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.

中文翻译：

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MicroRNA-25-5p通过靶向人脑微血管内皮细胞中的神经元生长调节剂1（NEGR1）来抵消氧化的LDL诱导的病理变化。

MicroRNA-25-5p（miR-25-5p）可能参与血管疾病的发病机理和过程。这项研究的目的是调查miR-25-5p在氧化的低密度脂蛋白（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。RT-qPCR和/或蛋白质印迹用于检测miR-25-5p和神经元生长调节剂1（NEGR1）的表达水平。分别通过CCK-8测定，transwell测定和流式细胞术测量miR-25-5p过表达和NEGR1沉默对HBMECs细胞增殖，迁移，凋亡和活性氧（ROS）产生的影响。用Western blot检测凋亡相关蛋白（裂解的caspase-3和caspase-3）的表达水平，并通过一氧化氮测定试剂盒测量一氧化氮（NO）的产生。在ox-LDL处理的HBMEC中，miR-25-5p的表达水平降低。与对照组相比，miR-25-5p过表达显着促进了用ox-LDL处理的HBMEC的增殖和迁移（p <0.01）。miR-25-5p的过表达显着抑制了ox-LDL诱导的HBMECs的细胞凋亡，ROS的产生和NO的减少（p <0.01）。另外，通过Targetscan在线分析预测miR-25-5p的靶基因为NEGR1。NEGR1沉默对ox-LDL诱导的HBMECs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沉默显着下调了JAK2和STAT3的蛋白表达水平。因此，miR-25-5p通过调节JAK / STA信号通路抑制NEGR1的表达，从而中和了ox-LDL对HBMECs多种功能的影响。