C-EBPβ mediates in cigarette/IL-17A-induced bronchial epithelial–mesenchymal transition in COPD mice

Background Cigarettes smoking and IL-17A contribute to chronic obstructive pulmonary disease (COPD), and have synergistical effect on bronchial epithelial cell proliferation. CCAAT/enhancer-binding protein β (C-EBPβ) could be induced by IL-17A and is up-regulated in COPD. We explored the effect of cigarettes and IL-17 on bronchial epithelial–mesenchymal transition (EMT) in COPD mice and potential mechanism involved with C-EBPβ in this study. Methods COPD model was established with mice by exposing to cigarettes. E-Cadherin, Vimentin, IL-17A and C-EBPβ distributions were detected in lung tissues. Primary bronchial epithelial cells were separated from health mice and cocultured with cigarette smoke extract (CSE) or/and IL-17A. E-Cadherin, Vimentin and IL-17 receptor (IL-17R) expressions in vitro were assessed. When C-EBPβ were silenced by siRNA in cells, E-Cadherin, Vimentin and C-EBPβ expressions were detected. Results E-Cadherin distribution was less and Vimentin distribution was more in bronchus of COPD mice than controls. IL-17A and C-EBPβ expressions were higher in lung tissues of COPD mice than controls. In vitro, C-EBPβ protein expression was highest in CSE + IL-17A group, followed by CSE and IL-17A groups. E-cadherin expression in vitro was lowest and Vimentin expression was highest in CSE + IL-17A group, followed by CSE or IL-17A group. Those could be inhibited by C-EBPβ silenced. Conclusions C-EBPβ mediates in cigarette/IL-17A-induced bronchial EMT in COPD mice. Our findings contribute to a better understanding on the progress from COPD to lung cancers, which will provide novel avenues in preventing tumorigenesis of airway in the context of cigarette smoking.

Dosing intact birch pollen grains at the air-liquid interface (ALI) to the immortalized human bronchial epithelial cell line BEAS-2B

In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.

Sodium Arsenite Induces Bronchial Epithelial Cell Cancer With Mitochondrial Dysfunction by Affecting Cell Cycle

Background: Arsenide, a kind of pollution widely existing in the environment, and the mechanism of occurrence and development of lung cancer leading by long-term arsenic exposure still needs further research up to present. We investigated how high and low doses of arsenic induce human bronchial epithelial cell transformation and the molecular mechanisms involvedMethods: After exposing human bronchial epithelial cells(Beas-2B) to different doses of sodium arsenite for 24 h, RNA-seq analysis was performed to detect arsenic-regulated genes; for cell viability and status and for cell cycle changes and mitochondrial function; long-term exposure to 1μM sodium arsenite after 20 consecutive passages for cell cycle changes and mitochondrial function.Results: In the study, human bronchial epithelial cells were exposed to varying high-dose sodium arsenite in short term or treated with low dose in a long term, and the data showed that both short and long terms treatment promoted G1/S transition of Beas-2B cells, inducing significant increases of expression of AKAP95, cyclin D1, cyclin D2 and cyclin E1. However, silencing AKAP95 by treating cells with siAKAP95 showed a protective function that inhibited G1/S transition, suggesting that regulatory mechanism of AKAP95 on cell cycle during cell malignant transformation induced by sodium arsenite. In addition, we also noticed some mitochondrial dysfunctions occurred during sodium arsenite exposure, including the disappearance of mitochondrial double membrane structure, the formation of vacuole structure, the decrease of mitochondrial membrane potential, the change of expression levels of mitochondrial related proteins Tim22, Tim23, Tom40 and OPA1, the release of lactate dehydrogenase (LDH) and the decrease of reduced glutathione.Beas-2B cells, which exposed to low-dose sodium arsenite for a long term, were subcultured for 20 generations, and it was found that the exposure time was positively proportional to the growth and migration rate of cells. The exposed cells were used in tumor bearing transplantation experiment (mice), and results showed that the longer the exposure time, the faster the tumor volume growth rate of As-Beas-2B cells. Tumor tissues were taken out for HE staining, and it was found that the cell morphology changed and the volume increasedConclusion: high and low doses of sodium arsenite induced malignant transformation of human bronchial epithelial cells by promoting G1/S turnover through AKAP95 with associated cyclins, accompanied by the development of mitochondrial dysfunction.

