BACKGROUND
plasma known as the fourth state of matter is partially or completely ionized gas including a mixture of electrons, ions, radicals, and energetic photons, which is typically generated under high temperature conditions.
due to the interdisciplinary effort of physics and biotechnology, recent technological progress in atmospheric plasmas has led to the creation of cold plasmas with an ion temperature close to the room temperature, which can be controlled or changed according to the desired applications.
moreover, that plasma can interact with organic materials without causing thermal damage to the cell surface, and can effectively inactivate different kinds of pathogens without adversely affecting the healthy tissues.
many other biological applications have also been explored.
accumulated evidence has shown that plasma can be generated and play an increasingly important role in various applications at room temperature, such as blood coagulation, wound healing, and tissue and device sterilization  <cit> .
furthermore, using endoscopic effect of non-thermal atmospheric pressure plasma has been developed in both clinical applications and disinfection of medical devices  <cit> , as a targeted and low invasive technique.
additionally, some researches have been performed in the utility of cold plasma for anti-tumor or cancer therapy in various tumor cell lines including several solid malignant  cells  and lymphoma cells  <cit> .
in particular, it is remarked here that non-thermal atmospheric pressure plasma  could treat some tumors under the superficial skin such as melanoma, as well as head and neck cancers  <cit> .
moreover, accumulated evidence has revealed that plasma treatment can induce various effects on multiple cancer cell types, including oxidative stress such as ros and rns mediated mitochondria-dependent apoptosis  <cit> , cell cycle arrest, cell growth inhibition  <cit>  and obstructed tumor invasion  <cit> .
the relationships between plasma exposure and cellular responses have been partially established in recent studies which demonstrated that plasma treatment of cells resulted in intense alterations of apoptotic signaling  <cit>  related transcription factors  <cit> , dna damages  <cit>  and atm/p <dig>  <cit>  pathways.
in particular, the reactive oxygen/nitrogen species including h2o <dig>  ox, no <dig>  nox, which lead to depolarization of the mitochondrial membrane and mitochondrial ros accumulation that displayed a compromised redox status evident from increased nadp+/nadph levels, reduced the gsh/gssg ratios and enhanced cytogenetic damages revealed by micronucleus formation assessment  <cit> .
moreover, yan and colleagues elaborated on the several stages involved in the induction of cancer cell death by cold atmospheric-pressure plasma through increased concentrations of no, ros and lipid peroxide  <cit> .
in fact, more factors affected the plasma effect on cancer cell viability.
chen defined the “plasma dosage” which characterized the relationship between the characteristics of the cold plasma  and cell viability  <cit> .
in reality, the capability of cell killing by plasma was even found to be sensitive to the concentration of fetal bovine serum in the media as well as the storage temperature for the media  <cit> .
however, the detailed mechanisms underlying these effects have not yet been fully identified.
an original research indicated that ntp executed killing effect of human lung cancer cell lines through mitochondrial dysfunction  <cit> .
moreover, recent studies confirmed these, which reported that ntp induced apoptosis of head and neck cancer cells by a mechanism involving mapk-dependent mitochondrial ros  <cit>  and that ros/rns triggered jnk and p <dig> pathways which promoted mitochondrial perturbation and apoptosis  <cit> .
to fundamentally understand and elucidate the effect of ntp on biological pathways, high-throughput microarrays, as an ideal screening tool for gene expression profiling analysis, could help us investigate the underlying genetic characteristic of processes involved in plasma-induced cellular or molecular responses.
the goal of the present study was to analyze the gene expression after ntp exposure that might be involved in cellular apoptosis, signaling transduction and stress response.
therefore, typical non-small cell lung cancer cells a <dig> were exposed to ntp with helium as the carrier gas.
with the high-density microarray technique, the transcriptome for genes regulated by ntp were screened.
the results showed different dose-dependent profile patterns upon various ntp exposure durations, and showed that the ntp exposure modulated the transcriptional factors , cytokines , leukocyte recruitment and activation factors , enzymes of phosphatase  and cyclooxygenase .
furthermore, several signaling pathways related to stress defensing mechanisms as well as key transcriptional processes were identified.
