Normal Human Bronchial Epithelial Cells Research Articles

MAP3K8 is a potential therapeutic target in airway epithelial inflammation

BackgroundWe have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1β stimulation. We analysed signalling transduction and global gene expression after IL-1β stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models.ResultsIL-1β significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1β stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1β stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1β stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1β following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose.ConclusionsThese results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.

Preparation, characterization, single-crystal analysis, and cytotoxic assessment of complexes formed by Schiff base and 8-hydroxyquinoline co-ligand with Co(II), Ni(II), and Mn(II) metal ions

This study focuses on the preparation of mixed ligand complexes of Ni(II), Co(II), and Mn(II) by combining the co-ligands 2-(((4-fluorophenyl)imino)methyl)-4-methoxy-6-nitrophenol (L1) and 8-hydroxyquinoline (L2). The chemical structures of the all compounds were examined using various techniques, including 1H and 13C NMR, FT-IR, UV–Vis, TGA, molar conductivity, LC-MS/MS, elemental analysis and magnetic susceptibility. The crystal structure of the ligand L1 was determined using single crystal X-ray analysis revealing that the ligand favors the phenol-imine tautomeric form in the solid state. All complexes are suggested to have an octahedral structure. Finally, the MTT assay was used to determine the toxicity level of the mixed ligand complexes in human non-small cell lung cancer (A549) and normal bronchial epithelial cell lines (BEAS-2B). The results showed that the [Ni(L1)(L2).(H2O)2] complex (IC50: 5.97 µM) exhibited high cytotoxic activity at a lower dose than cisplatin compared to the other compounds. In contrast, the [Ni(L1)(L2).(H2O)2] complex was shown to exhibit significantly lower cytotoxic activity (IC50 > 100 s µM) in the BEAS-2B (normal bronchial epithelial cell) cell line. The other compounds [Co(L1)(L2).(H2O)2] and [Mn(L1)(L2).(H2O)2] were shown to have no significant anticancer activity in A549 cells due to their cytotoxic activity at higher doses than cisplatin (IC50 > 100 s µM).

Chemical Composition of PM2.5-0.3 and PM0.3 Collected in Southern Lebanon and Assessment of Their Toxicity in BEAS-2B Cells

Fine particles (PM2.5) have generally been reported as the major contributor to the adverse health effects of air pollution. Lebanon is characterized by a high density of transport, the production of electricity by generators, and a problem of uncontrolled incineration of household waste. For the purpose of this paper, the physico-chemical properties of fine (PM2.5-0.3) and quasi-ultrafine (PM0.3) particulate matter sampled in Southern Lebanon, were studied. Then, an evaluation and comparison of the toxicity of the different extracted fractions from PM (i.e., native PM2.5-0.3 vs. organic extractable matter fraction (OEM2.5-0.3), and non-extractable matter fraction (NEM2.5-0.3)) was performed. Also, an examination of the toxicity of PM0.3 was conducted indirectly through the evaluation of the OEM0.3 harmfulness. The physico-chemical analysis showed that PM0.3 was much more concentrated than PM2.5-0.3 in organic compounds such as polycyclic aromatic hydrocarbons (PAHs) (28-fold) and their nitrated (N-PAHs, 14-fold) and oxygenated (O-PAHs, 10-fold) derivatives. Normal human bronchial epithelial cells (BEAS-2B) were exposed to PM2.5-0.3, its derived fractions (i.e., OEM2.5-0.3 and NEM2.5-0.3), and OEM0.3 before evaluating the global cytotoxicity, metabolic activation of organic compounds, genotoxicity, and inflammatory response. Different responses were observed depending on the considered fraction of particles. The global cytotoxicity showed a pronounced response related to ATP and LDH activities after exposure to the quasi-ultrafine organic extractable matter fraction (OEM0.3). There was no significant induction of the AhR cell-signaling pathway by NEM2.5-0.3. Despite the apparent difference in the kinetics of induction of the toxicological endpoints under study, OEM0.3 provoked a higher overall cytotoxicity and genotoxicity than OEM2.5-0.3 and total PM2.5-0.3. Taken together, these results clearly showed that the finest particles are more damaging to BEAS-2B cells than PM2.5-0.3 because they are richer in organic compounds, thereby inducing more remarkable toxic effects.

