Zhao Minghu, Yang Yuanyuan, Li Jingchao, Lu Min, Wu Yu
Department of Cardiovascular Comprehensive Ward II, Henan Provincial People's Hospital, Zhengzhou, China.
Front Cardiovasc Med. 2021 Mar 12;8:596506. doi: 10.3389/fcvm.2021.596506. eCollection 2021.
Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. LncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been found to be associated with the development of atherosclerosis. In this study, we further investigated the molecular basis of OIP5-AS1 in atherosclerosis pathogenesis. Oxidative low-density lipoprotein (ox-LDL) was used to treat human umbilical vein endothelial cells (HUVECs). The levels of OIP5-AS1, miR-135a-5p, and Krüppel-like factor 5 (KLF5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell viability, migration, and apoptosis were evaluated using the Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and malondialdehyde (MDA) were determined with enzyme-linked immunosorbent assay (ELISA). Targeted interactions among OIP5-AS1, miR-135a-5p, and KLF5 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Animal studies were performed to assess the role of OIP5-AS1 in atherosclerosis progression . Our data showed the significant upregulation of OIP5-AS1 in atherosclerosis serum and ox-LDL-stimulated HUVECs. The silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs and diminished lipids secretion in ApoE mice. Moreover, OIP5-AS1 functioned as a molecular sponge of miR-135a-5p, and miR-135a-5p was a functional mediator of OIP5-AS1 in regulating ox-LDL-induced HUVEC injury. KLF5 was a direct target of miR-135a-5p, and the increased expression of miR-135a-5p alleviated ox-LDL-induced cytotoxicity by downregulating KLF5. Furthermore, OIP5-AS1 influenced KLF5 expression through sponging miR-135a-5p. The current work identified that the silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs at least in part by influencing KLF5 expression via acting as a miR-135a-5p sponge.
长链非编码RNA（lncRNAs）与动脉粥样硬化的发病机制有关。已发现lncRNA OIP5反义RNA 1（OIP5-AS1）与动脉粥样硬化的发展有关。在本研究中，我们进一步探讨了OIP5-AS1在动脉粥样硬化发病机制中的分子基础。使用氧化型低密度脂蛋白（ox-LDL）处理人脐静脉内皮细胞（HUVECs）。通过定量实时聚合酶链反应（qRT-PCR）或蛋白质免疫印迹法检测OIP5-AS1、miR-135a-5p和Krüppel样因子5（KLF5）的水平。分别使用细胞计数试剂盒-8（CCK-8）、Transwell小室和流式细胞术评估细胞活力、迁移和凋亡。用酶联免疫吸附测定（ELISA）法测定白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和丙二醛（MDA）的水平。通过双荧光素酶报告基因和RNA免疫沉淀（RIP）实验证实OIP5-AS1、miR-135a-5p和KLF5之间的靶向相互作用。进行动物实验以评估OIP5-AS1在动脉粥样硬化进展中的作用。我们的数据显示，在动脉粥样硬化血清和ox-LDL刺激的HUVECs中，OIP5-AS显著上调。沉默OIP5-AS1可保护HUVECs免受ox-LDL诱导的细胞毒性，并减少ApoE小鼠的脂质分泌。此外，OIP5-AS1作为miR-135a-5p的分子海绵发挥作用，并且miR-135a-5p是OIP5-AS1调节ox-LDL诱导的HUVEC损伤的功能介质。KLF5是miR-135a-5p的直接靶点，miR-135a-5p表达增加通过下调KLF5减轻ox-LDL诱导的细胞毒性。此外，OIP5-AS1通过吸附miR-135a-5p影响KLF5的表达。目前的研究发现，沉默OIP5-AS1至少部分通过作为miR-135a-5p的海绵影响KLF5的表达，从而保护HUVECs免受ox-LDL诱导的细胞毒性。