RESULTS
non-thermal plasma-induced differential gene expression in a <dig> cell
using the microarray based approach, we analyzed the cellular gene expression profile of lung adenocarcinoma a <dig> cells upon treatment with non-thermal plasma, and focused on finding plasma-associated molecular signatures to elucidate the impact of ntp on the transcriptome of this tumor cell.
even though the survival of the 3-min treatment group decreased to only approximate  <dig> % at 4 h post exposure, when compared to the sham control , the rna integrity number  still showed that rna was not degraded and had sufficiently high quality for further analysis .fig.
<dig> the cell viability assessment of ntp on treated a <dig>  a photographs of a plasma jet and the plasma system.
b the assessment of cell viability of a <dig> cell after ntp exposure for 1 min right panel and 3 min left panel, respectively.
time points of  <dig>   <dig>   <dig> or 8 h for viability measurements after ntp exposure were adopted.
the mean and sd are shown for three independent experiments.
the t-test was applied for the comparative analysis between the treatment group and the control group.
the p-values were shown while ns indicated no significant difference
with the selection criteria mentioned above,  <dig> differentially expressed genes were obtained for all time points.
specifically, at 4 h after the 1-min ntp treatment,  <dig> genes  showed significant expression according to the preset criteria , whereas only  <dig> genes ,  <dig> genes  and  <dig> genes  expressed at  <dig>   <dig> and 4 h, respectively, after 3-min ntp exposures .
these data have been deposited in ncbi’s gene expression omnibus and are accessible through geo series accession number gse <dig>  and the complete overview on the differentially regulated genes could be achieved by geo2r or other software.fig.
<dig> the heatmap of differentially expressed genes.
patterns of changes in transcript abundance are shown on a heatmap for a robust set of  <dig> transcripts using a bh p-value of  <dig>  and fold changes >  <dig> .
red color represents relative increase in abundance, blue color represents relative decrease, and white color represents no change.
the color bars above the map indicate the time points and ntp doses.
gene ontology and pathway enrichment analysis
to retrieve the functional information on differentially expressed genes in each experimental group, co-regulated genes were further classified with bingo application with plugged-in cytoscape.
the individual expression products of the identified genes were distributed in different cellular compartments, functional classes, and involved numerous molecular and biological processes.
the bar charts  illustrated the affiliation of the top  <dig> terms of the regulated genes  with gene class ontologies identified for each group.
the whole go terms for each time point or group could be found in the additional file 1: table s <dig> fig.
<dig> the gene ontology category of each group at indicated time points.
the gene ontology analysis was performed by bingo plugged-in cytoscape.
the biological processes , molecular function mf and cellular components cc terms were involved.
to include only significant results, the fdr threshold was set to  <dig> .
the top  <dig> terms were selected, and represented as bar plots with gene number involved in these go terms .
moreover, the corrected p values were also obtained as indicated by the yellow dotted line, which had been transformed into log <dig> with reference to the lower y-axis.
the red dashed line indicated the threshold of adjusted p values .
firstly, the go terms of the whole transcriptome were compared with ntp to understand which categories in response to biological processes were enriched in plasma-treated cells.
at any time point after different ntp treatments, various genes were regulated and involved in a multitude of biological processes.
top hits among the biological process categories also comprised specific aspects of negative regulation of biological process, such as apoptosis and cell proliferation, response to stress and transcription factor activity, which concentrated on up-regulation of gene clusters.
meanwhile, chromosome organization and cell cycle terms were enriched in down-regulated gene clusters at 4 h in the 1-min ntp-exposed group .
in contrary to the 1-min ntp exposure, the 3-min ntp treatment caused differential go categories.
at 2 h post the 3-min ntp exposure, the up-regulated genes focused on transcription regulation, and down-regulated genes clustered into nucleotide, metabolic process and methyltransferase activity .
meanwhile, at 4 h after exposure, the top categories comprised nucleosome/chromatin assembly, dna behavior involved in conformation change and dna replication in up-regulated gene list, however most of the regulations involved in metabolic processes, nucleus and molecular binding were observed in the down-regulated genes .
