miR-374b-5p 在深静脉血栓形成中增加，并负向靶向作用于 IL-10。
miR-374b-5p is increased in deep vein thrombosis and negatively targets IL-10.
机构信息
School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, 18877 Jingshi Road, Jinan 250062, Shandong, China; Laboratory for Molecular Immunology, Institute of Basic Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 18877 Jingshi Road, Jinan 250062, Shandong, China.
Department of Peripheral Vascular Disease, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Bianque Building, 16369 Jingshi Road, Jinan 250014, Shandong, China.
出版信息
J Mol Cell Cardiol. 2020 Jul;144:97-108. doi: 10.1016/j.yjmcc.2020.05.011. Epub 2020 May 21.
BACKGROUND
Deep venous thrombosis (DVT) is one of the most common venous thromboembolic (VTE) disorders and the third leading cardiovascular complication. Accumulating evidence has shown that decreased interleukin-10 (IL-10) was involved in DVT. However, the underlying molecular mechanisms are still largely unknown. Here, we proposed that the epigenetic modification of IL-10 at the post-transcriptional level may be a crucial trigger for IL-10 down-regulation in DVT.
METHODS
miRNA expression in DVT was profiled by miRNA microarray analysis. The upstream miRNA regulators of IL-10 were predicted by in silico target prediction tools. The expression of IL-10 mRNA and miR-374b-5p were examined by quantitative real-time PCR (qRT-PCR) and the protein expression of IL-10 was detected by enzyme-linked immunoassay. Dual luciferase reporter assay was used to identify the interaction between miR-374b-5p and IL10. A murine model of DVT was developed and the localization of miR-374b-5p was visualized in vitro by fluorescence in situ hybridization. The biological effects of miR-374b-5p on IL-10 was examined both in vitro and in vivo.
RESULTS
Microarray and qRT-PCR results showed that the IL-10 expression was decreased while miR-374b-5p level was increased substantially in peripheral blood mononuclear cells of DVT patients, and there was significant negative correlation between miR-374b-5p and IL-10. Experiments in vitro showed that overexpressed miR-374b-5p reduced IL-10 expression, while miR-374b-5p knockdown increased IL-10 expression. Moreover, in vivo studies revealed that DVT mice with anti-IL-10 antibody or agomiR-374b-5p delivery resulted in decreased IL-10 expression and aggravated DVT formation, whereas antagomiR-374b-5p acted inversely. Dual luciferase reporter assay identified direct binding between miR-374b-5p and IL10.
CONCLUSIONS
These findings suggest that increased miR-374b-5p promotes DVT formation by downregulating IL-10 expression. miR-374b-5p may be explored as a promising diagnostic marker and therapeutic target for DVT.
背景
深静脉血栓形成（DVT）是最常见的静脉血栓栓塞（VTE）疾病之一，也是心血管并发症的第三大主要原因。越来越多的证据表明，白细胞介素-10（IL-10）的减少参与了 DVT 的发生。然而，其潜在的分子机制在很大程度上仍不清楚。在这里，我们提出，IL-10 在转录后水平的表观遗传修饰可能是 DVT 中 IL-10 下调的关键触发因素。
方法
通过 miRNA 微阵列分析来描绘 DVT 中的 miRNA 表达。通过计算机目标预测工具预测 IL-10 的上游 miRNA 调节剂。通过定量实时 PCR（qRT-PCR）检测 IL-10 mRNA 和 miR-374b-5p 的表达，通过酶联免疫吸附试验检测 IL-10 蛋白的表达。双荧光素酶报告基因实验用于鉴定 miR-374b-5p 与 IL10 之间的相互作用。建立 DVT 小鼠模型，通过荧光原位杂交技术在体外观察 miR-374b-5p 的定位。通过体外和体内实验研究 miR-374b-5p 对 IL-10 的生物学作用。
结果
微阵列和 qRT-PCR 结果显示，DVT 患者外周血单个核细胞中 IL-10 的表达降低，而 miR-374b-5p 水平显著升高，miR-374b-5p 与 IL-10 呈显著负相关。体外实验表明，过表达 miR-374b-5p 降低了 IL-10 的表达，而 miR-374b-5p 敲低则增加了 IL-10 的表达。此外，体内研究表明，用抗 IL-10 抗体或 agomiR-374b-5p 处理 DVT 小鼠导致 IL-10 表达降低和 DVT 形成加重，而反义 miR-374b-5p 则作用相反。双荧光素酶报告基因实验鉴定出 miR-374b-5p 与 IL10 之间的直接结合。
结论
这些发现表明，miR-374b-5p 通过下调 IL-10 表达促进 DVT 的形成。miR-374b-5p 可作为 DVT 的有前途的诊断标志物和治疗靶点进行探索。
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