Comprehensive Bioinformatics Analysis of Lipopolysaccharide-Induced Altered Autophagy in Acute Lung Injury and Construction of Underlying Competing Endogenous RNA Regulatory Mechanism

Background. Acute lung injury (ALI) is a fatal syndrome frequently induced by lipopolysaccharide (LPS) released from the bacterial cell wall. LPS could also trigger autophagy of lung bronchial epithelial cell to relieve the inflammation, while the overwhelming LPS would impair the balance of autophagy consequently inducing serious lung injury. Methods. We observed the autophagy variation of 16HBE, human bronchial epithelial cell, under exposure to different concentrations of LPS through western blot, immunofluorescence staining, and electron microscopy. Eight strands of 16HBE were divided into two groups upon 1000 ng/ml LPS stimulation or not, which were sent to be sequenced at whole transcriptome. Subsequently, we analyzed the sequencing data in functional enrichment, pathway analysis, and candidate gene selection and constructed a hsa-miR-663b-related competing endogenous RNA (ceRNA) network. Results. We set a series of concentrations of LPS to stimulate 16HBE and observed the variation of autophagy in related protein expression and autophagosome count. We found that the effective concentration of LPS was 1000 ng/ml at 12 hours of exposure and sequenced the 1000 ng/ml LPS-stimulated 16HBE. As a result, a total of 750 differentially expressed genes (DEGs), 449 differentially expressed lncRNAs (DElncRNAs), 76 differentially expressed circRNAs (DEcircRNAs), and 127 differentially expressed miRNAs (DEmiRNAs) were identified. We constructed the protein-protein interaction (PPI) network to visualize the interaction between DEGs and located 36 genes to comprehend the core discrepancy between LPS-stimulated 16HBE and the negative control group. In combined analysis of differentially expressed RNAs (DERNAs), we analyzed all the targeted relationships of ceRNA in DERNAs and figured hsa-miR-663b as a central mediator in the ceRNA network to play when LPS induced the variation of autophagy in 16HBE. Conclusion. Our research indicated that the hsa-miR-663b-related ceRNA network may contribute to the key regulatory mechanism in LPS-induced changes of autophagy and ALI.

MiRNA-21 Regulates Bronchial Epithelial Cell Proliferation by Activating Tgfβ1/Smad Signaling Pathway and Its Correlation with Asthma Severity in Children

Background: This research was designed to probe into the role of miRNA-21 in the pathogenesis of childhood asthma and its correlation with the severity. Methods: Fifty-four children with bronchial asthma admitted to the Third Affiliated Hospital of Qiqihar Medical University from Jun 2018 to Dec 2019 were included. Forty nine healthy children underwent physical examination at this time period were also enrolled. The miR-21 expression in peripheral blood serum was analyzed by qRT-PCR. The relationship between the expression and severity of asthma in children was explored by Spearman correlation analysis and ROC curve. Bronchial epithelial cell lines were cultured in vitro and divided into blank control group, negative control group and miR-21 inhibition and activation group. The changes of cell proliferation after treatment were detected by CCK-8 test in different groups. The expression of TGF-β1/Smad signaling pathway protein in cells was assessed by Western blot (WB). Results: Compared with that of healthy children, the miR-21 expression in peripheral blood serum of asthmatic children was higher (P<0.001). MiR-21 expression was positively correlated with the severity of illness (r=0.853, P<0.001). The results of cell experiments in vitro signified that miR-21 can promote the proliferation of bronchial epithelial cells, and may be involved in regulating the expression of TGF-β1/ Smad3 signaling pathway, thus affecting cell proliferation. Conclusion: miRNA-21 regulates the proliferation of bronchial epithelial cells by activating TGFβ1/Smad signaling pathway. And it is positively correlated with the severity of asthma in children.