MicroRNA-223 coordinates cholesterol homeostasis

Abstract

MicroRNAs (miRNAs) regulate a wide variety of biological processes and contribute to metabolic homeostasis. Here, we demonstrate that microRNA-223 (miR-223), an miRNA previously associated with inflammation, also controls multiple mechanisms associated with cholesterol metabolism. miR-223 promoter activity and mature levels were found to be linked to cellular cholesterol states in hepatoma cells. Moreover, hypercholesterolemia was associated with increased hepatic miR-223 levels in athero-prone mice. miR-223 was found to regulate high-density lipoprotein-cholesterol (HDL-C) uptake, through direct targeting and repression of scavenger receptor BI, and to inhibit cholesterol biosynthesis through the direct repression of sterol enzymes 3-hydroxy-3-methylglutaryl-CoA synthase 1 and methylsterol monooxygenase 1 in humans. Additionally, miR-223 was found to indirectly promote ATP-binding cassette transporter A1 expression (mRNA and protein) through Sp3, thereby enhancing cellular cholesterol efflux. Finally, genetic ablation of miR-223 in mice resulted in increased HDL-C levels and particle size, as well as increased hepatic and plasma total cholesterol levels. In summary, we identified a critical role for miR-223 in systemic cholesterol regulation by coordinated posttranscriptional control of multiple genes in lipoprotein and cholesterol metabolism.

Keywords: atherosclerosis; posttranscriptional gene regulation; reverse cholesterol transport.

Fig. 1.

miR-223 expression is linked to cholesterol. (A) miR-223 and miR-33 changes with cholesterol depletion (10% lipoprotein-depleted serum, LPDS) in Huh7 cells, as determined by real-time PCR. n = 5–6. h, hours. Mann–Whitney nonparametric tests. (B and C) Simvastatin (5 µM), cholesterol depletion (10% LPDS), LDL (100 µg/mL) treatments in J774 and Huh7 cells for 24 h. (B) miR-223 levels in J774 and Huh7 cells; n = 5. (C) miR-223 promoter activity. miR-223 promoter driving secretory Gaussia luciferase activity, which was normalized to secreted alkaline phosphatase for transfection control. Ratios are reported as fold change to FBS conditions; n = 6, Mann–Whitney nonparametric test. LPDS (lipoprotein-depleted serum) was switched to FBS at 24 h. (D) Total cholesterol levels in liver of apolipoprotein E-null mice (Apoe^−/−) mice on chow diet for 4 wk compared with wild-type (WT), as determined by total cholesterol colorimetric assays; n = 4. (E) Hepatic miR-223 levels in Apoe^−/− mice fed chow diet for 4 wk, as determined by real-time PCR; n = 3–4. (F) Total cholesterol levels in livers of Apoe^−/− mice on cocoa butter diet (CBD) for 4 wk, as determined by total cholesterol colorimetric assays; n = 4. (G) Hepatic miR-223 levels in Apoe^−/− mice fed CBD for 4 wk, as determined by real-time PCR; n = 4. (H) Hepatic Hmgcr, Hmgcs1, Sp3, Abca1, and Scarb1 (SR-BI) mRNA levels in Apoe^−/− mice on CBD for 4 wk, as determined by real-time PCR; n = 4. Data are mean ± SEM. Mann–Whitney nonparametric tests. *P < 0.05, **P < 0.0001.

Fig. 2.

miR-223 represses HDL-cholesterol uptake. (A) SR-BI mRNA levels with miR-223 overexpression and inhibition (hairpin inhibitor against miR-223, HI-223) in Huh7 cells (100 nM), as determined by real-time PCR. Control miR-151 (n = 4), control siRNA (n = 8), miR-223 (n = 14), siRNA SR-BI (n = 8), and HI-223 (n = 3) 100 nM. One-way analysis of variance with Newman–Keuls Multiple Comparison Post-Test. (B) ABCA1 and SR-BI protein levels in Huh7 cells after miR-223 or control miR-455-3p overexpression at 48 h and 72 h, as determined by Western blots. (C) Gene reporter (luciferase) assays in HEK293 cells. SR-BI 3′ UTR luciferase reporter (500 ng). Shown is plus or minus 3-base deletion in the predicted miR-223 target site; 50 nM miR-223 mimetic. Firefly luciferase (FL) normalized to transfection control Renilla luciferase (RL); n = 3–9. Mann–Whitney nonparametric test. (D) HDL-C [³H-cholesteryl ester (CE)] uptake reported as fold change uptake in Huh7 cells. miR-223, siRNA SR-BI, or control miR-455-3p (100 nM); n = 3. Unpaired Student t test. *P < 0.05, **P < 0.0001.

