LINC00612/miR-31-5p/Notch1 Axis Regulates Apoptosis, Inflammation, and Oxidative Stress in Human Pulmonary Microvascular Endothelial Cells Induced by Cigarette Smoke Extract

Abstract

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-α, 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.