Tan Lina, Wang Lingjun, Liu Jiajun, Yu Yue
Department of Neurology, The First Affiliated Hospital of Qiqihar Medical University, Hengtai B District Fularji District, Qiqihar, 161000, Heilongjiang, China.
Department of Cardiovascular Medicine, QiqiharNO.1 Hospital, Qiqihar, Heilongjiang, China.
Exp Brain Res. 2023 Mar;241(3):781-791. doi: 10.1007/s00221-023-06555-3. Epub 2023 Feb 3.
The pivotal regulatory role of circular RNAs (circRNAs) in ischemic stroke (IS) has been expounded. The study aimed to probe the exact role and underlying mechanism of circVRK1 in oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cells (HBMECs) injury. HBMECs challenged by OGD were used as in vitro models of IS. Quantitative real-time PCR was used to examine the levels of circVRK1, vaccinia-related kinase 1 (VRK1), miR-150-5p and MLLT1 mRNA. Cell viability, migration angiogenesis ability and death were evaluated by Cell counting kit-8 assay, transwell assay, wound-healing assay, tube formation assay and flow cytometry analysis. All the protein levels were monitored by western blot assay. Enzyme-linked immunosorbent assay was conducted for examining cell oxidative stress. Dual-luciferase reporter assay, RIP assay and RNA pull-down assay were performed to verify the combination between miR-150-5p and circVRK1 or MLLT1. CircVRK1 was upregulated in OGD-treated HBMECs. CircVRK1 knockdown alleviated OGD-caused effects on HBMECs migration, angiogenesis, death, inflammatory response and oxidative stress. Furthermore, circVRK1 could sponge miR-150-5p, and miR-150-5p silencing also mitigated the impact of circVRK1 deficiency on OGD-evoked injury. Besides, MLLT1 acted as a molecular target of miR-150-5p, and the protective influence of miR-150-5p on OGD-induced cell damage was overturned by MLLT1 introduction. CircVRK1 knockdown weakened OGD-evoked injury in HBMECs through modulating miR-150-5p/MLLT1 pathway, and this might supply new insights and probable targets for IS treatment.
环状RNA（circRNAs）在缺血性脑卒中（IS）中的关键调控作用已得到阐述。本研究旨在探究circVRK1在氧糖剥夺（OGD）诱导的人脑微血管内皮细胞（HBMECs）损伤中的具体作用及潜在机制。受OGD刺激的HBMECs被用作IS的体外模型。采用定量实时聚合酶链反应检测circVRK1、痘苗相关激酶1（VRK1）、miR-150-5p和MLLT1 mRNA的水平。通过细胞计数试剂盒-8检测、Transwell检测、伤口愈合检测、管腔形成检测和流式细胞术分析评估细胞活力、迁移血管生成能力和细胞死亡情况。所有蛋白质水平均通过蛋白质免疫印迹法监测。采用酶联免疫吸附测定法检测细胞氧化应激。进行双荧光素酶报告基因检测、RNA免疫沉淀检测和RNA下拉检测以验证miR-150-5p与circVRK1或MLLT1之间的结合。circVRK1在OGD处理的HBMECs中上调。敲低circVRK1可减轻OGD对HBMECs迁移、血管生成、细胞死亡、炎症反应和氧化应激的影响。此外，circVRK1可以吸附miR-150-5p，沉默miR-150-5p也可减轻circVRK1缺陷对OGD诱发损伤的影响。此外，MLLT1是miR-150-5p的分子靶点，引入MLLT1可逆转miR-150-5p对OGD诱导的细胞损伤的保护作用。敲低circVRK1通过调节miR-150-5p/MLLT1通路减弱OGD诱发的HBMECs损伤，这可能为IS治疗提供新的见解和潜在靶点。
