Department of Cardiology, Zaozhuang Municipal Hospital, Zaozhuang, Shandong 277100, P.R. China.
Department of Cardiology, Zaozhuang Hospital of Zaozhuang Mining Group, Zaozhuang, Shandong 277100, P.R. China.
Int J Mol Med. 2021 Apr;47(4). doi: 10.3892/ijmm.2021.4873. Epub 2021 Feb 12.
The dysfunctions of human umbilical vein endothelial cells (HUVECs) are important features of atherosclerosis (AS). Circular RNAs (circRNAs) are regulators of a wide range of human diseases, including AS. The present study aimed to investigate the role of circUSP36 in the ectopic phenotype of HUVECs and to provide evidence of the involvement of circUSP36 in the pathogenesis of AS. AS cell models were established using HUVECs exposed to oxidized low‑density lipoprotein (ox‑LDL). Cell viability, cell cycle progression and apoptosis, and cell migration and invasion were assessed by cell counting kit‑8 (CCK‑8) assay, flow cytometric assay and Transwell assay, respectively. The expression levels or releases of pro‑inflammatory factors were detected by western blot analysis or enzyme‑linked immunosorbent assay (ELISA). The mRNA expression of circUSP36, miR‑20a‑5p and Rho‑associated coiled‑coil kinase 2 (ROCK2) was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and the protein level of ROCK2 was detected by western blot analysis. The targeted association between miR‑20a‑5p and circUSP36 or ROCK2 was verified by dual‑luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The results revealed that circUSP36 was highly expressed in ox‑LDL‑exposed HUVECs. CircUSP36 knockdown attenuated ox‑LDL‑induced cell cycle arrest, cell apoptosis and inflammatory responses, and promoted cell migration and invasion which had been blocked by ox‑LDL. miR‑20a‑5p was found to be a target of circUSP36, and miR‑20a‑5p inhibition reversed the effects of circUSP36 knockdown. Moreover, miR‑20a‑5p directly bound to ROCK2, and miR‑20a‑5p inhibition aggravated ox‑LDL‑induced injury by increasing the ROCK2 level. More importantly, circUSP36 targeted miR‑20a‑5p to regulate the expression of ROCK2. On the whole, the present study demonstrates that, circUSP36 regulates ox‑LDL‑induced HUVEC injury and inflammation by modulating ROCK2 via competitively targeting miR‑20a‑5p.
人脐静脉内皮细胞（HUVEC）功能障碍是动脉粥样硬化（AS）的重要特征。环状 RNA（circRNA）是广泛涉及人类疾病的调节剂，包括 AS。本研究旨在探讨 circUSP36 在 HUVEC 异位表型中的作用，并为 circUSP36 参与 AS 发病机制提供证据。使用暴露于氧化低密度脂蛋白（ox-LDL）的 HUVEC 建立 AS 细胞模型。通过细胞计数试剂盒（CCK-8）测定、流式细胞术分析和 Transwell 测定分别评估细胞活力、细胞周期进程和细胞凋亡以及细胞迁移和侵袭。通过 Western blot 分析或酶联免疫吸附测定（ELISA）检测促炎因子的表达水平或释放。通过逆转录-定量聚合酶链反应（RT-qPCR）检测 circUSP36、miR-20a-5p 和 Rho 相关卷曲螺旋激酶 2（ROCK2）的 mRNA 表达，通过 Western blot 分析检测 ROCK2 的蛋白水平。通过双荧光素酶报告基因测定和 RNA 免疫沉淀（RIP）测定验证 miR-20a-5p 与 circUSP36 或 ROCK2 之间的靶向关联。结果表明，circUSP36 在 ox-LDL 暴露的 HUVEC 中高表达。circUSP36 敲低可减弱 ox-LDL 诱导的细胞周期阻滞、细胞凋亡和炎症反应，并促进细胞迁移和侵袭，而 ox-LDL 可阻断这些作用。发现 miR-20a-5p 是 circUSP36 的靶标，而 miR-20a-5p 抑制可逆转 circUSP36 敲低的作用。此外，miR-20a-5p 直接与 ROCK2 结合，miR-20a-5p 抑制通过增加 ROCK2 水平加剧 ox-LDL 诱导的损伤。更重要的是，circUSP36 通过竞争性靶向 miR-20a-5p 来调节 ROCK2 的表达。总之，本研究表明，circUSP36 通过调节 ROCK2 来调节 ox-LDL 诱导的 HUVEC 损伤和炎症，从而发挥作用。
