MicroRNA­27a­3p regulates the proliferation and chemotaxis of pulmonary macrophages in non­small cell lung carcinoma tissues through CXCL2.

The present study aimed to investigate microRNA (miRNA)-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with early non-small cell lung carcinoma (NSCLC) and its regulatory effect on the infiltration of pulmonary macrophages into cancer tissues and invasion of NSCLC cells. Blood specimens were withdrawn from 36 patients with NSCLC and 29 healthy subjects. NSCLC tissues and cancer-adjacent tissues were both obtained from patients with NSCLC; furthermore, certain tissue samples were used to extract macrophages. The levels of miRNA-27a-3p and C-X-C motif ligand chemokine 2 (CXCL2) mRNA were detected by reverse transcription-quantitative PCR and the levels of CXCL2 protein were measured by ELISA and western blot analysis. A dual-luciferase reporter assay was performed to determine the interactions between miRNA and mRNA. An MTT assay was employed to examine the viability of transfected cells and macrophages and a Transwell assay was performed to assess chemotaxis. The differential expression of miRNA-27a-3p in NSCLC tissues, pulmonary macrophages and peripheral blood indicated that miRNA-27a-3p exerted different roles in these specimens. CXCL2 was upregulated in NSCLC tissues at both transcriptional and translational levels. In addition, the untranslated region of CXCL2 was confirmed to be directly targeted by miRNA-27a-3p prior to its transcriptional activation. Furthermore, miRNA-27a-3p regulated CXCL2 expression, thereby affecting the proliferation of human pulmonary macrophages. The present study highlights that miRNA-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with NSCLC is downregulated, while its target gene CXCL2 is upregulated. miRNA-27a-3p may regulate the viability and chemotaxis of macrophages in tumor tissues of patients with NSCLC through CXCL2 and is expected to become a genetic marker of this disease.

Effect of transfected induced pluripotent stem cells with Decorin gene on control of lung remodeling in allergic asthma.

Asthma is a complex respiratory disease, which is controlled by genetic and environmental factors. Type 2-dominant immune response is responsible for asthma. Decorin (Dcn) and stem cells have modulatory effect on immune system and may control tissue remodeling and asthma pathophysiology. In this study, immunomodulatory effect of transduced induced pluripotent stem cells (iPSCs) with expression of Dcn gene on allergic asthma pathophysiology was evaluated. After transduction of iPSCs with Dcn gene, allergic asthma mice were treated with iPSCs and transduced iPSCs via intrabronchial. Then, airway hyperresponsiveness (AHR), levels of interleukin (IL)-4, IL-5, IL-13, IL-33, total IgE, leukotrienes (LTs) B4, C4, hydroxyproline (HP) content, and transforming growth factor-beta (TGF-ß) were measured. Also, lung histopathology study was done. AHR, levels of IL-4, IL-5, IL-13, IL-33, total IgE, LTs B4, C4, TGF-ß, HP content, mucus secretion, goblet cell hyperplasia, and eosinophilic inflammation were controlled by iPSCs and transduced iPSCs treatment. Therapeutic effect of iPSCs could control main allergic asthma symptoms and related pathophysiologic mechanisms and the effect can be increased when applied with Dcn expression gene.

Bag-1L Protects against Cell Apoptosis in an In Vitro Model of Lung Ischemia-Reperfusion Injury through the C-Terminal "Bag" Domain.

Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional and antiapoptotic protein that binds to the antiapoptosis regulator Bcl-2 and promotes cell survival. To investigate the protective function of Bag-1, we examined the effects of Bag-1L, one isoform of Bag-1, in an in vitro cell culture model of lung ischemia-reperfusion injury (LIRI) generated by treatment of A549 cells with hypoxia/reoxygenation. Overexpression of full-length Bag-1L increased the viability of A549 cells and reduced cell apoptosis in response to 6 h of hypoxia/reoxygenation treatment. Furthermore, Bag-1L overexpression enhanced the heat shock protein 70 (HSP70) and Bcl-2 protein levels, increased the phosphorylation of AKT, decreased Bax and cleaved caspase-3 levels, and was able to overcome cell cycle arrest. These effects were not observed in A549 cells overexpressing a truncated form of Bag-1L lacking the "Bag domain," denoted Bag-1L△C. The "Bag domain" is the C-terminal 47 amino acids. Taken together, the results of this study suggest that Bag-1L overexpression can protect against oxidative stress and apoptosis in an in vitro LIRI model, with a dependence on the Bag domain.

Bag-1 Silence Sensitizes Non-Small Cell Lung Cancer Cells To Cisplatin Through Multiple Gene Pathways.

PURPOSE: B-cell lymphoma-2 (Bcl-2) associated athanogene 1 (Bag-1) is a multifunctional protein, and Bag -1 overexpression is associated with progression, metastasis, and drug resistance in lung cancer. This study assessed the effects of Bag-1 siRNA on sensitization of cisplatin on non-small cell lung cancer (NSCLC) cells. MATERIAL AND METHODS: NSCLC A549 cell line was transfected with Bag-1 or negative control siRNA and then treated with cisplatin for cell viability, CCK-8, LDH, and flow cytometry assays. The Ca2+ levels were analyzed using Fluo-3/AM fluorescence staining, and the protein levels were assessed using Western blot analysis. RESULTS: Bag-1 siRNA significantly knocked down Bag-1 expression and inhibited cell invasion versus the negative control siRNA, while Bag-1 silence sensitized cisplatin to induce A549 cells to apoptosis by induction of cell cycle G1 arrest. At protein level, Bag-1 silence reduced the expression ratio of Bcl-2 to Bcl-2 associated X protein (Bax), downregulated activity of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, and potently upregulated the calcium signaling-mediated pathway. CONCLUSION: This study demonstrated that Bag-1 silencing sensitized A549 to cisplatin to enhance A549 cell apoptosis by modified multiple gene pathways. Further study will evaluate the usefulness of Bag-1 siRNA as a potential targeting therapy for NSCLC.

HSP70 silencing aggravates apoptosis induced by hypoxia/reoxygenation in vitro.

Lung ischemia-reperfusion can cause acute lung injury, which is closely associated with apoptosis. Heat shock protein 70 (HSP70) is an anti-apoptotic protein that promotes cell survival under a variety of different stress conditions. However, the role and mechanism of HSP70 in lung ischemia-reperfusion injury is yet to be fully elucidated. In the present study, an in vitro hypoxia/reoxygenation model of A549 cells was established to simulate lung ischemia-reperfusion and HSP70 was silenced by transfecting A549 cells with an shRNA sequence targeting HSP70. Western blotting, reverse transcription-quantitative polymerase chain reaction, Cell Counting kit-8 and flow cytometry were used to detect protein levels, RNA expression, cell activity and apoptosis. The results revealed that silencing HSP70 reduced cell viability, aggravated apoptosis, increased lactate dehydrogenase levels and induced a G2/M blockade in a hypoxia-reoxygenation A549 cell model. Furthermore, silencing HSP70 decreased the phosphorylation levels of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK); however, the total AKT and ERK levels did not change significantly. Pretreating A549 cells with the AKT pathway inhibitor, LY294002 and the ERK pathway inhibitor, U0216 led to a decrease in HSP70 expression. These results indicate that silencing HSP70 may aggravate apoptosis in hypoxia-reoxygenation cell models, potentially via the mitogen-activated protein kinase/ERK and phosphoinositide 3-kinase/AKT signaling pathways.