in the next step, we utilized gene ontology enrichment analysis to concentrate on the go categories that were really pivotal at the indicated time point of each group.
briefly, different ntp exposure durations led to distinct time-dependent profiling patterns.
specifically, the down-regulated genes after the 1-min ntp exposure were mainly related to cellular metabolism including chromosome processes, cell cycle, atpase activity, and nucleoside-triphosphatase activity.
meanwhile, the up-regulated genes were related to kinase activity, transcription factor activity, and response to reactive oxygen species .
interestingly, the gene list for 2 h post 3-min ntp exposure focused on methyltransferase activity, which was a unique gene set among all groups.
the enrichment gene sets at 4 h after the 3-min ntp exposure was mostly related to negative or down regulation of cellular processes.
these genes were involved in the macromolecule metabolic process, methylation, nucleoplasm, cell cycle, and dna metabolic process and atp energy metabolism .
all the genes mentioned above are known to be important in the stimulation of cell proliferation and recruitment.fig.
<dig> the gene ontology and pathway enrichment results at indicated time points for ntp treatment for 1 min and 3 min.
the biological processes bp, molecular function mf and cellular components cc terms were performed for go enrichment analysis and the kegg, reactome and wikipathways databases were performed for pathway enrichment analysis of each differential group genes.
the statistical test was enrichment/depletion two-side hypergeometric test, and the benjamini and hochberg false-discovery rate was set to  <dig> .
terms with up/down regulated genes are shown in red/green, respectively.
the color gradient shows the gene proportion of each cluster associated with the term.
equal proportions of the two clusters are represented in gray.
the resulting corrected p values are included in the network visualization by the size of nodes.
furthermore, in the pathway enrichment analysis, the regulated genes for 1-min ntp exposure were clustered into biological groups which were different from those corresponding to the 3-min ntp exposure.
for instance, it was found that the 1-min ntp exposure significantly repressed the expression of genes involved in mitotic telophase/cytokinesis and prometaphase as well as cell cycle, including dna damage response and mirna mediated regulation.
moreover, the 1-min ntp exposure also activated some of pathways including the il- <dig> signaling pathway, p <dig> mediated signaling pathway, mapk, tgf-β and tnf signaling pathways .
however, the 3-min ntp exposure caused up-regulated genes like histones which played a role in hats acetylate histones and rna polymerase involved in transcription regulation processes.
in contrast, the 1-min ntp exposure reduced the expression of genes involved in regulation of the il- <dig> signaling pathway and the tgf-β signaling pathway .
however, the effect of extended ntp processing time on gene profiles and cellular behavior and metabolism was not clear.
involved hub transcription factors
considering that some factors play critical roles in biological processes and interact with abundant related genes or proteins, the top hits proteins were sorted and assessed from protein-protein interaction  network.
apparently, the 1-min ntp exposure induced specific patterns that included some specifically up-regulated genes.
these genes were involved in gtp and gdp energy metabolism , and ca2+ ion flow or release  .
moreover, some transcription factors that played important roles in transcription and regulation were also significantly enhanced .
jun and fos were thought to play important roles in signal transduction, cell proliferation and differentiation.
ccaat/enhancer binding protein   were also important transcriptional activators that regulated expression of genes involved in immune and inflammatory responses.
they could bind to regulatory regions of several acute-phase and cytokines genes, and probably played a role in the regulation of acute-phase reaction and inflammation.
nfkbia inhibited the activity of nf-κb/rel complex by trapping rel dimers in the cytoplasm through masking their nuclear localization signals.
additionally, other processes were also present, which involved tyrosine-kinase-based signaling related to cell adhesion , and cell cycle phase transition .fig.
<dig> the selected genes from deg based on protein network betweenness and degree.
the genes selected from different expression gene lists, and sorted by top  <dig> % genes in the protein interaction database according to pid betweenness and degree.
red color represents relative increase in abundance, blue color represents relative decrease, and white represents no change.
fig.
