MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis - PubMed (original) (raw)

MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis

Svenja Meiler et al. Arterioscler Thromb Vasc Biol. 2015 Feb.

Abstract

Objective: Macrophage foam cell formation is a key feature of atherosclerosis. Recent studies have shown that specific microRNAs (miRs) are regulated in modified low-density lipoprotein-treated macrophages, which can affect the cellular cholesterol homeostasis. Undertaking a genome-wide screen of miRs regulated in primary macrophages by modified low-density lipoprotein, miR-302a emerged as a potential candidate that may play a key role in macrophage cholesterol homeostasis.

Approach and results: The objective of this study was to assess the involvement of miR-302a in macrophage lipid homeostasis and if it can influence circulating lipid levels and atherosclerotic development when it is inhibited in a murine atherosclerosis model. We found that transfection of primary macrophages with either miR-302a or anti-miR-302a regulated the expression of ATP-binding cassette (ABC) transporter ABCA1 mRNA and protein. Luciferase reporter assays showed that miR-302a repressed the 3' untranslated regions (UTR) activity of mouse Abca1 by 48% and human ABCA1 by 45%. In addition, transfection of murine macrophages with miR-302a attenuated cholesterol efflux to apolipoprotein A-1 (apoA-1) by 38%. Long-term in vivo administration of anti-miR-302a to mice with low-density lipoprotein receptor deficiency (Ldlr(-/-)) fed an atherogenic diet led to an increase in ABCA1 in the liver and aorta as well as an increase in circulating plasma high-density lipoprotein levels by 35% compared with that of control mice. The anti-miR-302a-treated mice also displayed reduced atherosclerotic plaque size by ≈25% and a more stable plaque morphology with reduced signs of inflammation.

Conclusions: These studies identify miR-302a as a novel modulator of cholesterol efflux and a potential therapeutic target for suppressing atherosclerosis.

Keywords: ABCA1 protein; HDL cholesterol; atherosclerosis; cholesterol-efflux regulatory protein; macrophages; microRNA.

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Conflict of interest statement

DISCLOSURES

There are no conflicts of interest for any of the authors.

Figures

Figure 1. MiR-302a is inversely correlated with ABCA1 and ABCG1

Real time PCR analysis of miR-302a, Abca1 and Abcg1 expression in primary mouse macrophages (A) or primary human macrophages (B). Macrophages were either untreated or loaded with cholesterol by either AcLDL (10 μg/ml) or oxLDL (10 μg/ml) treatment for 6 hours (n=4 independent experiments). In addition, mouse macrophages were treated with 10 μg/ml native LDL. *P<0.05, **P<0.005, ***P<0.0005. (C) Real time PCR analysis of miR-302a, Abca1 and Abcg1 expression in aorta of _Ldlr_−/− mice (n=4 per group) fed either a normal chow or an atherogenic diet for 8, 12 and 24 weeks. (A) to (C), data are expressed as mean ± SEM. ANOVA with Bonferroni’s multiple comparison test was used.

Figure 2. Molecular characteristics of miR-302a

(A) Schematic overview of the LARP7 gene locus, showing the miR-302a coding sequence within intron 8. (B) Expression profile of miR-302a and Larp7 host gene in liver, aorta and spleen of C57BL/6 mice (n=4 per group) and primary mouse macrophages.. (C) Real time PCR analysis of miR-302a and Larp7 in liver and aorta of _Ldlr_−/− mice (n=3 per group) fed an atherogenic diet for 12 weeks and primary mouse macrophages loaded with cholesterol by AcLDL treatment. As controls (white bars), _Ldlr_−/− mice fed a normal chow and primary macrophages without cholesterol loading were used. (B) and (C), data are expressed as mean ± SEM. **P<0.005, ***P<0.0005. (D) Schematic representation of the miR-302a stem-loop sequence and its conservation among species. (E&F) Schematic overview demonstrating the predicted target site of miR-302a in the 3′UTR region of human ABCA1 (E) and mouse Abca1 (F). ANOVA with Bonferroni’s multiple comparison test was used.

