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目的探讨miR-19b对三磷酸腺苷结合盒转运体A1(ABCA1)介导巨噬细胞胆固醇流出的影响及其机制。方法将miR-19b mimic或miR-19b inhibitor转染入THP-1源性巨噬细胞,液体闪烁计数仪检测细胞内胆固醇流出,油红O染色观察细胞内脂滴情况,生物信息学分析miR-19b与ABCA1 3’UTR的结合关系,荧光素酶报告基因检测miR-19b与ABCA1的结合情况,Western blot检测ABCA1蛋白水平。结果 miR-19b抑制巨噬细胞胆固醇流出,增加胞内脂质蓄积和泡沫细胞形成,而anti-miR-19b则刚好出现相反的结果。miR-19b与ABCA1 3’UTR的3110-3116位结合,且二者结合的自由能较低。miR-19b显著抑制荧光素酶活性及其巨噬细胞中ABCA1蛋白的表达。结论 miR-19b靶向沉默ABCA1进而抑制其介导的胆固醇流出,增加巨噬细胞内脂质蓄积。
Objective To investigate the effect and mechanism of miR-19b on cholesterol efflux from macrophage mediated by adenosine triphosphate binding cassette A1 (ABCA1). Methods miR-19b mimic or miR-19b inhibitor was transfected into THP-1-derived macrophages, and the intracellular cholesterol efflux was detected by liquid scintillation counter. The intracellular lipid droplets were observed by oil red O staining. The bioinformatics analysis of miR- 19b and ABCA1 3’UTR. The luciferase reporter gene assayed the binding of miR-19b to ABCA1 and the level of ABCA1 protein by Western blot. Results miR-19b inhibited macrophage cholesterol efflux, increased intracellular lipid accumulation and foam cell formation, while anti-miR-19b appeared to have the opposite result. miR-19b binds to positions 3110-3116 of ABCA1 3’UTR, and the binding energy of both is low. miR-19b significantly inhibited luciferase activity and ABCA1 protein expression in macrophages. Conclusion Targeted silencing of ABCA1 by miR-19b inhibits its cholesterol efflux and increases lipid accumulation in macrophages.