<dig> the details of selected pathways after ntp exposure.
the illustration showed gene correlation of indicated pathway enrichment in details by cluepedia plugged-in cytoscape.
the nodes represented genes in each pathway, with red and green colors of nodes indicating up- or down-regulated genes, respectively.
the green line and yellow line represented activation and expression relationship in gene interaction enrichment, respectively.
responded multiple pathways
differential gene expression in response to the ntp exposure was assessed to elucidate effects of diverse pathways on the cancer cell.
pathway enrichment analysis and pid top hits genes categorized several genes involved in the response to ntp exposure, which comprised transcription factors , negative intracellular signaling factors , leukocyte recruitment and activation factors , enzymes of phosphatase  and cyclooxygenase .
in the pathway enrichment results, the up-regulated pathways included il- <dig>  tnf, tgf-β and mapk signaling pathways, which constituted a network with centralization of jun, fos and jak critical transcription factors .
among these, the transcription factors were up-regulated at least 8-fold in the 1-min plasma-treated cells.
next, we focused on changes in expression values of genes whose transcription levels increased under stressful growth arrest conditions.
the p <dig> was a hub of response to stress signals, as shown in the right box of fig.
<dig>  gadd <dig> was a dna-damage inducing gene, mediating the activation of the p38/juk pathway via mtk1/mekk <dig> kinase.
dusp also negatively regulated members of map kinase superfamily , which was associated with cellular proliferation and differentiation.
all the genes mentioned above displayed enhanced mrna expression.fig.
<dig> western blot analysis and validation of several key proteins from microarray results.
a <dig> cells were collected and lysed after 1-min ntp treatment at different time points, namely,  <dig>   <dig>   <dig>   <dig> and 24 h. with sds-page gel electrophoresis, the expressions of caspase <dig>  parp, mtor, phospho-mtor, akt, phospho-akt, nf-κb, phospho-iκbα, jnk, phospho-jnk, p <dig> mapk, phospho-p <dig> mapk, c-jun, junb, c-fos, and actb were measured.
qpcr and western blot validation
to further validate some of the microarray profiles, seven representative candidate genes were selected for analyzing the gene expression levels via the qpcr method.
the regulation patterns for all selected genes were consistent with the microarray data and only had marginal differences in the relative fold changes, except the egr <dig> and fos genes which presented extremely high mrna expression levels by qpcr when compared to microarray analysis .
moreover, among the microarray results, we focused on and selected several candidate proteins for multiple pathways, which included apoptosis, mapk signaling pathway, jnk, akt and nf-κb pathways, and some critical transcriptional factors .
among the western blot results, the caspase- <dig> and parp degraded over the time points, which indicated the classical apoptosis cascade.
mtor and phosphorylated mtor levels peaked at 8 h post ntp treatment and descended thereafter.
mtor is a serine/threonine protein kinase which is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy and stress signals  <cit> .
it plays a critical role in the phosphorylation of akt  <cit> , which has also been validated in our western blot result in that the expression of phospho-akt was increased till 8 h after exposure.
after that, the akt regulated the nf-κb signaling pathway by inducing release of nf-κb through phosphorylation and degradation of iκbα .
ntp stimuli activated jnk, and phosphorylation of jnk on the site of thr183/tyr <dig> was increased in the time course, which modified the activities of numerous proteins that resided at the mitochondria or acted in the nucleus, and were involved in apoptosis, cell differentiation and proliferation.
moreover, the jnk activated the ap- <dig> transcriptional factors   <cit> .
only junb showed continued enhancement with in the time course after ntp treatment, while the expression levels of c-jun and c-fos remained stable.
overall, from the translational level results, ntp treatment induced cell apoptosis and cell cycle arrest to assist processes for cell repair.table  <dig> verification of gene expression changes in 1-min ntp treatment at 4 h after exposure
note: the results were shown as mean ± sd in the 2nd and 3ed columns, and asterisks indicated significant differences between two methods found using t-test
discussion
the impact of ntp on cancer cells has not been investigated using the microarray approach in detail to date.
