Can We Get the Full Picture? Comparison of a Series of Volatile Chemicals’ Action on Gene Expression in A549 Cells as Part of a Read-Across Approach

ABSTRACTAdenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells.IMPORTANCEViral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

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Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.271.6.l963 ◽

1996 ◽

Vol 271 (6) ◽

pp. L963-L971 ◽

Cited By ~ 11

Author(s):

M. A. Fiedler ◽

K. Wernke-Dollries ◽

J. M. Stark

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Viral Replication ◽

Transcriptional Activation ◽

Mutational Analysis ◽

A549 Cells ◽

Nf Kappa B ◽

Mobility Shift ◽

Syncytial Virus ◽

Time Point

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

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Respiratory syncytial virus increases IL-8 gene expression and protein release in A549 cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.269.6.l865 ◽

1995 ◽

Vol 269 (6) ◽

pp. L865-L872 ◽

Cited By ~ 13

Author(s):

M. A. Fiedler ◽

K. Wernke-Dollries ◽

J. M. Stark

Keyword(s):

Gene Expression ◽

Respiratory Syncytial Virus ◽

Viral Replication ◽

Mrna Expression ◽

A549 Cells ◽

Protein Release ◽

Tnf Alpha ◽

Epithelial Cell Line ◽

Airway Epithelial ◽

Syncytial Virus

The mechanism of respiratory syncytial virus (RSV)-induced inflammation in the airways of infants and children is not fully understood. We hypothesized that RSV directly induces interleukin (IL)-8 gene expression in airway epithelial cells, independent of IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) production. Exposure of A549 cells (an airway epithelial cell line) to RSV resulted in increased IL-8 mRNA expression and IL-8 protein release from the cells as early as 2 h after treatment. Neither IL-1 beta nor TNF-alpha (mRNA or protein) were detected. Viral replication was not necessary for the effects of RSV on IL-8 mRNA expression and protein release early in the infectious process. However, sustained levels of increased IL-8 production required RSV replication. A dose-response relationship was observed between the multiplicity of infection and IL-8 production with both active and nonreplicative RSV at the 2-h time point. Both active RSV and nonreplicative RSV increased the transcriptional activity of the 1.6-kb 5' flanking region of the IL-8 gene. Neither active RSV nor nonreplicative RSV increased the stability of the IL-8 mRNA in A549 cells. We conclude that RSV increases IL-8 gene expression in A549 cells in a biphasic pattern independent of viral replication early (2 h) but dependent on viral replication late (24 h).

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Role of oxidants in influenza virus-induced gene expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.1.l134 ◽

1998 ◽

Vol 274 (1) ◽

pp. L134-L142 ◽

Cited By ~ 13

Author(s):

Katharine Knobil ◽

Augustine M. K. Choi ◽

Gordon W. Weigand ◽

David B. Jacoby

Keyword(s):

Gene Expression ◽

Cell Death ◽

Influenza Virus ◽

Virus Infection ◽

A549 Cells ◽

Influenza Virus Infection ◽

Pyrrolidine Dithiocarbamate ◽

Oxygen Intermediates ◽

Mrna Induction

Influenza virus-induced epithelial damage may be mediated, in part, by reactive oxygen intermediates (ROIs). In this study, we investigated the role of ROIs in the influenza virus-induced gene expression of antioxidant enzymes and in the activation of nuclear factor-κB (NF-κB), an oxidant-sensitive transcriptional factor. Influenza virus infection increased production of intracellular ROIs in A549 pulmonary epithelial cells. Induction of manganese superoxide dismutase (MnSOD) mRNA correlated with increased MnSOD protein and enzyme activity. Influenza virus infection also activated NF-κB binding as determined by an electrophoretic mobility shift assay. Pretreatment of A549 cells with N-acetyl-l-cysteine attenuated virus-induced NF-κB activation and interleukin (IL)-8 mRNA induction but did not block induction of MnSOD mRNA. In contrast, pyrrolidine dithiocarbamate blocked activation of NF-κB and induction of MnSOD and IL-8 mRNAs. Treatment with pyrrolidine dithiocarbamate also markedly decreased virus-induced cell death. Thus oxidants are involved in influenza virus-induced activation of NF-κB, in the expression of IL-8 and MnSOD, and in virus-induced cell death.

