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

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. 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. 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. 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.

Luo J ，Li L ，Hu D ，Zhang X ... -  《International Journal of Chronic Obstructive Pulmonary Disease》

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

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. 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. 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. 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.

Wang Z ，Zuo Y ，Gao Z 《International Journal of Chronic Obstructive Pulmonary Disease》

Hsa_circ_0006872 promotes cigarette smoke-induced apoptosis, inflammation and oxidative stress in HPMECs and BEAS-2B cells through the miR-145-5p/NF-κB axis.

Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Circular RNAs (circRNAs) are involved in regulating various biological processes. This study aimed to explore the role and molecular basis of hsa_circ_0006872 in cigarette smoke extract (CSE)-induced cell injury. HPMECs and BEAS-2B cells were treated with CSE to mimic COPD in vitro. The levels of hsa_circ_0006872 and miR-145-5p were measured by quantitative real-time polymerase chain reaction. Cell proliferation was assessed via Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was used to evaluate apoptosis and cell cycle. The levels of inflammatory factors were assayed via enzyme-linked immunosorbent assay (ELISA). The levels of oxidative stress markers were determined via commercial kits. The interaction between hsa_circ_0006872 and miR-145-5p was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Protein expression was measured using Western blot assay. Hsa_circ_0006872 level was elevated in COPD patients and was negatively correlated with miR-145-5p level. CSE exposure promoted apoptosis, inflammation and oxidative stress of HPMECs and BEAS-2B cells, while hsa_circ_0006872 down-regulation undermined the effects. In addition, hsa_circ_0006872 silencing inhibited CSE-induced cell injury via regulating miR-145-5p. Moreover, CSE contributed to the activation of NF-κB pathway through hsa_circ_0006872/miR-145-5p axis. Hsa_circ_0006872 facilitated CSE-triggered apoptosis, inflammation and oxidative stress in HPMECs and BEAS-2B cells by regulating miR-145-5p/NF-κB pathway.

Xue M ，Peng N ，Zhu X ，Zhang H ... -  《-》

LncRNA MIR155HG contributes to smoke-related chronic obstructive pulmonary disease by targeting miR-128-5p/BRD4 axis.

Chronic obstructive pulmonary disease (COPD) is a common airway disease characterized by an exaggerated pulmonary inflammatory response. Long noncoding MIR155 host gene (lncRNA MIR155HG) has been identified to be related to the macrophage polarization in COPD. However, the detailed function of MIR155HG in cigarette smoke (CS)-mediated COPD remains largely unknown. The expression level of MIR155HG was elevated while miR-218-5p was decreased in lung tissues of smokers without or with COPD, especially in smokers with COPD, and cigarette smoke extract (CSE)-treated human pulmonary microvascular endothelial cell (HPMECs) in a dose- and time-dependent manner. Then, functional experiments showed that MIR155HG deletion could reverse CSE exposure-induced apoptosis and inflammation in HPMECs. MiR-218-5p was confirmed to be a target of MIR155HG and rescue assay showed miR-218-5p inhibitor attenuated the inhibitory action of MIR155HG knockdown on CSE-induced HPMECs. Subsequently, miR-218-5p was found to target bromodomain containing 4 (BRD4) directly, and miR-218-5p overexpression overturned CSE-induced injury of HPMECs via regulating BRD4. Additionally, co-expression analysis indicated MIR155HG indirectly regulated BRD4 expression in HPMECs via miR-218-5p. Thus, we concluded that MIR155HG contributed to the apoptosis and inflammation of HPMECs in smoke-related COPD by regulating miR-128-5p/BRD4 axis, providing a novel insight on the pathogenesis of COPD and a therapeutic strategy on COPD treatments.

Song J ，Wang Q ，Zong L 《-》

lncRNA TUG1 regulates human pulmonary microvascular endothelial cell apoptosis via sponging of the miR-9a-5p/BCL2L11 axis in chronic obstructive pulmonary disease.

The aim of the present study was to investigate the function of long non-coding RNA taurine-upregulated gene 1 (lncRNA TUG1) in chronic obstructive pulmonary disease and further assess the underlying molecular mechanisms. Flow cytometry analysis was performed to detect cell apoptosis of human pulmonary microvascular endothelial cells (HPMECs) treated with 1% cigarette smoke extract (CSE). The activity of caspase-3 was measured using a Caspase-3 Activity assay kit and the protein expression of cleaved caspase-3, caspase-3 and Bcl-2 like 11 (BCL2L11) were measured using western blotting. Reverse transcription-quantitative PCR (RT-qPCR) was performed to measure the expression of TUG1 mRNA levels in the treated cells. The association between TUG1, the miR-9a-5p/BCL2L11 axis and with miR-9a-5p were predicted and verified using a dual luciferase reporter assay system. The mRNA expression of miR-9a-5p and BCL2L11, and the transfection efficiency were measured by RT-qPCR. The results showed that CSE induced cell apoptosis and increased lncRNA TUG1 expression in HPMECs. CSE significantly reduced the expression of miR-9a-5p in HPMECs compared with the control group. TUG1-short hairpin RNA relieved cell apoptosis induced by CSE by upregulating miR-9a-5p in HPMECs. The present study predicted and verified that BCL2L11 is a direct target of miR-9a-5p. The mRNA expression of BCL2L11 was increased in HPMECs following CSE treatment compared with the control group. miR-9a-5p mimic and BCL2L11-plasmid markedly increased the expression of miR-9a-5p and BCL2L11, respectively. miR-9a-5p mimic reversed the increase in cell apoptosis induced by CSE by inhibiting BCL2L11 expression in HPMECs. To conclude, the present study demonstrated that lncRNA TUG1 exerted roles in cell apoptosis induced by CSE through modulating the miR-9a-5p/BCL2L11 axis.

Chen X ，Mao M ，Shen Y ，Jiang X ，Yin Z ... -  《-》