the present work was the first to employ this high-throughput technique to elucidate the responses of nsclc tumor cells to a particular ntp treatment in vitro.
the microarray technique was applied to determine the activities of thousands of genes at once to create a partial picture of cellular functions of a <dig> cells following ntp exposure.
in a previous study  <cit> , the microarray technique had been used and to examine the therapeutic potential of a plasma jet on cancer cell lines and tumors, with a focus on the capability of selective tumor cell eradication and on deregulation of signaling pathways.
these selective effects of ntp on different cell types can find important applications in cancer treatment  <cit> .
besides the apoptotic effects induced by long-time  ntp exposures to a <dig> cells, we also focused on the biological effects induced by short-time  ntp exposures, which were below the levels that induced apoptosis in most of the cells.
the time points of  <dig>   <dig> and 4 h post cell treatment were selected for detection of early effects in terms of ntp modulated gene activities, which were indeed confirmed by the microarray data.
the expressions of more than  <dig> genes were modulated by ntp exposures, with at least  <dig> -fold induction or repression when compared to those of non-treated cells.
our results showed that the affected genes differed significantly between the control and treated cells depending on the conditions.
both sub-groups  contained differentially expressed genes encoding for molecules known to participate in the apoptotic process, cellular response to stress, chromosome organization, cell cycle, dna damage response and mirna regulation.
the signaling and network analysis by cluego/cluepedia revealed that certain significantly regulated genes were involved in pathways which led to the p <dig> signaling pathway, mapk signaling pathway, tgf and tnf signaling pathways, and il-4/il- <dig> signaling pathways.
likewise, the majority of the biological processes that decreased the expressions were mainly related to chromosome organization/assembly, cell cycle and metabolic processes.
the most striking effect of ntp in the gene expression profiling analysis was the modulation of a large set of genes that were involved in multiple signaling pathways, including pi3k/akt and nf-κb mediated tnf signaling, mapk, jak-stat, tgf-β and vegf pathways.
to better understand the influence of plasma-induced reactive species and potentially beneficial mechanisms of ntp on a <dig> cells, we examined the protein expression of apoptosis and related transcriptional factors which were mostly up-regulated or down-regulated after ntp exposure.
a number of transcripts were identified to be significantly modulated for transcriptional factors.
in this regard, cluego predicted the involvement of fos/jun/p <dig> in intracellular signaling in our experimental setup.
further analysis of our data suggested that ntp might activate a broader range of p53-relevant processes.
it is known that p <dig> is a critical transcription factor which participates in transcription as well as signal transduction, including cell cycle arrest  <cit> , apoptosis  <cit> , dna repair  <cit>  and damage prevention, cellular senescence and p <dig> negative feedback.
the western blot assay also confirmed the multiple pathways involved in these complicated processes.
the mapk, jnk and nf-κb pathways were all involved, and played critical roles in the regulation of responses to external stresses.
in summary, the large number of negatively regulated genes involved in biological or cellular processes reflected the cellular responses to ntp, and helped confirm specific pathways or stress responses to ntp.
although the details regarding the activation of specific pathways remained to be elucidated, our data presented new evidence that ntp treatment of a <dig> cells modulated the transcription of a variety of genes, which might center on p <dig> and mapk and tnf signaling pathways, etc.
our data further indicated that it was possible to modulate cellular signaling as well as metabolism and/or apoptosis processes in ntp treated tumor cells on a genetic level.
CONCLUSIONS
overall, non-thermal plasma treated cells exhibited a pattern typical of damage responses, thereby validating several critical pathways/regulators, presumably mediated through multiplex signaling pathways.
further investigation of these genes in other cell lines may reveal comprehensive mechanisms of plasma induced effects.

