MicroRNA-24 通过抑制清道夫受体 B 类 I 的转录后抑制从高密度脂蛋白胆固醇中选择性摄取脂质，从而加剧动脉粥样硬化。

MicroRNA-24 aggravates atherosclerosis by inhibiting selective lipid uptake from HDL cholesterol via the post-transcriptional repression of scavenger receptor class B type I.

机构信息

Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, University of South China, Hengyang City, 421001, Hunan Province, China.

Department of Histology and Embryology, University of South China, Hengyang, Hunan, 421001, China.

出版信息

Atherosclerosis. 2018 Mar;270:57-67. doi: 10.1016/j.atherosclerosis.2018.01.045. Epub 2018 Feb 4.

DOI:10.1016/j.atherosclerosis.2018.01.045

PMID:29407889

Abstract

BACKGROUND AND AIMS

Liver scavenger receptor class B type I (SR-BI) exerts atheroprotective effects through selective lipid uptake (SLU) from high-density lipoprotein cholesterol (HDL-C). Low hepatic SR-BI expression leads to high HDL-C levels in the circulation and an increased risk of atherosclerosis. Furthermore, macrophage SR-BI mediates bidirectional cholesterol flux and may protect against atherogenesis. Previous studies have revealed that miR-24 is closely related to cardiovascular disease (CVD) progression. We aimed to investigate the molecular mechanisms by which miR-24 participates in SR-BI-mediated selective HDL cholesteryl ester (HDL-CE) uptake and further atherogenesis in apoE mice.

METHODS

Bioinformatic predictions and luciferase reporter assays were utilized to detect the association between miR-24 and the SR-BI 3' untranslated region (3' UTR), and RT-PCR and western blotting were used to evaluate SR-BI mRNA and protein expression, respectively. The effects of miR-24 on Dil-HDL uptake were determined by flow cytometry assay. Double-radiolabeled HDL (I-TC-/[H] CEt-HDL) was utilized to measure the effects of miR-24 on HDL and CE binding and SLU in HepG2 and PMA-treated THP-1 cells. In addition, total cholesterol (TC) levels in HepG2 cells were analyzed using enzymatic methods, and macrophage lipid content was evaluated by high-performance liquid chromatography (HPLC) assay. Small interfering RNA (siRNA) and pcDNA3.1(-)-hSR-BI plasmid transfection procedures were utilized to confirm the role of SR-BI in the effects of miR-24 on Dil-HDL uptake, SLU and cholesterol levels in both cell types. Hepatic SR-BI level in apoE mice was measured by western blotting. Liver TC, FC and CE levels and plasma triglycerides (TG), TC and HDL-C levels were evaluated enzymatically using commercial test kits. Atherosclerotic lesion sizes were measured using Oil Red O and hematoxylin-eosin staining.

RESULTS

miR-24 directly repressed SR-BI expression by targeting its 3'UTR. In addition, miR-24 decreased Dil-HDL uptake and SLU in HepG2 and THP-1 macrophages. In the presence of HDL, miR-24 decreased TC levels in HepG2 cells and TC, free cholesterol (FC) and CE levels in macrophages. Overexpression and down-regulation assays showed that SR-BI mediated the effects of miR-24 on Dil-HDL uptake, SLU and cholesterol levels. Lastly, miR-24 administration decreased hepatic SR-BI expression and promoted atheromatous plaque formation in apoE mice, findings in line with those of our in vitro studies.

CONCLUSIONS

These findings indicate that miR-24 accelerates atherogenesis by repressing SR-BI-mediated SLU from HDL-C.

摘要

背景与目的

肝 X 受体 B 型 I 型（SR-BI）通过从高密度脂蛋白胆固醇（HDL-C）选择性摄取脂质（SLU）发挥抗动脉粥样硬化作用。肝内 SR-BI 表达降低导致循环中 HDL-C 水平升高，动脉粥样硬化风险增加。此外，巨噬细胞 SR-BI 介导胆固醇的双向流动，可能有助于防止动脉粥样形成。先前的研究表明，miR-24 与心血管疾病（CVD）的进展密切相关。本研究旨在探讨 miR-24 参与 SR-BI 介导的选择性 HDL 胆甾醇酯（HDL-CE）摄取以及进一步在载脂蛋白 E （apoE）小鼠中发生动脉粥样硬化的分子机制。

方法

生物信息学预测和荧光素酶报告基因检测用于检测 miR-24 与 SR-BI 3'非翻译区（3'UTR）之间的关联，并用 RT-PCR 和 Western blot 分别评估 SR-BI mRNA 和蛋白表达。用流式细胞术检测 miR-24 对 Dil-HDL 摄取的影响。用双放射性标记的 HDL（I-TC-/[H]CEt-HDL）测量 miR-24 对 HepG2 和 PMA 处理的 THP-1 细胞中 HDL 和 CE 结合和 SLU 的影响。此外，用酶法分析 HepG2 细胞中的总胆固醇（TC）水平，用高效液相色谱法（HPLC）分析巨噬细胞脂质含量。用小干扰 RNA（siRNA）和 pcDNA3.1（-）-hSR-BI 质粒转染程序来确认 SR-BI 在 miR-24 对 Dil-HDL 摄取、SLU 和两种细胞类型中的胆固醇水平的影响中的作用。用 Western blot 检测 apoE 小鼠肝内 SR-BI 水平。用商业试剂盒用酶法测定肝 TC、FC 和 CE 水平以及血浆甘油三酯（TG）、TC 和 HDL-C 水平。用油红 O 和苏木精-伊红染色测量动脉粥样硬化病变大小。

结果

miR-24 通过靶向其 3'UTR 直接抑制 SR-BI 的表达。此外，miR-24 降低了 HepG2 和 THP-1 巨噬细胞中的 Dil-HDL 摄取和 SLU。在 HDL 存在的情况下，miR-24 降低了 HepG2 细胞中的 TC 水平以及巨噬细胞中的 TC、游离胆固醇（FC）和 CE 水平。过表达和下调实验表明，SR-BI 介导了 miR-24 对 Dil-HDL 摄取、SLU 和胆固醇水平的影响。最后，miR-24 给药降低了肝内 SR-BI 的表达并促进了 apoE 小鼠的动脉粥样硬化斑块形成，这与我们的体外研究结果一致。