Chemical Composition of PM2.5-0.3 and PM0.3 Collected in Southern Lebanon and Assessment of Their Toxicity in BEAS-2B Cells

Fine particles (PM2.5) have generally been reported as the major contributor to the adverse health effects of air pollution. Lebanon is characterized by a high density of transport, the production of electricity by generators, and a problem of uncontrolled incineration of household waste. For the purpose of this paper, the physico-chemical properties of fine (PM2.5-0.3) and quasi-ultrafine (PM0.3) particulate matter sampled in Southern Lebanon, were studied. Then, an evaluation and comparison of the toxicity of the different extracted fractions from PM (i.e., native PM2.5-0.3 vs. organic extractable matter fraction (OEM2.5-0.3), and non-extractable matter fraction (NEM2.5-0.3)) was performed. Also, an examination of the toxicity of PM0.3 was conducted indirectly through the evaluation of the OEM0.3 harmfulness. The physico-chemical analysis showed that PM0.3 was much more concentrated than PM2.5-0.3 in organic compounds such as polycyclic aromatic hydrocarbons (PAHs) (28-fold) and their nitrated (N-PAHs, 14-fold) and oxygenated (O-PAHs, 10-fold) derivatives. Normal human bronchial epithelial cells (BEAS-2B) were exposed to PM2.5-0.3, its derived fractions (i.e., OEM2.5-0.3 and NEM2.5-0.3), and OEM0.3 before evaluating the global cytotoxicity, metabolic activation of organic compounds, genotoxicity, and inflammatory response. Different responses were observed depending on the considered fraction of particles. The global cytotoxicity showed a pronounced response related to ATP and LDH activities after exposure to the quasi-ultrafine organic extractable matter fraction (OEM0.3). There was no significant induction of the AhR cell-signaling pathway by NEM2.5-0.3. Despite the apparent difference in the kinetics of induction of the toxicological endpoints under study, OEM0.3 provoked a higher overall cytotoxicity and genotoxicity than OEM2.5-0.3 and total PM2.5-0.3. Taken together, these results clearly showed that the finest particles are more damaging to BEAS-2B cells than PM2.5-0.3 because they are richer in organic compounds, thereby inducing more remarkable toxic effects.

PGAP3 regulates human bronchial epithelial cell mRNAs present in asthma and respiratory virus reference data sets.

PGAP3 is a glycosylphosphatidylinositol (GPI) phospholipase gene localized within chromosome 17q12-21, a region highly linked to asthma. Although much is known about the function of other chromosome 17q12-21 genes expressed at increased levels in bronchial epithelium such as ORMDL3 and GSDMB, little is known about the function of increased PGAP3 expression in bronchial epithelium in the context of asthma. The aim of this study was therefore to determine whether increased PGAP3 expression in human bronchial epithelial cells regulated expression of mRNA pathways important to the pathogenesis of asthma by utilizing RNA-sequencing and bioinformatic analysis. We performed RNA-sequencing on normal human bronchial epithelial cells transfected with PGAP3 for 24 and 48 hours. PGAP3 regulated genes were compared to asthma and respiratory virus (influenza A, rhinovirus, respiratory syncytial virus) reference data sets to identify PGAP3 target genes and pathways. Approximately 9% of the upregulated PGAP3-induced genes were found in an asthma reference data set, 41% in a rhinovirus reference data set, 33% in an influenza A reference data set, and 3% in a respiratory syncytial virus reference data set. PGAP3 significantly upregulated the expression of several genes associated with the innate immune response and viral signatures of respiratory viruses associated with asthma exacerbations. Two of the highest expressed genes induced by PGAP3 are RSAD2, OASL, and IFN-λ, which are anti-viral genes associated with asthma. PGAP3 also upregulated the antiviral gene BST2, which like PGAP3 is a GPI-anchored protein. We conclude that PGAP3 expression in human bronchial epithelial cells regulates expression of genes known to be linked to asthma, and also regulates the bronchial epithelial expression of genes pertinent to the pathogenesis of respiratory viral triggered asthma exacerbations.