 plasma known as the fourth state of matter is partially or completely ionized gas including a mixture of electrons, ions, radicals, and energetic photons, which is typically generated under high temperature conditions. due to the interdisciplinary effort of physics and biotechnology, recent technological progress in atmospheric plasmas has led to the creation of cold plasmas with an ion temperature close to the room temperature, which can be controlled or changed according to the desired applications. moreover, that plasma can interact with organic materials without causing thermal damage to the cell surface, and can effectively inactivate different kinds of pathogens without adversely affecting the healthy tissues. many other biological applications have also been explored. accumulated evidence has shown that plasma can be generated and play an increasingly important role in various applications at room temperature, such as blood coagulation, wound healing, and tissue and device sterilization. furthermore, using endoscopic effect of non-thermal atmospheric pressure plasma has been developed in both clinical applications and disinfection of medical devices, as a targeted and low invasive technique. additionally, some researches have been performed in the utility of cold plasma for anti-tumor or cancer therapy in various tumor cell lines including several solid malignant cells and lymphoma cells. in particular, it is remarked here that non-thermal atmospheric pressure plasma could treat some tumors under the superficial skin such as melanoma, as well as head and neck cancers. moreover, accumulated evidence has revealed that plasma treatment can induce various effects on multiple cancer cell types, including oxidative stress such as ros and rns mediated mitochondria-dependent apoptosis, cell cycle arrest, cell growth inhibition and obstructed tumor invasion. the relationships between plasma exposure and cellular responses have been partially established in recent studies which demonstrated that plasma treatment of cells resulted in intense alterations of apoptotic signaling related transcription factors, dna damages and atm/p pathways. in particular, the reactive oxygen/nitrogen species including h2o ox, no nox, which lead to depolarization of the mitochondrial membrane and mitochondrial ros accumulation that displayed a compromised redox status evident from increased nadp+/nadph levels, reduced the gsh/gssg ratios and enhanced cytogenetic damages revealed by micronucleus formation assessment. moreover, yan and colleagues elaborated on the several stages involved in the induction of cancer cell death by cold atmospheric-pressure plasma through increased concentrations of no, ros and lipid peroxide. in fact, more factors affected the plasma effect on cancer cell viability. chen defined the “plasma dosage” which characterized the relationship between the characteristics of the cold plasma and cell viability. in reality, the capability of cell killing by plasma was even found to be sensitive to the concentration of fetal bovine serum in the media as well as the storage temperature for the media. however, the detailed mechanisms underlying these effects have not yet been fully identified. an original research indicated that ntp executed killing effect of human lung cancer cell lines through mitochondrial dysfunction. moreover, recent studies confirmed these, which reported that ntp induced apoptosis of head and neck cancer cells by a mechanism involving mapk-dependent mitochondrial ros and that ros/rns triggered jnk and p pathways which promoted mitochondrial perturbation and apoptosis. to fundamentally understand and elucidate the effect of ntp on biological pathways, high-throughput microarrays, as an ideal screening tool for gene expression profiling analysis, could help us investigate the underlying genetic characteristic of processes involved in plasma-induced cellular or molecular responses. the goal of the present study was to analyze the gene expression after ntp exposure that might be involved in cellular apoptosis, signaling transduction and stress response. therefore, typical non-small cell lung cancer cells a were exposed to ntp with helium as the carrier gas. with the high-density microarray technique, the transcriptome for genes regulated by ntp were screened. the results showed different dose-dependent profile patterns upon various ntp exposure durations, and showed that the ntp exposure modulated the transcriptional factors, cytokines, leukocyte recruitment and activation factors, enzymes of phosphatase and cyclooxygenase. furthermore, several signaling pathways related to stress defensing mechanisms as well as key transcriptional processes were identified.  