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Gene expression profiling of cancer stem cell in human lung adenocarcinoma A549 cells

Molecular Cancer ◽

10.1186/1476-4598-6-75 ◽

2007 ◽

Vol 6 (1) ◽

pp. 75 ◽

Cited By ~ 55

Author(s):

Dong-Cheol Seo ◽

Ji-Min Sung ◽

Hee-Jung Cho ◽

Hee Yi ◽

Kun-Ho Seo ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Cancer Stem Cell ◽

Lung Adenocarcinoma ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Human Lung ◽

A549 Cells ◽

Human Lung Adenocarcinoma ◽

Lung Adenocarcinoma A549

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Study on the antitumor activity and mechanism of novel targeted nanodrugs based on miRNA in cervical cancer

Materials Express ◽

10.1166/mex.2020.1797 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1218-1223

Author(s):

Xinping Chen ◽

Zhichao Ma ◽

Juan Zhu ◽

Weihua Xu ◽

Junjie Hu ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Antitumor Activity ◽

A549 Cell ◽

Cell Apoptosis ◽

Caspase 3 ◽

A549 Cells ◽

Dependent Manner ◽

Double Staining ◽

Dose Dependent

The aim of this study was to investigate the effect of different concentrations of novel targeted nanodrugs based on miRNA on the antitumor activity and mechanism in cervical carcinoma A549 cells. The MTT method was used to determine the effect of different concentrations of novel targeted nanodrugs based on miRNA on A549 cell proliferation, and annexin V FITC/PI double staining flow cytometry was performed to analyze the effect of these nanodrugs on A549 cell apoptosis. Western blotting was performed to observe the effect of these nanodrugs on the expression of Bax, Bcl-2, and caspase-3-related genes involved in A549 cell apoptosis. Compared with the control group, the novel targeted nanodrugs based on miRNA significantly inhibited the proliferation of A549 cells in a time- and dose-dependent manner. Results of double staining flow cytometry demonstrated that these nanodrugs could increase the apoptotic rate of A549 cells in a dose-dependent manner 48 h later. Western blotting revealed that these nanodrugs could upregulate the expression of Bax and caspase3 genes and downregulate the expression of Bcl-2 gene. Nanodrugs display an obvious antitumor activity in vitro, and the underlying mechanism may be associated with the upregulation of Bax and caspase-3 gene expression and the downregulation of Bcl-2 gene expression.

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Burkholderia pseudomallei Down-Regulates Host Defense Gene Expression in Non-phagocytic A549 Cells

International Journal of Infectious Diseases ◽

10.1016/j.ijid.2008.05.553 ◽

2008 ◽

Vol 12 ◽

pp. e223

Author(s):

S.J. Chai ◽

R. Mohamed ◽

S. Nathan

Keyword(s):

Gene Expression ◽

Host Defense ◽

Burkholderia Pseudomallei ◽

A549 Cells ◽

Defense Gene Expression ◽

Defense Gene

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Glucocorticoids inhibit IL-1β-induced GM-CSF expression at multiple levels: roles for the ERK pathway and repression by MKP-1

Biochemical Journal ◽

10.1042/bj20091038 ◽

2010 ◽

Vol 427 (1) ◽

pp. 113-124 ◽

Cited By ~ 32

Author(s):

Robert Newton ◽

Elizabeth M. King ◽

Wei Gong ◽

Christopher F. Rider ◽

Karl J. Staples ◽

...