ACE-2 Blockade & TMPRSS2 Inhibition Mitigate SARS-CoV-2 Severity Following Cigarette Smoke Exposure in Airway Epithelial Cells In Vitro.

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

Lack of cytotoxic and genotoxic effects of mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium despite cellular uptake in cancerous and noncancerous lung cells

Cytotoxic and genotoxic effects of novel mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium (Mg0.1-γ-Fe2O3(mPEG-silane)0.5) have been investigated on human adenocarcinomic alveolar basal epithelial (A549) and human normal bronchial epithelial (BEAS-2B) cells. In the studies several molecular and cellular targets addressing to cell membrane, cytoplasm organelles and nucleus components were served as toxicological endpoints. The as-synthesized nanoparticles were found to be stable in the cell culture media and were examined for different concentration and exposure times. No cytotoxicity of the tested nanoparticles was found although these nanoparticles slightly increased reactive oxygen species in both cell types studied. Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles did not produce any DNA strand breaks and oxidative DNA damages in A549 and BEAS-2B cells. Different concentration of Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles and different incubation time did not affect cell migration. The lung cancer cells' uptake of the nanoparticles was more effective than in normal lung cells. Altogether, the results evidence that mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium do not elucidate any deleterious effects on human normal and cancerous lung cells despite cellular uptake of these nanoparticles. Therefore, it seems reasonable to conclude that these novel biocompatible nanoparticles are promising candidates for further development towards medical applications.

Curcumin Inhibits the Progression of Non-small Cell Lung Cancer by Regulating DMRT3/SLC7A11 Axis.

Non-small cell lung cancer (NSCLC) is a fatal malignancy all over the world. Emerging studies have shownthat curcumin might repress NSCLC progression by regulating ferroptosis, but the underlying mechanism remains unclear. 16HBE, LK-2, and H1650 cell viability was detected using Cell Counting Kit-8 assay. LK-2 and H1650 cell proliferation, apoptosis, and angiopoiesis were measured using 5-ethynyl-2'-deoxyuridine, flow cytometry, and tube formation assay. Superoxide dismutase, Malondialdehyde, Glutathione, and lactate dehydrogenase levels in LK-2 and H1650 cells were examined using special assay kits. Fe+ level was assessed using an iron assay kit. Doublesex and Mab-3 related Transcription Factor 3 (DMRT3) and solutecarrierfamily7member11(SLC7A11) protein levels were detected using western in NSCLC tissues, adjacent matched normal tissues, 16HBE cells, LK-2 cells, H1650 cells, and xenograft tumor tissues. Glutathione peroxidase 4, Acyl-CoA Synthetase Long Chain Family Member 4, and transferrinreceptor1 protein levels in LK-2 and H1650 cells were examined by western blot assay. DMRT3 and SLC7A11 levels were determined using real-time quantitative polymerase chain reaction. After JASPAR prediction, binding between DMRT3 and SLC7A11 promoter was verified using Chromatin immunoprecipitation and dual-luciferase reporter assays in LK-2 and H1650 cells. Role of curcumin on NSCLC tumor growth was assessed using the xenograft tumor model in vivo. Curcumin blocked NSCLC cell proliferation and angiopoiesis, and induced apoptosis and ferroptosis. DMRT3 or SLC7A11 upregulation partly abolished the suppressive role of curcumin on NSCLC development. In mechanism, DMRT3 was a transcription factor of SLC7A11 and increased the transcription of SLC7A11 via binding to its promoter region. Curcumin inhibited NSCLC growth in vivo by modulating DMRT3. Curcumin might constrain NSCLC cell malignant phenotypes partly through the DMRT3/SLC7A11 axis, providing a promising therapeutic strategy for NSCLC.

BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Activation of ERK/NF-kB Pathways Contributes to the Inflammatory Response in Epithelial Cells and Macrophages Following Manganese Exposure.

