Dataset Information

CircANKRD11 Knockdown Protects HPMECs from Cigarette Smoke Extract-Induced Injury by Regulating miR-145-5p/BRD4 Axis.

ABSTRACT:

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.

SUBMITTER: Wang Z

PROVIDER: S-EPMC8021255 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Long non-coding RNAs (lncRNAs) have been reported as key regulators in chronic obstructive pulmonary disease (COPD). However, the precise role of LINC00612 remains unclear. Methods:The real-time quantitative polymerase chain reaction (RT-qPCR) was used to quantify the expression levels of LINC00612, miR-31-5p, and Notch homolog 1 (Notch1) in lung tissues and cells. Under a cigarette smoke extract (CSE) stimulation condition, the apoptosis was analyzed by flow cytometry assay. Caspase-3 activity was examined with a caspase-3 activity assay kit; besides, inflammation and oxidative stress were assessed by measuring interleukin-6, tumor necrosis factor-?, glutathione/oxidized glutathione, reactive oxygen species, malondialdehyde, and superoxide dismutase activity. The interaction relationship between miR-31-5p and LINC00612 or Notch1 was predicted by bioinformatics databases, while dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays were performed to confirm prediction. Eventually, the related protein expression was estimated with western blot assay. Results:LINC00612 was downregulated in COPD tissues when compared with controls. Consistently, CSE inhibited LINC00612 expression in HPMECs with a dose/time-dependent method. Gain-of-function experiments indicated that the upregulation of LINC00612 could repress cell apoptosis, inflammation, and oxidative stress in HPMECs induced by CSE. In addition, miR-31-5p was negatively regulated by LINC00612 in HPMECs treated with CSE. The overexpression of miR-31-5p could abolish LINC00612-induced effects on HPMECs exposed to CSE. Importantly, LINC00612 could weaken CSE-induced cell apoptosis, inflammation, and oxidative stress in HPMECs by regulating the miR-31-5p/Notch1 signaling pathway. Conclusion:Current findings suggest that CSE-mediated cell apoptosis, inflammation, and oxidative stress in HPMECs were abolished by upregulation of LINC00612. Furthermore, the LINC00612/miR-31-5p/Notch1 axis may represent a novel regulator of apoptosis, inflammation, and oxidative stress of HPMECs, which may be a potential therapeutic target for COPD in the future.