miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DEAD-box helicase 6 (DDX6) under hypoxia

Stroke is a main cause of disability and death worldwide, and ischemic stroke accounts for most stroke cases. Recently, microRNAs (miRNAs) have been verified to play critical roles in the development of stroke. Herein, we explored effects of miR-152-3p on vascular endothelial cell functions under hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia to mimic cell injury in vitro. Reverse transcription quantitative polymerase chain reaction revealed that miR-152-3p exhibited high expression in HUVECs treated with hypoxia. The inhibition of miR-152-3p reversed hypoxia-induced decrease in cell viability and the increase in angiogenesis, according to the results of cell counting kit-8 assays and tube formation assays. miR-152-3p inhibition reversed the increase in endothelial cell permeability mediated by hypoxia, as shown by endothelial cell permeability in vitro assays. In addition, the increase in protein levels of angiogenetic markers and the decrease in levels of tight junction proteins induced by hypoxia were reversed by miR-152-3p inhibition. Mechanistically, miR-152-3p directly targets 3ʹ-untranslated region of DEAD-box helicase 6 (DDX6), which was confirmed by luciferase reporter assays. DDX6 is lowly expressed in HUVECs under hypoxic condition, and mRNA expression and protein level of DDX6 were upregulated in HUVECs due to miR-152-3p inhibition. Rescue assays showed that DDX6 knockdown reversed effects of miR-152-3p on cell viability, angiogenesis and endothelial permeability. The results demonstrated that miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DDX6 under hypoxia.脑卒中（Stroke）是全球范围内导致残疾与死亡的主要病因，其中缺血性脑卒中（ischemic stroke）占脑卒中病例的绝大多数。近期研究证实，微小RNA（microRNAs, miRNAs）在脑卒中的发生发展中发挥关键调控作用。本研究探讨了miR-152-3p在缺氧条件下对血管内皮细胞功能的影响。我们采用缺氧处理人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）以体外模拟细胞损伤模型。逆转录定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction）结果显示，缺氧处理的HUVECs中miR-152-3p呈高表达状态。通过细胞计数试剂盒-8（cell counting kit-8）实验与管形成实验可知，抑制miR-152-3p可逆转缺氧诱导的细胞活力降低与血管生成增多现象。体外内皮细胞通透性实验结果表明，抑制miR-152-3p可逆转缺氧介导的内皮细胞通透性升高。此外，缺氧诱导的血管生成标志物蛋白水平升高、紧密连接蛋白（tight junction proteins）水平降低，均能被miR-152-3p抑制所逆转。机制层面，荧光素酶报告基因实验（luciferase reporter assays）证实，miR-152-3p可直接靶向DEAD-box解旋酶6（DEAD-box helicase 6, DDX6）的3'非翻译区（3'-untranslated region）。缺氧条件下HUVECs中DDX6呈低表达，而抑制miR-152-3p可上调HUVECs中DDX6的mRNA表达与蛋白水平。挽救实验（rescue assays）结果显示，敲低DDX6可逆转miR-152-3p对细胞活力、血管生成及内皮通透性的调控作用。本研究结果表明，在缺氧条件下，miR-152-3p通过靶向DDX6加重血管内皮细胞功能障碍。