Zheng Zhanqiang, Zhang Guanglin, Liang Xiaodong, Li Tianxiao
Department of Neurosurgery, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China.
Department of Neurointervention, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China.
Mol Cell Biochem. 2021 Jan;476(1):443-455. doi: 10.1007/s11010-020-03921-5. Epub 2020 Sep 29.
Cerebrovascular diseases have a high mortality and disability rate in developed countries. Endothelial cell injury is the main cause of atherosclerosis and cerebrovascular disease. Long non-coding RNA (lncRNA) has been proved to participate in the progression of endothelial cell. Our study aimed to develop the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury. The expression of OIP5-AS1, miR-98-5p and High-mobility group protein box-1 (HMGB1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect the cell proliferation and apoptosis. The levels of cyclinD1, Bcl-2 Associated X Protein (Bax), Cleaved-caspase-3, Toll like receptors 4 (TLR4), phosphorylation of p65 (p-P65), phosphorylation of nuclear factor-kappa B inhibitor α (p-IκB-α) and HMGB1 were measured by Western blot. The concentrations of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were detected by Enzyme-linked immunosorbent assay (ELISA). The production of Reactive oxygen species (ROS), Superoxide Dismutase (SOD) and malondialdehyde (MDA) was detected by the corresponding kit. The targets of OIP5-AS and miR-98-5p were predicted by starBase 3.0 and TargetScan and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of OIP5-AS1 was upregulated, while miR-98-5p was downregulated in ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, inflammatory injury and oxidative stress injury in ox-LDL-induced HUVEC cells. Interestingly, miR-98-5p was a target of OIP5-AS1 and miR-98-5p inhibition abolished the effects of OIP5-AS1 downregulation on ox-LDL-induced HUVECs injury. More importantly, miR-98-5p directly targeted HMGB1, and OIP5-AS1 regulated the expression of HMGB1 by sponging miR-98-5p. Finally, OIP5-AS1 regulated the TLR4/nuclear factor-kappa B (NF-κB) signaling pathway through miR-98-5p/HMGB1 axis. LncRNA OIP5-AS1 accelerates ox-LDL-induced endothelial cell injury through regulating HMGB1 mediated by miR-98-5p via the TLR4/NF-κB signaling pathway.
在发达国家,脑血管疾病具有较高的死亡率和致残率。内皮细胞损伤是动脉粥样硬化和脑血管疾病的主要原因。长链非编码RNA(lncRNA)已被证明参与内皮细胞的进展。我们的研究旨在探究长链非编码RNA opa相互作用蛋白5反义RNA 1(OIP5-AS1)在氧化型低密度脂蛋白(ox-LDL)诱导的内皮细胞损伤中的作用。通过定量实时聚合酶链反应(qRT-PCR)检测OIP5-AS1、miR-98-5p和高迁移率族蛋白盒1(HMGB1)的表达。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴盐(MTT)法和流式细胞术检测细胞增殖和凋亡。通过蛋白质免疫印迹法检测细胞周期蛋白D1、Bcl-2相关X蛋白(Bax)、裂解的半胱天冬酶-3、Toll样受体4(TLR4)、p65的磷酸化(p-P65)、核因子-κB抑制因子α的磷酸化(p-IκB-α)和HMGB1的水平。采用酶联免疫吸附测定(ELISA)检测白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的浓度。通过相应试剂盒检测活性氧(ROS)、超氧化物歧化酶(SOD)和丙二醛(MDA)的产生。通过starBase 3.0和TargetScan预测OIP5-AS和miR-98-5p的靶标,并通过双荧光素酶报告基因测定和RNA免疫沉淀(RIP)测定进行验证。在ox-LDL诱导的人脐静脉内皮细胞(HUVECs)中,OIP5-AS1的表达上调,而miR-98-5p的表达下调。在功能上,敲低OIP5-AS1可诱导ox-LDL诱导的HUVEC细胞增殖,并抑制其凋亡、炎症损伤和氧化应激损伤。有趣的是,miR-98-5p是OIP5-AS1的靶标,抑制miR-98-5p可消除OIP5-AS1下调对ox-LDL诱导的HUVECs损伤的影响。更重要的是,miR-98-5p直接靶向HMGB1,OIP5-AS1通过海绵吸附miR-98-5p来调节HMGB1的表达。最后,OIP5-AS1通过miR-98-5p/HMGB1轴调节TLR4/核因子-κB(NF-κB)信号通路。长链非编码RNA OIP5-AS1通过TLR4/NF-κB信号通路,经由miR-98-5p介导调节HMGB1,从而加速ox-LDL诱导的内皮细胞损伤。