Li Sufang, Chen Hong, Ren Jingyi, Geng Qiang, Song Junxian, Lee Chongyou, Cao Chengfu, Zhang Jing, Xu Ning
Department of Cardiology, Peking University People's Hospital, No 11. Xizhimen South Street, Xicheng District, Beijing 100044, China.
Department of Cardiology, Peking University People's Hospital, No 11. Xizhimen South Street, Xicheng District, Beijing 100044, China.
Atherosclerosis. 2014 Dec;237(2):514-20. doi: 10.1016/j.atherosclerosis.2014.09.033. Epub 2014 Oct 18.
Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs.
Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3'-untranslated regions (3'UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation.
MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.
动脉粥样硬化是一种慢性炎症过程，在此过程中，血管内皮细胞（ECs）因炎症因子和生长因子等化学物质的作用而功能失调。组织因子（TF）的表达由上述化学物质在活化的内皮细胞中诱导产生。TF在破裂的动脉粥样硬化斑块上引发血栓形成，这在急性冠状动脉综合征（ACS）的发病过程中起着至关重要的作用。越来越多的证据表明，微小RNA作为动脉粥样硬化疾病的表观遗传调节因子发挥着重要作用。我们研究的目的是确定微小RNA-223（miR-223）是否在ECs中靶向TF。
生物信息学分析表明，TF是miR-223的一个潜在靶标。蛋白质印迹分析显示，肿瘤坏死因子α（TNF-α）处理4小时后，C57BL/6J小鼠主动脉及培养的内皮细胞（EA.hy926细胞和人脐静脉内皮细胞（HUVEC））中TF表达增加。在TNF-α处理的内皮细胞中，通过实时定量PCR检测发现TF mRNA也增加。实时定量PCR结果显示，在TNF-α处理的C57BL/6J小鼠主动脉及培养的内皮细胞中，miR-223水平下调。用miR-223模拟物或miR-223抑制剂转染内皮细胞，在mRNA和蛋白质水平上均改变了TF的表达。荧光素酶报告基因检测证实，miR-223通过与TF 3'非翻译区（3'UTR）序列结合来抑制TF表达。无论有无TNF-α刺激，过表达miR-223均可抑制TF的促凝血活性。
miR-223介导的TF表达抑制为凝血级联反应调控提供了一种新的分子机制，并提示了动脉粥样硬化斑块破裂过程中抗血栓形成的线索。