Different types of metals, including manganese (Mn), are constantly encountered in various environmental matrices due to natural and anthropogenic activities. They induce a sustained inflammatory response in various organs, which is considered to be an important priming event in the pathogenesis of several diseases. Mn-induced neuroinflammation and subsequent neurodegeneration are well recognized. However, emerging data suggest that occupationally and environmentally relevant levels may affect various organs, including the lungs. Therefore, the present study was carried out to investigate the effects of Mn (as Mn2+) exposure on the inflammatory response in human normal bronchial (BEAS-2B) and adenocarcinoma alveolar basal (A549) epithelial cells, as well as in murine macrophages (J774). Mn2+ exposure significantly induced mRNA and protein expression of various pro-inflammatory mediators (cytokines and chemokines) in all cells compared to corresponding vehicle controls. Furthermore, Mn2+ treatment also led to increased phosphorylation of extracellular-signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-kB) p65 in both epithelial cells and macrophages. As expected, cells treated with inhibitors of ERK1/2 (PD98059) and NF-kB p65 (IMD0354) effectively mitigated the expression of various pro-inflammatory mediators induced by Mn2+, suggesting that ERK/NF-kB pathways have a critical role in the Mn2+-induced inflammatory response. Further, in vivo studies are required to confirm these in vitro findings to support clinical translation.

Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

Coinfection of Influenza A and B and Human OC43 Coronavirus in Normal Human Bronchial Epithelial Cells.

Influenza viruses and seasonal coronaviruses are pathogens transmitted via an airborne route that can cause respiratory diseases in humans that have similar symptoms such as fever, cough, and pneumonia. These two viruses can infect similar human tissues, such as the respiratory tract and nasal, bronchial, and alveolar epithelial cells. Influenza virus and seasonal coronavirus coinfections are poorly understood. Here, we coinfected normal human bronchial epithelial (NHBE) cells with influenza A/California/04/09 (IAV) or B/Victoria/504/2000 (IBV) strains and the seasonal human beta-coronavirus OC43 and evaluated viral replication capacities. We also examined changes in the expression of various cytokines/chemokines by qPCR and Luminex assay. We observed that the replication of IAV and IBV was not affected by coinfection with OC43. However, coinfection reduced OC43 titers (~3-fold) compared with infection with OC43 alone. Select cytokine/chemokine expression was increased in coinfected cells compared with all single infections with greater differences seen between coinfected cells and cells infected with OC43 alone compared with IAV- or IBV-infected cells. In addition, IL-8 and IL-1RA showed the highest expression among a panel of 22 cytokines by Luminex. As the rate of influenza and seasonal coronavirus coinfection continue to increase, our findings may help set guidelines for the treatments of the individuals coinfected with both viruses.

Transcriptional and phenotypical alterations associated with a gradual benzo[a]pyrene-induced transition of human bronchial epithelial cells into mesenchymal-like cells

The role of benzo[a]pyrene (BaP), a prominent genotoxic carcinogen and aryl hydrocarbon receptor (AhR) ligand, in tumor progression remains poorly characterized. We investigated the impact of BaP on the process of epithelial-mesenchymal transition (EMT) in normal human bronchial epithelial HBEC-12KT cells. Early morphological changes after 2-week exposure were accompanied with induction of SERPINB2, IL1, CDKN1A/p21 (linked with cell cycle delay) and chemokine CXCL5. After 8-week exposure, induction of cell migration and EMT-related pattern of markers/regulators led to induction of further pro-inflammatory cytokines or non-canonical Wnt pathway ligand WNT5A. This trend of up-regulation of pro-inflammatory genes and non-canonical Wnt pathway constituents was observed also in the BaP-transformed HBEC-12KT-B1 cells. In general, transcriptional effects of BaP differed from those of TGFβ1, a prototypical EMT inducer, or a model non-genotoxic AhR ligand, TCDD. Carcinogenic polycyclic aromatic hydrocarbons could thus induce a unique set of molecular changes linked with EMT and cancer progression.

Forced expression of microRNA-221-3p exerts protective effects against manganese-induced cytotoxicity in human lung epithelial cells