non-thermal plasma-induced differential gene expression in a cell using the microarray based approach, we analyzed the cellular gene expression profile of lung adenocarcinoma a cells upon treatment with non-thermal plasma, and focused on finding plasma-associated molecular signatures to elucidate the impact of ntp on the transcriptome of this tumor cell. even though the survival of the 3-min treatment group decreased to only approximate % at 4 h post exposure, when compared to the sham control, the rna integrity number still showed that rna was not degraded and had sufficiently high quality for further analysis.fig. the cell viability assessment of ntp on treated a a photographs of a plasma jet and the plasma system. b the assessment of cell viability of a cell after ntp exposure for 1 min right panel and 3 min left panel, respectively. time points of or 8 h for viability measurements after ntp exposure were adopted. the mean and sd are shown for three independent experiments. the t-test was applied for the comparative analysis between the treatment group and the control group. the p-values were shown while ns indicated no significant difference with the selection criteria mentioned above, differentially expressed genes were obtained for all time points. specifically, at 4 h after the 1-min ntp treatment, genes showed significant expression according to the preset criteria, whereas only genes, genes and genes expressed at and 4 h, respectively, after 3-min ntp exposures. these data have been deposited in ncbi’s gene expression omnibus and are accessible through geo series accession number gse and the complete overview on the differentially regulated genes could be achieved by geo2r or other software.fig. the heatmap of differentially expressed genes. patterns of changes in transcript abundance are shown on a heatmap for a robust set of transcripts using a bh p-value of and fold changes > . red color represents relative increase in abundance, blue color represents relative decrease, and white color represents no change. the color bars above the map indicate the time points and ntp doses. gene ontology and pathway enrichment analysis to retrieve the functional information on differentially expressed genes in each experimental group, co-regulated genes were further classified with bingo application with plugged-in cytoscape. the individual expression products of the identified genes were distributed in different cellular compartments, functional classes, and involved numerous molecular and biological processes. the bar charts illustrated the affiliation of the top terms of the regulated genes with gene class ontologies identified for each group. the whole go terms for each time point or group could be found in the additional file 1: table s fig. the gene ontology category of each group at indicated time points. the gene ontology analysis was performed by bingo plugged-in cytoscape. the biological processes, molecular function mf and cellular components cc terms were involved. to include only significant results, the fdr threshold was set to. the top terms were selected, and represented as bar plots with gene number involved in these go terms. moreover, the corrected p values were also obtained as indicated by the yellow dotted line, which had been transformed into log with reference to the lower y-axis. the red dashed line indicated the threshold of adjusted p values. firstly, the go terms of the whole transcriptome were compared with ntp to understand which categories in response to biological processes were enriched in plasma-treated cells. at any time point after different ntp treatments, various genes were regulated and involved in a multitude of biological processes. top hits among the biological process categories also comprised specific aspects of negative regulation of biological process, such as apoptosis and cell proliferation, response to stress and transcription factor activity, which concentrated on up-regulation of gene clusters. meanwhile, chromosome organization and cell cycle terms were enriched in down-regulated gene clusters at 4 h in the 1-min ntp-exposed group. in contrary to the 1-min ntp exposure, the 3-min ntp treatment caused differential go categories. at 2 h post the 3-min ntp exposure, the up-regulated genes focused on transcription regulation, and down-regulated genes clustered into nucleotide, metabolic process and methyltransferase activity. meanwhile, at 4 h after exposure, the top categories comprised nucleosome/chromatin assembly, dna behavior involved in conformation change and dna replication in up-regulated gene list, however most of the regulations involved in metabolic processes, nucleus and molecular binding were observed in the down-regulated genes. in the next step, we utilized gene ontology enrichment analysis to concentrate on the go categories that were really pivotal at the indicated time point of each group. briefly, different ntp exposure durations led to distinct time-dependent profiling patterns. specifically, the down-regulated genes after the 1-min ntp exposure were mainly related to cellular metabolism including chromosome processes, cell cycle, atpase activity, and nucleoside-triphosphatase activity. meanwhile, the up-regulated genes were related to kinase activity, transcription factor activity, and response to reactive oxygen species. interestingly, the gene list for 2 h post 3-min ntp exposure focused on methyltransferase activity, which was a unique gene set among all groups. the enrichment gene sets at 4 h after the 3-min ntp exposure was mostly related to negative or down regulation of cellular processes. these genes were involved in the macromolecule metabolic process, methylation, nucleoplasm, cell cycle, and dna metabolic process and atp energy metabolism. all the genes mentioned above are known to be important in the stimulation of