Department of Cerebrovascular Diseases, Blue Cross Brain Hospital Affiliated to Tongji University, Shanghai, 201101, China.
Department of Cerebrovascular Diseases, Blue Cross Brain Hospital Affiliated to Tongji University, Shanghai, 201101, China.
Biochimie. 2019 Oct;165:141-149. doi: 10.1016/j.biochi.2019.07.020. Epub 2019 Jul 28.
MicroRNA-25-5p (miR-25-5p) may be involved in the pathogenesis and processes of vascular diseases. The aim of this study was to investigate the role of miR-25-5p in oxidized low-density lipoprotein (ox-LDL)-treated human brain microvessel endothelial cells (HBMECs) and the underlying mechanisms. RT-qPCR and/or Western blot were used to detect the expression levels of miR-25-5p and neuronal growth regulator 1 (NEGR1). The effect of miR-25-5p overexpression and NEGR1 silencing on cell proliferation, migration, apoptosis and reactive oxygen species (ROS) production of HBMECs were measured by using CCK-8 assay, transwell assay and flow cytometry, respectively. The expression levels of apoptosis-related protein (cleaved caspase-3 and pro-caspase-3) were detected using Western blot, and the nitric oxide (NO) production was measured by a nitric oxide assay kit. The expression level of miR-25-5p was decreased in HBMECs treated with ox-LDL. Compared with the control group, miR-25-5p overexpression significantly promoted the proliferation and migration of HBMECs treated with ox-LDL (p < 0.01). Overexpression of miR-25-5p significantly suppressed cell apoptosis, ROS production and NO reduction of ox-LDL-induced HBMECs (p < 0.01). In addition, the target gene of miR-25-5p was predicted to be NEGR1 through Targetscan online analysis. The effect of NEGR1 silencing on cell proliferation, migration, apoptosis, ROS and NO production of ox-LDL-induced HBMECs was similar to that of miR-25-5p overexpression. Furthermore, miR-25-5p overexpression and NEGR1 silencing significantly downregulated the protein expression levels of JAK2 and STAT3. Thus, miR-25-5p neutralizes the effects of ox-LDL on multiple functions of HBMECs through suppressing the expression of NEGR1 via regulating the JAK/STA signaling pathway.
微小 RNA-25-5p（miR-25-5p）可能参与血管疾病的发病机制和进程。本研究旨在探讨 miR-25-5p 在氧化型低密度脂蛋白（ox-LDL）处理的人脑微血管内皮细胞（HBMEC）中的作用及其潜在机制。使用 RT-qPCR 和/或 Western blot 检测 miR-25-5p 和神经元生长调节剂 1（NEGR1）的表达水平。通过 CCK-8 测定、Transwell 测定和流式细胞术分别检测 miR-25-5p 过表达和 NEGR1 沉默对 HBMEC 细胞增殖、迁移、凋亡和活性氧（ROS）产生的影响。Western blot 检测凋亡相关蛋白（cleaved caspase-3 和 pro-caspase-3）的表达水平，用一氧化氮（NO）测定试剂盒测定 NO 的产生。ox-LDL 处理的 HBMEC 中 miR-25-5p 的表达水平降低。与对照组相比，ox-LDL 处理的 HBMEC 中 miR-25-5p 过表达显著促进细胞增殖和迁移（p<0.01）。miR-25-5p 过表达显著抑制 ox-LDL 诱导的 HBMEC 细胞凋亡、ROS 产生和 NO 减少（p<0.01）。此外，通过 Targetscan 在线分析预测 miR-25-5p 的靶基因是 NEGR1。NEGR1 沉默对 ox-LDL 诱导的 HBMEC 细胞增殖、迁移、凋亡、ROS 和 NO 产生的影响与 miR-25-5p 过表达相似。此外，miR-25-5p 过表达和 NEGR1 沉默显著下调了 JAK2 和 STAT3 蛋白表达水平。因此，miR-25-5p 通过调节 JAK/STAT 信号通路抑制 NEGR1 的表达，从而中和 ox-LDL 对 HBMEC 多种功能的影响。