Fig. 3.

miR-223 inhibits cholesterol biosynthesis. (A) Shown are 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) mRNA levels in Huh7 cells. Mock n = 4, control miRNA n = 4, miR-223 n = 4, and hairpin inhibitor-223 (HI-223) n = 4. One-way analysis of variance with Newman–Keuls Multiple Comparison Post-Test. (B) Methylsterol monooxygenase 1 (SC4MOL) mRNA levels in Huh7 cells. Mock n = 3, control miRNA n = 3, miR-223 n = 3, and hairpin inhibitor-223 (HI-223) n = 3. Unpaired t test. (C) Gene reporter (luciferase) assays in HEK293 cells. HMGCS1 3′ UTR luciferase reporter (500 ng). Plus or minus 3-base deletion in the predicted miR-223 target site (500 ng); 50 nM miR-223 mimetic. Firefly luciferase (FL) normalized to transfection control Renilla luciferase (RL); n = 6; Mann–Whitney nonparametric test. (D) Cholesterol biosynthesis in Huh7 cells, as determined by radiolabeled (³H-acetic acid) acetate incorporation assays. Control miR-106a n = 6 and miR-223 n = 5. Mann–Whitney nonparametric test. Data are mean ± SEM; *P < 0.05, **P < 0.01.

Fig. 4.

miR-223 promotes cholesterol efflux through ABCA1. (A) ABCA1 mRNA levels in Huh7 cells, as quantified by real-time PCR. Mock n = 6, control miR-151 n = 6, and miR-223 n = 9. Mann–Whitney nonparametric tests. (B) Cholesterol efflux to apoA-I (10 µg/mL) in Huh7 cells. Mock n = 11 and miR-223 n = 12. Percent efflux [media counts/total counts (media plus cell lysates)] reported as fold change Mann–Whitney nonparametric tests. (C) Sp3 transcription factor mRNA levels in Huh7 cells, as determined by microarray (Mock n = 6, miR-223 n = 6) and real-time PCR analysis [Mock n = 7, miR-223 n = 7, and hairpin inhibitor (HI-223) n = 7]. Mann–Whitney nonparametric tests. One-way analysis of variance with Newman–Keuls Multiple Comparison Post-Test. (D) Gene reporter (luciferase) assays in HEK293 cells. Sp3 3′ UTR luciferase reporter (500 ng). Firefly-Sp3 3′ UTR reporter with 2-base deletion in the predicted miR-223 target site (500 ng); 1 nM miR-223 mimetic. Firefly luciferase (FL) normalized to transfection control Renilla luciferase (RL); n = 21. Mann–Whitney nonparametric test. (E) Nuclear Sp3 transcriptional activity in Huh7 cells, as determined by Sp3 transcription factor binding assays. Mock n = 4, siRNA Sp3 n = 2, miR-223 n = 4, hairpin inhibitor miR-223 (HI-223) n = 3. Mann–Whitney nonparametric tests. (F) miR-223 expression after siRNA knockdown of Sp3 in Huh7 cells, as quantified by real-time PCR. Mock n = 6 and siRNA Sp3 n = 6. Mann–Whitney nonparametric tests. (G) Schematic of miR-223 efflux regulation. Data are mean ± SEM. *P < 0.05, **P < 0.0001.

Fig. 5.

miR-223 controls cholesterol homeostasis in vivo. (A) Significant differential gene (mRNA) expression changes in miR-223^−/− livers compared with wild-type (WT) controls. Benjamini–Hochberg false discovery rate corrected P < 0.05; absolute fold change > 1.5; n = 6. Volcano plot illustrating significant down-regulated and up-regulated genes (black dots). (B) Plasma total cholesterol (mg/dL) in miR-223^−/− and WT mice. WT n = 4, miR-223^−/− n = 21. Mann–Whitney nonparametric tests. (C) Fast Protein Liquid Chromatography profile of total cholesterol (mg/dL) from WT and miR-223^−/− mice. Pooled samples for each condition. (D and E) Colorimetric cholesterol assays on lipids extracted from miR-223^−/− and WT mouse livers; mg/dL reported as fold change; n = 9–10; Mann–Whitney Nonparametric test. (D) Total cholesterol, (E) free cholesterol, and (F) cholesteryl ester. (G) Schematic of miR-223 targets and regulatory modules associated with cholesterol metabolism. Cholesterol biosynthesis intermediates: a, acetoacetyl-CoA; b, beta-hydroxy-beta-methylglutaryl-CoA; c, 4,4-dimethylcholesta-8(9),24-dien-3beta-ol; d, 4-methyl,4-carboxycholesta-8(9) 24-dien-3beta-ol. Data are mean ± SEM. *P < 0.05.