Figure 3. MiR-302a regulates ABCA1

(A,B) Real time PCR analysis of Abca1 and Abcg1 gene expression in BMDM transfected with (A) control miR and 200 nM miR-302a, or (B) control miR and 200 nM anti-miR-302a. 24 hours after transfection, primary macrophages were either untreated or stimulated with 40 μg/ml AcLDL and/or 10 μM T0901317 for an additional 24 hours (n=4 independent experiments). Data are expressed as mean ± SEM. *P<0.05, **P<0.005. (C,D) Western blot analysis of Abca1 protein expression in primary mouse cells after transfection with (C) control miR-302a and 200 nM miR-302a, or (D) control miR and 200 nM anti-miR-302a. Macrophages were either untreated or treated with 40 μg/ml AcLDL and/or 10 μM T0901317 for 48 hours 24 hours after transfection. Representative blots are shown. (E) Gene expression analysis of inflammation markers (Arginase-I (Arg-I), Arginase-II (Arg-II), Interleukin-10 (Il-10), Interleukin-6 (Il-6) and Interferon gamma (Ifn-γ)) in BMDM after transfection with either control miR or 200 nM anti-miR-302a using real time PCR. Data are expressed as mean ± SEM. *P<0.05, ***P<0.0005. ANOVA with Bonferroni’s multiple comparison test was used.

Figure 4. MiR-302a targets the 3′UTR of ABCA1 and regulates cellular cholesterol efflux in vitro

(A) Luciferase reporter activity in COS-7 cells co-transfected with the indicated 3′UTR luciferase reporter vectors and increasing concentrations (0, 50, 100 and 200 nM) of control miR or miR-302a of mouse Abca1 (mAbca1) and human ABCA1 (hABCA1). (B,C) Activity of luciferase reporter constructs fused to the 3′UTR of mAbca1 (B) and hABCA1 (C) in COS-7 cells transfected with 200 nM control miR or miR-302a. As control, mAbca1 3′UTR and hABCA1 3′UTR containing the indicated point mutations (PM)(bold) in the miR-302a target site (represented in red) were used. Data are expressed as mean ± SEM and are representative of 4 independent experiments. *P<0.05. (D–G) Cholesterol efflux to apoA-1 in primary mouse (D) and primary human (F) macrophages stimulated with 50 μg/ml AcLDL and transfected with control miR or 100 nM miR-302a (n=4 independent experiments). Cholesterol efflux to apoA-1 in primary mouse (E) and primary human (G) macrophages stimulated with 50 μg/ml AcLDL and transfected with control miR or 200 nM anti-miR-302a (n=4 independent experiments). Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005. ANOVA with Bonferroni’s multiple comparison test was used.

Figure 5. Anti-miR-302a treatment in vivo increases Abca1 expression in aorta and liver of Ldlr−/− mice

(A) Real time PCR analysis of Abca1 gene expression in liver and aorta of _Ldlr_−/− mice after in vivo delivery of control anti-miR and anti-miR-302a (i.p. injections of 10 mg/kg per week over a period of 8 weeks). (B) Western blot analysis of Abca-1 protein expression in liver lysates of _Ldlr_−/− mice after in vivo delivery of control anti-miR and anti-miR-302a (i.p. injections of 10 mg/kg per week over a period of 8 weeks). Data are expressed as mean ± SEM. *P<0.05, **P<0.005. 2-tailed Student’s t test was used.

Figure 6. Anti-miR-302a treatment increases circulating HDL levels and reduces athersclerosis progression in Ldlr−/− mice inducing a stable plaque phenotype with less degree of inflammation

(A) HPLC lipoprotein profiles from plasma of Ldlr−/− mice treated with control anti-miR or anti-miR-302a (i.p. injections of 10 mg/kg per week over a period of 8 weeks) (n=7 mice per group). (B,C) Quantification of Oil Red O+ lipid depositions in the aorta (B) and aortic root (C) of Ldlr−/− mice treated with either control anti-miR or anti-miR-302a. Fluorescence microscopy was used to analyze levels of MOMA-2-positive macrophages (D) and smoothelin-positive smooth muscle cells (SMC) (E) in the aortic root of Ldlr−/− mice treated with either control anti-miR or anti-miR-302a. Representative images are shown. (F) Quantification of necrotic cores within aortic root lesion of Ldlr−/− mice with either control anti-miR or anti-miR-302a. Data are expressed as mean ± SEM. *P<0.05, **P<0.005. 2-tailed Student’s t test was used.

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MicroRNA 302a is a novel modulator of cholesterol homeostasis and atherosclerosis - PubMed (original) (raw)