MicroRNA-10a/b靶向调控ABCA1和ABCG1对胆固醇流出的影响

The ATP binding cassette transporter A1 (ABCA1) and ABCG1 are major regulators of reverse cholesterol transport. miR-33 is the only miRNA that can regulate the ABCA1 and ABCG1. Bioinformatics prediction has become an important method in search for miRNA target genes. Many authoritative bioinformatics website analysis shows that miR-10a/b is highly conserved, a high degree of integration with ABCA1 and ABCG1. Moreover, Preliminary experiments show that miR-10b combines the 3′UTR of ABCA1 and ABCG1. miR-10b decreases the expression of ABCA1 and ABCG1 in THP-1 macrophage-derived foam cells; Serological detection pointed out the clinical role of miR-10a/b is similar to miR-33. So we propose the following hypothesis that miR-10a/b can inhibit cholesterol efflux and promote atherosclerosis, through targeting regulate the expression of ABCA1 and ABCG1. In this study, we will adopt luciferase reporter assay to clear whether miR-10a/b can directly combine with the ABCA1; Overexpression or silencing of THP-1 and RAW264.7 cells of miR-10a/b, we intend to observe the expression of ABCA1 and ABCG1, lipid droplets, cholesterol efflux and cholesterol ester content; Overexpression or silencing apoE-/- mice of miR-10a/b, we will detect the expression of ABCA1 and ABCG1, HDL, plaque area and the efficiency of reverse cholesterol transport impact. We expect to reveal the molecular mechanisms of miR-10a/b leading to atherosclerosis and find a new target for regulation of ABCA1 and ABCG1.

ABCA1/G1是胆固醇逆转运（RCT）的关键蛋白，可抗AS。miR-33是唯一对ABCA1/G1同时起作用的miRNA。多个靶点预测网站分析：miR-10a/b高度保守，可与ABCA1/G1结合。预实验结果示：miR-10b可与ABCA1/G1靶向结合；miR-10b可降低泡沫细胞ABCA1/G1表达；血清学检测示miR-10a/b与miR-33临床意义相似。为此，我们提出假说：miR-10a/b可靶向沉默ABCA1/G1，抑制RCT。本项目拟采用荧光素酶报告基因实验分析miR-10a/b对ABCA1/G1的直接作用；过表达或沉默泡沫细胞miR-10a/b，观察对细胞ABCA1/G1，胆固醇含量，胆固醇流出的影响；过表达或沉默apoE-/-鼠miR-10a/b，观察对ABCA1/G1，HDL，AS斑块面积，胆固醇逆转运效率的影响。有望发现调控ABCA1/G1新靶点，为AS防治提供新途径。

1本项目主要研究“miR-10a/b靶向沉默ABCA1和ABCG1对胆固醇逆转运的影响”。研究内容分为三部分：1、利用荧光素酶报告基因实验，研究miR-10a/b与ABCA1和ABCG1的靶向结合。结果显示，miR-10a/b可降低野生型ABCA1 和ABCG1的3’UTR荧光素酶活性；当ABCA1 和ABCG1的 3’UTR 突变两个序列后，miR-10a/b不能降低ABCA1 3’UTR荧光素酶活性；Con miR不能降低野生型和突变后ABCA1 和ABCG1的3’UTR荧光素酶活性。说明miR-10a/b可靶向结合ABCA1和ABCG1的mRNA 3’UTR。2、细胞水平研究miR-10a/b可靶向沉默ABCA1和ABCG1的表达，并对RCT的作用。结果显示，与对照组相比，miR-10a/b可降低THP-1源性泡沫细胞ABCA1和ABCG1的 mRNA和蛋白的表达；miR-10a/b inhibitor 可升高ABCA1和ABCG1的 mRNA和蛋白的表达。从而，miR-10a/b降低THP-1源性泡沫细胞的胆固醇流出率。3、利用ApoE基因敲除小鼠研究miR-10a/b对AS的影响。结果显示，miR-10a/b可加重apoE-/-小鼠的动脉粥样硬化病变，而anti-miR-10a/b 可减轻apoE-/-小鼠的动脉粥样硬化病变；miR-10a/b 可减少apoE-/-小鼠 HDL的含量，升高LDL，而anti-miR-10a/b可增加HDL的含量，降低LDL含量。总之，miR-10a/b可抑制ABCA1和ABCG1的表达，降低胆固醇流出率，升高细胞内胆固醇，加重动脉粥样硬化。miR-10a/b inhibitor作用与miR10a/b相反。本项目研究成果，发表SCI 论文1篇，IF5.5分，本项目培养硕士研究生1名。.