环状ANKRD11 敲低通过调控 miR-145-5p/BRD4 轴保护人肺动脉内皮细胞免受香烟烟雾提取物诱导的损伤。
CircANKRD11 Knockdown Protects HPMECs from Cigarette Smoke Extract-Induced Injury by Regulating miR-145-5p/BRD4 Axis.
机构信息
Department of Respiratory Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, People's Republic of China.
Department of Physical Examination Center, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, People's Republic of China.
出版信息
Int J Chron Obstruct Pulmon Dis. 2021 Apr 1;16:887-899. doi: 10.2147/COPD.S300332. eCollection 2021.
BACKGROUND
Chronic obstructive pulmonary disease (COPD) is a major cause of death because of its high incidence and mortality, which is chiefly resulted from cigarette smoke exposure. A large number of studies show that circular RNA (circRNA) participates in regulating COPD process. This study aims to reveal the role of circRNA ankyrin repeat domain 11 (circANKRD11) in cigarette smoke extract (CSE)-induced cell apoptosis, inflammation, and oxidative stress.
METHODS
The expression of circANKRD11, microRNA-145-5p (miR-145-5p) and bromodomain-containing 4 (BRD4) mRNA was detected by quantitative real-time polymerase chain reaction. The expression of apoptosis-related proteins and BRD4 protein was determined by Western blot. Cell apoptosis was detected by flow cytometry and Western blot. Cell inflammation was demonstrated by determining the levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) through enzyme-linked immunosorbent assay. Oxidative stress was investigated by the reactive oxygen species (ROS) and malondialdehyde (MDA) determination assays as well as superoxide dismutase (SOD) activity assay. The binding relationship between miR-145-5p and circANKRD11 or BRD4 was predicted by circinteractome or MicroT_CDS online database, and identified by dual-luciferase reporter, RNA immunoprecipitation or RNA pull-down assay.
RESULTS
CircANKRD11 and BRD4 expression were increased, whereas miR-145-5p expression was decreased in the lung tissues of smokers with or without COPD and CSE-induced HPMECs compared with the lung tissues of non-smokers as well as untreated HPMECs, respectively. CircANKRD11 silencing ameliorated CSE-induced cell apoptosis, inflammation, and oxidative stress. CircANKRD11 acted as a sponge of miR-145-5p, and regulated CSE-induced cell injury via sponging miR-145-5p. Additionally, miR-145-5p mimics protected against CSE-induced cell injury through targeting BRD4.
CONCLUSION
CircANKRD11 absence protected HPMECs from CSE-induced injury by regulating BRD4 through associating with miR-145-5p, which demonstrated that circANKRD11 had the potential to act as a diagnosis biomarker for smoker-caused COPD.
背景
慢性阻塞性肺疾病（COPD）是一种主要的死亡原因，因为它的发病率和死亡率很高，主要是由于吸烟暴露。大量研究表明，环状 RNA（circRNA）参与调节 COPD 过程。本研究旨在揭示环状 RNA 锚蛋白重复域 11（circANKRD11）在香烟烟雾提取物（CSE）诱导的细胞凋亡、炎症和氧化应激中的作用。
方法
通过定量实时聚合酶链反应检测 circANKRD11、微小 RNA-145-5p（miR-145-5p）和溴结构域包含 4（BRD4）mRNA 的表达。通过 Western blot 检测凋亡相关蛋白和 BRD4 蛋白的表达。通过流式细胞术和 Western blot 检测细胞凋亡。通过酶联免疫吸附试验测定白细胞介素-1β（IL-1β）、白细胞介素-6 和肿瘤坏死因子-α（TNF-α）的水平来证明细胞炎症。通过测定活性氧（ROS）和丙二醛（MDA）以及超氧化物歧化酶（SOD）活性来研究氧化应激。通过 circinteractome 或 MicroT_CDS 在线数据库预测 miR-145-5p 与 circANKRD11 或 BRD4 的结合关系，并通过双荧光素酶报告基因、RNA 免疫沉淀或 RNA 下拉实验进行验证。
结果
与非吸烟者和未经处理的 HPMECs 相比，吸烟者和有或没有 COPD 的吸烟者的肺组织以及 CSE 诱导的 HPMECs 中 circANKRD11 和 BRD4 的表达增加，而 miR-145-5p 的表达降低。沉默 circANKRD11 可改善 CSE 诱导的细胞凋亡、炎症和氧化应激。circANKRD11 作为 miR-145-5p 的海绵，通过海绵 miR-145-5p 调节 CSE 诱导的细胞损伤。此外，miR-145-5p 模拟物通过靶向 BRD4 来保护细胞免受 CSE 诱导的损伤。
结论
circANKRD11 缺失通过与 miR-145-5p 结合调节 BRD4，从而保护 HPMEC 免受 CSE 诱导的损伤，这表明 circANKRD11 有潜力作为由吸烟者引起的 COPD 的诊断生物标志物。
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