Circ-HACE1 Aggravates Cigarette Smoke Extract-Induced Injury in Human Bronchial Epithelial Cells via Regulating Toll-Like Receptor 4 by Sponging miR-485-3p

Abstract

Background Smoking is the most common cause of chronic obstructive pulmonary disease (COPD), and the early diagnosis for COPD remains poor. Exploring the molecular mechanism and finding feasible biomarkers will be beneficial for clinical management of COPD. Circular RNAs (circRNAs) are noncoding RNAs that act as miRNA sponges to regulate the expression levels of genes, leading to the changes of cellular phenotypes and disease progression. CircRNA HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (circ-HACE1) was abnormally expressed after the induction of cigarette smoke extract (CSE) in cell model. This study was performed to explore its function and mechanism in COPD. Methods Circ-HACE1, microRNA-485-3p (miR-485-3p) and toll-like receptor 4 (TLR4) detection was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis/cell cycle were respectively examined using cell counting kit-8 (CCK-8) and flow cytometry. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was evaluated through the measurement of malondialdehyde (MDA) and superoxide dismutase (SOD). The target binding analysis was conducted via dual-luciferase reporter assay. Western blot was employed for protein expression detection of TLR4. Results Circ-HACE1 was overexpressed in smokers or smokers with COPD and CSE upregulated circ-HACE1 expression in 16HBE cells. Knockdown of circ-HACE1 attenuated CSE-stimulated cell viability and cell cycle repression, as well as the enhancement of cell apoptosis, inflammatory response and oxidative stress. MiR-485-3p was a target of circ-HACE1. Circ-HACE1 regulated CSE-induced cell injury via targeting miR-485-3p. TLR4 was a downstream target of miR-485-3p, and miR-485-3p inhibited the CSE-induced cell damages by directly downregulating the level of TLR4. Circ-HACE1/miR-485-3p regulated TLR4 expression in CSE-treated 16HBE cells, and TLR4 overexpression also reversed all effects of si-circ-HACE1 on CSE-treated 16HBE cells. Conclusion These findings elucidated that circ-HACE1 contributed to the CSE-induced cell damages in COPD cell models via regulating TLR4 by acting as the sponge of miR-485-3p.

Cellular and Molecular Signatures of Oxidative Stress in Bronchial Epithelial Cell Models Injured by Cigarette Smoke Extract

“…Oxidative stress can also be measured indirectly, by analyzing the degree of lipid peroxidation, a phenomenon that occurs in the presence of elevated concentrations of oxidants. Malondialdehyde (MDA) is one of the products of lipid peroxidation, and can be easily detected by colorimetric or fluorometric analysis [28,29,33,39,70,71]. Alternatively, it is possible to measure the levels of 4-hydroxynonenale (4-HNE) protein adducts by ELISA [22,24].…”

“…As is well known, the activation of these pathways leads to increased levels of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8. Of note, these cytokines are key mediators of CS-induced lung inflammation as well [17,19,22,24,25,[27][28][29][30]32,33,36,37,40,42,[44][45][46][69][70][71]74,75,[77][78][79][80][81]83,90,98,99,102,104,112,113]. ROS, increased by CS, enhance the activation of the IκB kinase (IKK) complex (IKKβ, IKKα, and NF-κB essential modulator (NEMO), leading to the phosphorylation, ubiquitination and proteasomal degradation of IκB, in particular IκBα.…”

“…Non-coding RNAs regulate gene expression through direct binding to their target mRNAs, leading to inhibition of translation. CSE induces the upregulation of several non-coding RNAs in bronchial epithelial cells, including miR-21, SCAL1, circRNA oxysterol binding protein like 2 (circOSBPL2) and circRNA HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (circ-HACE1) [70,71,74,102]. miR-21 is a small non-coding RNA involved in the regulation of oxidative stress-dependent inflammatory reactions and cell proliferation.…”

“…Induction of miR-21 by CSE occurs via TLR4, and it is reverted by treatment with the antioxidant apigenin [74]. CircOSBPL2 and circ-HACE1 increase in the lung of smokers and COPD subjects [70,71]. In 16HBE cells exposed to CSE, circOSBPL2 and circHACE1 are upregulated and contribute to apoptosis, inflammation and oxidative stress by acting as a sponge for miR-193a-5p and miR-485-3p, respectively [70].…”

Long non-coding RNA OIP5-AS1 regulates smoke-related chronic obstructive pulmonary disease via targeting micro RNA -410-3p/IL-13

“…Human bronchial epithelial cell 16HBE was commonly used in the studies of COPD [ 17 , 18 ]. 16HBE cells were purchased from Procell (Wuhan, China) and fostered in the RPMI-1640 culture with 10% FBS and 1% P/S.…”