Manganese (Mn)-induced pulmonary toxicity and the underlying molecular mechanisms remain largely enigmatic. Further, in recent years, microRNAs (miRNAs) have emerged as regulators of several pollutants-mediated toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in manganese (II) chloride (MnCl2) (Mn2+)-induced cytotoxicity in lung epithelial cells. Growth inhibition of Mn2+ towards normal human bronchial epithelial (BEAS-2B) and adenocarcinomic human alveolar basal epithelial (A549) cells was analyzed by MTT assay following 24 or 48 h treatment. Reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), cell cycle arrest, and apoptosis were evaluated by flow cytometry. RT-qPCR and Western blot were performed to analyze the expression of cyclins, anti-oxidant genes, and miRNAs. We used small RNA sequencing to investigate Mn2+-induced changes in miRNA expression patterns. In both cell lines, Mn2+ treatment inhibited growth in a dose-dependent manner. Further, compared with vehicle-treated cells, Mn2+ (250 μM) treatment induced ROS generation, cell cycle arrest, apoptosis, and decreased ΔΨm as well as altered the expression of cyclins and anti-oxidant genes. Sequencing data revealed that totally 296 miRNAs were differentially expressed in Mn2+-treated cells. Among them, miR-221-3p was one of the topmost down-regulated miRNAs in Mn2+-treated cells. We further confirmed this association in A549 cells. In addition, transient transfection was performed to study gain-of-function experiments. Forced expression of miR-221-3p significantly improved cell viability and reduced Mn2+-induced cell cycle arrest and apoptosis in BEAS-2B cells. In conclusion, miR-221-3p may be the most likely target that accounts for the cytotoxicity of Mn2+−exposed lung epithelial cells.

The Diagnostic Value of ACSL1, ACSL4, and ACSL5 and the Clinical Potential of an ACSL Inhibitor in Non-Small-Cell Lung Cancer.

Abnormal expression of ACSL members 1, 3, 4, 5, and 6 is frequently seen in human cancer; however, their clinical relevance is unclear. In this study, we analyzed the expression of ACSLs and investigated the effects of the ACSL inhibitor Triacsin C (TC) in lung cancer. We found that, compared to normal human bronchial epithelial (NHBE) cells, ACSL1, ACSL4, and ACSL6 were highly expressed, while ACSL3 and ACSL5 were lost in the majority of lung cancer cell lines. ACSL activity was associated with the expression levels of the ACSLs. In primary lung tumors, a higher expression of ACSL1, ACSL4, and ACSL5 was significantly correlated with adenocarcinoma (ADC). Moreover, ACSL5 was significantly reversely related to the proliferation marker Ki67 in low-grade tumors, while ACSL3 was positively associated with Ki67 in high-grade tumors. Combination therapy with TC and Gemcitabine enhanced the growth-inhibitory effect in EGFR wild-type cells, while TC combined with EGFR-TKIs sensitized the EGFR-mutant cells to EGFR-TKI treatment. Taken together, the data suggest that ACSL1 may be a biomarker for lung ADC, and ACSL1, ACSL4, and ACSL5 may be involved in lung cancer differentiation, and TC, in combination with chemotherapy or EGFR-TKIs, may help patients overcome drug resistance.

TMEM2 suppresses TLR3-mediated IFN-β/ISG56/CXCL10 expression in BEAS-2B bronchial epithelial cells.

Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-β and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-β. The expression of TMEM2, IFN-β, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-β, CXCL10, and ISG56, while IFN-β-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. TMEM2 knockdown enhanced TLR3-mediated IFN-β, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.

CircABCB10 Promotes the Apoptosis and Inflammatory Response of 16HBE Cells by Cigarette Smoke Extract by Targeting miR-130a/PTEN Axis.

Chronic obstructive pulmonary disease (COPD) has become a global public health problem due to its high mortality. So there is an urgent need to find an effective treatment. The targeting relationship among circABCB10, miR-130a and PTEN was predicted by the targetscan database (TargetScanHuman 8.0, https://www.targetscan.org/vert_80/). A total of 60 patients which were from the second affiliated hospital of Qiqihar Medical University from 2022 to 2023 were enrolled. The lung condition was detected by CT (Computed Tomography). The expression levels of circABCB10, miR-130a and PTEN in lung tissues were determined by qRT-PCR. The COPD model was established by stimulating normal and silenced 16HBE cells in circABCB10 genes with cigarette smoke extract (CSE) at different concentrations. qRT-PCR was conducted for the expression levels of circABCB10, miR-130a and PTEN, WB for the expression levels of apoptotic proteins, ELISA for the content of inflammatory factors, and CCK8 for the effect of CSE on the proliferation of cells. CircABCB10 expression increased in lung tissues from patients with COPD and in 16HBE cells treated with CSE. The stimulation on cells with CSE increased the expression of inflammatory factors, while knocking down circABCB10 could reverse this response. The inflammatory response to the knockdown of circABCB10 was reversed by miR-130a inhibitor, which increased the expression of c-caspase 3. The targetscan database predicted the target factor downstream miR-130a was PTEN. Transfecting OE-PTEN reversed the inflammation of knocking down circABCB10, and increased the apoptosis and inflammation. CircABCB10 can cause the inflammatory response by targeting miR-130a/PTEN axis, which is a mechanism that may lead to the occurrence and development of COPD.