Keyword(s):

Gene Expression ◽

Protein Synthesis ◽

Transcriptional Repression ◽

Actinomycin D ◽

De Novo ◽

A549 Cells ◽

Mitogen Activated Protein Kinase ◽

Erk Pathway ◽

Gm Csf ◽

Erk Phosphorylation

In the present study, IL (interleukin)-1β increased GM-CSF (granulocyte/macrophage colony-stimulating factor) expression from pulmonary A549 cells and primary HBE (human bronchial epithelial) cells. These responses were repressed by the glucocorticoid dexamethasone, allowing the use of A549 cells as a relevant model. IL-1β induced GM-CSF release into the culture medium by 6 h and in cell lysates (cytosolic) at 2 h. These effects were profoundly inhibited by dexamethasone, yet IL-1β-induced GM-CSF mRNA and unspliced nRNA (nuclear RNA; a surrogate of transcription rate) were modestly inhibited by dexamethasone at times up to 2 h. Although this indicates an effect on protein synthesis, actinomycin D chase experiments also indicated post-transcriptional repression by dexamethasone. Dexamethasone-dependent mRNA repression increased with time and was prevented by translational blockade. In addition, dexamethasone and the dissociated steroid RU24858 repressed GM-CSF release in an actinomycin D-sensitive manner, thereby implicating glucocorticoid-induced gene expression. At 2 h, IL-1β-induced expression of GM-CSF protein, but not mRNA, was sensitive to the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitors PD098059 and U0126. Although this indicates a role for the MEK/ERK pathway in GM-CSF translation, PD098059 subsequently destabilized GM-CSF mRNA. Dexamethasone and RU24858 both reduced IL-1β-induced ERK phosphorylation and increased MKP-1 (MAPK phosphatase-1) expression. Inhibition of ERK phosphorylation was reproduced by MKP-1 overexpression and prevented by MKP-1-targeting siRNA (small interfering RNA). Since MKP-1 prevented GM-CSF expression by transcriptional, post-transcriptional and translational processes, we propose that glucocorticoids induce MKP-1 expression to reduce both MEK/ERK activation and GM-CSF protein synthesis. Thus de novo gene expression, particularly of MKP-1, is involved in the repressive effects of glucocorticoids.

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Novel Disulfide-Containing Poly(β-amino ester)-Functionalised Magnetic Nanoparticles for Efficient Gene Delivery

Australian Journal of Chemistry ◽

10.1071/ch15293 ◽

2016 ◽

Vol 69 (3) ◽

pp. 349 ◽

Cited By ~ 1

Author(s):

Yucheng Liu ◽

Shufeng Li ◽

Liandong Feng ◽

Hao Yu ◽

Xiaoliang Qi ◽

...

Keyword(s):

Gene Expression ◽

Magnetic Nanoparticles ◽

Disulfide Bonds ◽

Transfection Efficiency ◽

A549 Cells ◽

Weight Ratio ◽

Cell Types ◽

Amino Ester ◽

Gene Complexes

Poly(β-amino ester)s (PBAEs) have been proved to effectively transfer DNA to various cell types. However, PBAEs with high molecular weights also show considerable toxicities, partly resulting from inadequate degradation of their polyester backbone. In this study, we created novel poly(β-amino ester)s (SF-1, 2, 3, and 4; notation SFs refers to all the four polymers) which were characterised by the cleavable disulfide bonds. Moreover, a new technique, termed magnetofection that uses magnetic nanoparticles to enhance gene expression, has recently been well developed. The negatively charged magnetic nanoparticles (MNPs) with good biocompatibility in vitro were prepared here to subsequently combine with SFs and DNA via electrostatic interaction, leading to the formation of the magnetic gene complexes MNP/SFs/DNA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and transfection experiments were performed in A549 cells to investigate all the resulting complexes. Studies indicated that the synthesised PBAEs exhibited good biodegradation and regulated release of DNA as a result of the reductive cleavage of the disulfide bonds, giving higher transfection efficiency along with much lower cytotoxicity compared with commercially available transfection agent polyethylenimine (Mw 25 kDa). Furthermore, when MNP was involved at a MNP/DNA weight ratio of 0.5, the magnetic gene complexes MNP/SFs/DNA showed enhanced levels of gene expression while maintaining low cytotoxicity.

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