the PPARγ- and LXRα-dependent pathways.
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Discussion
The transformation of macrophages to foam cells plays a fundamental role in the
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pathogenesis of atherosclerosis [3]. An improved understanding of the mechanisms by which macrophage foam cell formation is regulated should lead to new approaches to atherosclerosis prevention and treatment. In this study, we showed that miR-150 was
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upregulated in THP-1 macrophages in response to oxLDL treatment, suggesting its
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involvement in macrophage foam cell production. Both loss- and gain-of function experiments validated miR-150 as a suppressor of macrophage foam cell formation. Our data demonstrated that miR-150 overexpression antagonized lipid accumulation in oxLDL-exposed THP-1 macrophages. In contrast, inhibition of miR-150 enhanced
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lipid deposition in THP-1 macrophages after oxLDL treatment. A previous study has reported that miR-150 can be packaged into microvesicles (MVs) and secreted from monocytes [22]. Clinical analysis revealed that MVs isolated from the plasma of
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patients with atherosclerosis have greater amounts of miR-150 than those from
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healthy donors [22]. This finding can be explained that during the pathogenesis atherosclerosis, recruited monocytes release via MVs suppressive miRNAs such as miR-150, which prevent lipid accumulation and foam cell formation, thereby leading to an increase in miR-150-containing MVs in the circulation.
Excessive uptake of oxLDL and/or reduced cholesterol efflux contribute to the transformation of macrophages into foam cells. Several miRNAs such as miR-27a/b
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ACCEPTED MANUSCRIPT [23], miR-155 [13], and miR-33 [24] have been found to coordinate cholesterol influx and/or efflux in macrophage foam cell formation. In this study, we demonstrated that miR-150 promoted macrophage cholesterol efflux to apoA-I and HDL and that this
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response was accompanied by increased expression of ABCA1 and ABCG1. However, miR-150 overexpression or inhibition seemed not to affect oxLDL uptake by macrophages. At the molecular level, the key genes involved in cholesterol uptake
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(SR-A and CD36) were not altered by either miR-150 upregulation or downregulation.
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These data highlight an important role for miR-150 in the regulation of cholesterol homeostasis in macrophages. miR-150-mediated inhibition of macrophage foam cell formation is largely ascribed to enhanced cholesterol efflux.
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A number of target genes of miR-150 have been identified, such as Elk1, Etf1 and Myb [15], angiopoetin-2 [25], and FOXO4 [26]. In different biological processes, miR-150 seems to target different transcripts [15,25,26]. Using luciferase reporter
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assays, we showed that miR-150 repressed the expression of AdipoR2 via interaction
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with the putative site within the AdipoR2 3′-UTR. Moreover, overexpression of miR-150 downregulated the endogenous expression of AdipoR2 in macrophages. Our data identified AdipoR2 as a direct target of miR-150 in macrophages. In high fat-induced experimental atherosclerosis, AdipoR2 deficiency yielded beneficial effects, as evidenced by decreased plaque area in the brachiocephalic artery [21]. Moreover, absence of AdipoR2 was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages, suggesting its implication in the regulation
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THP-1 macrophages, which was accompanied by upregulation of ABCA1 and ABCG1. Moreover, AdipoR2 silencing upregulated the expression of PPARγ and LXRα. Pharmacological inhibition of PPARγ or LXRα abolished the induction of
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ABCA1 and ABCG1 in AdipoR2-depleted cells. Taken together, AdipoR2 controls
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cholesterol efflux in oxLDL-treated THP-1 macrophages through regulation of PPARγ and LXRα signaling. It has been reported that activation of PPARγ signaling induces the expression of AdipoR2 in macrophages [27] and hepatocytes [28].
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Therefore, there is a complex interaction between PPARγ and AdipoR2.
In conclusion, we demonstrate that miR-150 can hinder lipid accumulation in oxLDL-treated
macrophages
through
promotion
of
cholesterol
efflux.
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miR-150-mediated inhibition of macrophage foam cell formation is causally linked to
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targeting of AdipoR2. These results suggest that delivery of miR-150 may represent a promising strategy for prevention of macrophage foam cell formation in the development of atherosclerosis.
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Figure legends
Fig. 1. miR-150 prevents the transformation of oxLDL-treated macrophages into foam
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cells. (A) qRT-PCR analysis of miR-150 expression in THP-1 macrophages after treatment with 50 µg/ml oxLDL for indicated times. *P < 0.05, compared to cells without oxLDL treatment. (B) THP-1 macrophages transfected with indicated
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Red O staining. Stained lipids were extracted with isopropanol and absorbance was measured at 510 nm. (C) Measurement of cholesterol contents in THP-1 macrophages transfected with indicated constructs. Bar graphs represent the results of three
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Fig. 2. miR-150 facilitates cholesterol efflux from oxLDL-laden macrophages. (A) THP-1 macrophages transfected with miR-150, anti-miR-150, or their controls were
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loaded with [3H]cholesterol. After incubation with apoA-I or HDL, cholesterol efflux
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was measured. (B) THP-1 macrophages were transfected with indicated constructs and then incubated with Dil-oxLDL. Mean fluorescence intensity was determined by flow cytometry. (C and D) THP-1 macrophages were transfected with negative control miRNA or miR-150 and treated with 50 µg/ml oxLDL or medium. The mRNA (C) and protein (D) levels of ABCA1 and ABCG1 were measured. Bar graphs represent the results of three independent experiments. *P < 0.05.
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ACCEPTED MANUSCRIPT Fig. 3. AdipoR2 is a functional target of miR-150. (A) A potential target site for miR-150 in the 3′-UTR of human AdipoR2 mRNA is shown. To confirm miR-150 binding to the putative site, the predicted site was mutated. (B) HEK293 cells were
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co-transfected with miR-150 or control miRNA together with wild-type or mutant AdipoR2 3′-UTR constructs and subjected to luciferase reporter assays. (C and D) THP-1 macrophages were transfected with miR-150 or control miRNA and tested for
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the protein (C) and mRNA (D) levels of AdipoR2. *P < 0.05.
Fig. 4. Downregulation of AdipoR2 phenocopies the effects of miR-150 on macrophage foam cell formation. (A) THP-1 macrophages transfected with AdipoR2 siRNA or control siRNA were treated with 50 µg/ml oxLDL and tested for lipid
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deposition by Oil Red O staining. (B) Measurement of cholesterol efflux in THP-1 macrophages with indicated treatments and incubated with apoA-I or HDL. (C) THP-1 macrophages were transfected with AdipoR2 siRNA or control siRNA and
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were pretreated with GGPP (10 µM) or GW9662 (5 µM) for 1 h before transfection of AdipoR2 or control siRNA and examined for the mRNA expression of ABCA1 and ABCG1. Bar graphs represent the results of three independent experiments. *P < 0.05 vs. control siRNA; #P < 0.05 vs. AdipoR2 siRNA alone.
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Highlights
miR-150 inhibits macrophage foam cell formation.
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miR-150 accelerates cholesterol efflux from oxLDL-laden macrophages.
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miR-150 suppresses macrophage foam cell formation by targeting AdipoR2.