Department of Cardiovascular Surgery Intensive Care Unit, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China.
Department of Critical Care Medicine, The Second Affiliated Hospital of Hainan Medical University, No. 368 Yehai Avenue, Longhua District, Haikou, Hainan, China.
Neurochem Res. 2021 Nov;46(11):2885-2896. doi: 10.1007/s11064-021-03387-x. Epub 2021 Jul 5.
Brain microvascular endothelial cells (BMECs) injury is one of the main causes of cerebrovascular diseases. Circular RNA (circRNA) has been found to be involved in the regulation of cerebrovascular diseases progression. However, the role and mechanism of circ_0003423 in cerebrovascular diseases is still unclear. In our study, oxidized low density lipoprotein (ox-LDL)-induced HBMEC-IM cells were used to construct cerebrovascular cell injury model in vitro. Quantitative real-time PCR was used to determine the expression levels of circ_0003423, miR-589-5p and Ten-eleven translocation 2 (TET2). The interactions between miR-589-5p and circ_0003423 or TET2 were confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Cell viability, angiogenesis and apoptosis were measured using cell counting kit 8 assay, tube formation assay and flow cytometry. Cell oxidative stress was evaluated by detecting the levels of reactive oxygen species and lactate dehydrogenase. The protein levels were examined by western blot analysis. Our results showed that circ_0003423 was a downregulated circRNA in ox-LDL-induced HBMEC-IM cells. In the terms of mechanism, circ_0003423 was found to be a sponge of miR-589-5p. Function analysis showed that circ_0003423 overexpression could relieve ox-LDL-induced HBMEC-IM cell injury, and this effect could be reversed by miR-589-5p mimic. In addition, TET2 was confirmed to be a target of miR-589-5p, and its overexpression could alleviate ox-LDL-induced HBMEC-IM cell injury. Moreover, the rescue experiments also confirmed that TET2 silencing could abolish the inhibition effect of anti-miR-589-5p on ox-LDL-induced HBMEC-IM cell injury. In summary, our data showed that circ_0003423 alleviated ox-LDL-induced HBMEC-IM cells injury through regulating the miR-589-5p/TET2 axis.
脑微血管内皮细胞（BMEC）损伤是脑血管疾病的主要原因之一。环状 RNA（circRNA）已被发现参与了脑血管疾病进展的调控。然而，circ_0003423 在脑血管疾病中的作用和机制尚不清楚。在我们的研究中，使用氧化型低密度脂蛋白（ox-LDL）诱导的 HBMEC-IM 细胞构建了体外脑血管细胞损伤模型。定量实时 PCR 用于测定 circ_0003423、miR-589-5p 和 Ten-eleven 转位酶 2（TET2）的表达水平。通过双荧光素酶报告基因检测、RIP 检测和 RNA 下拉实验证实了 miR-589-5p 与 circ_0003423 或 TET2 之间的相互作用。通过细胞计数试剂盒 8 检测、管形成检测和流式细胞术检测细胞活力、血管生成和细胞凋亡。通过检测活性氧和乳酸脱氢酶的水平评估细胞氧化应激。通过 Western blot 分析检测蛋白水平。结果显示，circ_0003423 在 ox-LDL 诱导的 HBMEC-IM 细胞中呈下调表达。就机制而言，circ_0003423 被发现是 miR-589-5p 的海绵。功能分析表明，circ_0003423 的过表达可以减轻 ox-LDL 诱导的 HBMEC-IM 细胞损伤，而 miR-589-5p 模拟物可以逆转这种作用。此外，TET2 被证实是 miR-589-5p 的靶标，其过表达可以减轻 ox-LDL 诱导的 HBMEC-IM 细胞损伤。此外，挽救实验还证实，沉默 TET2 可以消除抗 miR-589-5p 对 ox-LDL 诱导的 HBMEC-IM 细胞损伤的抑制作用。综上所述，我们的数据表明，circ_0003423 通过调节 miR-589-5p/TET2 轴减轻 ox-LDL 诱导的 HBMEC-IM 细胞损伤。
