Dataset Information

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-?, 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.

SUBMITTER: Luo J

PROVIDER: S-EPMC7457876 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Luo Jun J Li Li L Hu Die D Zhang Xian X

International journal of chronic obstructive pulmonary disease 20200826

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 32921999

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Project description:BackgroundChronic 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.MethodsThe 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.ResultsCircANKRD11 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.ConclusionCircANKRD11 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.

Project description:This study investigated whether the mitochondrial-targeted peptide SS-31 can protect against cigarette smoke- (CS-) induced airway inflammation and oxidative stress in vitro and in vivo. Mice were exposed to CS for 4 weeks to establish a CS-induced airway inflammation model, and those in the experimental group were pretreated with SS-31 1 h before CS exposure. Pathologic changes and oxidative stress in lung tissue, inflammatory cell counts, and proinflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) were examined. The mechanistic basis for the effects of SS-31 on CS extract- (CSE-) induced airway inflammation and oxidative stress was investigated using BEAS-2B bronchial epithelial cells and by RNA sequencing and western blot analysis of lung tissues. SS-31 attenuated CS-induced inflammatory injury of the airway and reduced total cell, neutrophil, and macrophage counts and tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, and matrix metalloproteinase (MMP) 9 levels in BALF. SS-31 also attenuated CS-induced oxidative stress by decreasing malondialdehyde (MDA) and myeloperoxidase (MPO) activities and increasing that of superoxide dismutase (SOD). It also reversed CS-induced changes in the expression of mitochondrial fission protein (MFF) and optic atrophy (OPA) 1 and reduced the amount of cytochrome c released into the cytosol. Pretreatment with SS-31 normalized TNF-α, IL-6, and MMP9 expression, MDA and SOD activities, and ROS generation in CSE-treated BEAS-2B cells and reversed the changes in MFF and OPA1 expression. RNA sequencing and western blot analysis showed that SS-31 inhibited CS-induced activation of the mitogen-activated protein kinase (MAPK) signaling pathway in vitro and in vivo. Thus, SS-31 alleviates CS-induced airway inflammation and oxidative stress via modulation of mitochondrial function and regulation of MAPK signaling and thus has therapeutic potential for the treatment of airway disorders caused by smoking.

Project description:Tobacco smoking and hemodynamic forces are key stimuli in the development of endothelial dysfunction and atherosclerosis. High laminar flow has an atheroprotective effect on the endothelium and leads to a reduced response of endothelial cells to cardiovascular risk factors compared to regions with disturbed or low laminar flow. We hypothesize that the atheroprotective effect of high laminar flow could delay the development of endothelial dysfunction caused by cigarette smoking. Primary human endothelial cells were stimulated with increasing dosages of aqueous cigarette smoke extract (CSEaq). CSEaq reduced cell viability in a dose-dependent manner. The main mediator of cellular adaption to oxidative stress, nuclear factor erythroid 2-related factor 2 (NRF2) and its target genes heme oxygenase (decycling) 1 (HMOX1) or NAD(P)H quinone dehydrogenase 1 (NQO1) were strongly increased by CSEaq in a dose-dependent manner. High laminar flow induced elongation of endothelial cells in the direction of flow, activated the AKT/eNOS pathway, increased eNOS expression, phosphorylation and NO release. These increases were inhibited by CSEaq. Pro-inflammatory adhesion molecules intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1), selectin E (SELE) and chemokine (C-C motif) ligand 2 (CCL2/MCP-1) were increased by CSEaq. Low laminar flow induced VCAM1 and SELE compared to high laminar flow. High laminar flow improved endothelial wound healing. This protective effect was inhibited by CSEaq in a dose-dependent manner through the AKT/eNOS pathway. Low as well as high laminar flow decreased adhesion of monocytes to endothelial cells. Whereas, monocyte adhesion was increased by CSEaq under low laminar flow, this was not evident under high laminar flow. This study shows the activation of major atherosclerotic key parameters by CSEaq. Within this process, high laminar flow is likely to reduce the harmful effects of CSEaq to a certain degree. The identified molecular mechanisms might be useful for development of alternative therapy concepts.

Project description:The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

Dataset Information

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

Publications

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

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