German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 invivo.

IncRNA AC004943.2 regulates miR-135a-5p and PTK2/P13K axis to promote laryngeal squamous cell carcinoma progression.

Long noncoding RNAs (lncRNAs) are involved in regulatory processes in laryngeal squamous cell carcinoma (LSCC) at posttranscriptional epigenetic modification level. Yet, the function and underlying mechanism behind lncRNA AC004943.2 in LSCC is still obscure. Therefore, the potential role of AC004943.2 in LSCC progression was investigated. The expression of gene or protein was tested by real-time quantitative polymerase chain reaction and western blot. MTT, colony formation, wound healing, and transwell experiments were applied to detect LSCC cell viability, proliferation, migration and invasion, respectively. The interaction among AC004943.2, miR-135a-5p, and protein tyrosine kinase 2 (PTK2) were analyzed by bioinformatics prediction and luciferase assay. AC004943.2 was highly expressed in LSCC cells compared with normal human bronchial epithelial cells, while miR-135a-5p was lowly expressed. AC004943.2 knockdown or miR-135a-5p overexpression inhibited LSCC cell viability, proliferation, migration and invasion. Mechanistically, AC004943.2 increased PTK2 expression in LSCC cells by sponging miR-135a-5p. Furthermore, miR-135a-5p knockdown inverted the inhibitory effect of AC004943.2 silencing on LSCC cell malignant behaviors. MiR-135a-5p upregulation attenuated the PTK2/PI3K pathway to inhibit progression of LSCC. AC004943.2 facilitated the cancerous phenotypes of LSCC cells by activating the PTK2/PI3K pathway through targeting miR-135a-5p, which furnished a therapeutic candidate for LSCC treatment.

Nrf2/HO-1 signaling activation alleviates cigarette smoke-induced inflammation in chronic obstructive pulmonary disease by suppressing NLRP3-mediated pyroptosis

BackgroundThis study examined the effect of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway on chronic obstructive pulmonary disease (COPD) and the potential molecular mechanism.MethodsA COPD mouse model was established by cigarette smoke exposure and administered with either ML385 or dimethyl fumarate (DMF). Airway resistance of mice was detected. IL-1β and IL-6 levels in mice alveolar lavage fluid were examined by enzyme-linked immunosorbent assay. Hematoxylin and eosin staining and immunohistochemical of lung tissues were utilized to detect lung injury and NLRP3 expression. DMF was used to treat COPD cell model constructed by exposing normal human bronchial epithelial (NHBE) cells to cigarette smoke extract. NHBE cells were transfected by NLRP3-expression vectors. Expression of proteins was detected by Western blot.ResultsCOPD mice showed the enhanced airway resistance, the inactivated Nrf2/HO-1 pathway and the overexpressed NLRP3, Caspase-1 and GSDMD-N proteins in lung tissues, and the increased IL-1β and IL-6 levels in alveolar lavage fluid. ML385 treatment augmented these indicators and lung injury in COPD mice. However, DMF intervention attenuated these indicators and lung injury in COPD mice. Nrf2/HO-1 pathway inactivation and overexpression of NLRP3, Caspase-1 and GSDMD-N proteins were observed in COPD cells. DMF intervention activated Nrf2/HO-1 pathway and down-regulated NLRP3, Caspase-1 and GSDMD-N proteins in COPD cells. However, NLRP3 overexpression abolished the effect of DMF on COPD cells.ConclusionNrf2/HO-1 pathway activation may alleviate inflammation in COPD by suppressing the NLRP3-related pyroptosis. Activating the Nrf2/HO-1 pathway may be an effective method to treat COPD.