cell proliferation and recruitment.fig. the gene ontology and pathway enrichment results at indicated time points for ntp treatment for 1 min and 3 min. the biological processes bp, molecular function mf and cellular components cc terms were performed for go enrichment analysis and the kegg, reactome and wikipathways databases were performed for pathway enrichment analysis of each differential group genes. the statistical test was enrichment/depletion two-side hypergeometric test, and the benjamini and hochberg false-discovery rate was set to. terms with up/down regulated genes are shown in red/green, respectively. the color gradient shows the gene proportion of each cluster associated with the term. equal proportions of the two clusters are represented in gray. the resulting corrected p values are included in the network visualization by the size of nodes. furthermore, in the pathway enrichment analysis, the regulated genes for 1-min ntp exposure were clustered into biological groups which were different from those corresponding to the 3-min ntp exposure. for instance, it was found that the 1-min ntp exposure significantly repressed the expression of genes involved in mitotic telophase/cytokinesis and prometaphase as well as cell cycle, including dna damage response and mirna mediated regulation. moreover, the 1-min ntp exposure also activated some of pathways including the il- signaling pathway, p mediated signaling pathway, mapk, tgf-β and tnf signaling pathways. however, the 3-min ntp exposure caused up-regulated genes like histones which played a role in hats acetylate histones and rna polymerase involved in transcription regulation processes. in contrast, the 1-min ntp exposure reduced the expression of genes involved in regulation of the il- signaling pathway and the tgf-β signaling pathway. however, the effect of extended ntp processing time on gene profiles and cellular behavior and metabolism was not clear. involved hub transcription factors considering that some factors play critical roles in biological processes and interact with abundant related genes or proteins, the top hits proteins were sorted and assessed from protein-protein interaction network. apparently, the 1-min ntp exposure induced specific patterns that included some specifically up-regulated genes. these genes were involved in gtp and gdp energy metabolism, and ca2+ ion flow or release. moreover, some transcription factors that played important roles in transcription and regulation were also significantly enhanced. jun and fos were thought to play important roles in signal transduction, cell proliferation and differentiation. ccaat/enhancer binding protein were also important transcriptional activators that regulated expression of genes involved in immune and inflammatory responses. they could bind to regulatory regions of several acute-phase and cytokines genes, and probably played a role in the regulation of acute-phase reaction and inflammation. nfkbia inhibited the activity of nf-κb/rel complex by trapping rel dimers in the cytoplasm through masking their nuclear localization signals. additionally, other processes were also present, which involved tyrosine-kinase-based signaling related to cell adhesion, and cell cycle phase transition.fig. the selected genes from deg based on protein network betweenness and degree. the genes selected from different expression gene lists, and sorted by top % genes in the protein interaction database according to pid betweenness and degree. red color represents relative increase in abundance, blue color represents relative decrease, and white represents no change. fig. the details of selected pathways after ntp exposure. the illustration showed gene correlation of indicated pathway enrichment in details by cluepedia plugged-in cytoscape. the nodes represented genes in each pathway, with red and green colors of nodes indicating up- or down-regulated genes, respectively. the green line and yellow line represented activation and expression relationship in gene interaction enrichment, respectively. responded multiple pathways differential gene expression in response to the ntp exposure was assessed to elucidate effects of diverse pathways on the cancer cell. pathway enrichment analysis and pid top hits genes categorized several genes involved in the response to ntp exposure, which comprised transcription factors, negative intracellular signaling factors, leukocyte recruitment and activation factors, enzymes of phosphatase and cyclooxygenase. in the pathway enrichment results, the up-regulated pathways included il- tnf, tgf-β and mapk signaling pathways, which constituted a network with centralization of jun, fos and jak critical transcription factors. among these, the transcription factors were up-regulated at least 8-fold in the 1-min plasma-treated cells. next, we focused on changes in expression values of genes whose transcription levels increased under stressful growth arrest conditions. the p was a hub of response to stress signals, as shown in the right box of fig.  gadd was a dna-damage inducing gene, mediating the activation of the p38/juk pathway via mtk1/mekk kinase. dusp also negatively regulated members of map kinase superfamily, which was associated with cellular proliferation and differentiation. all the genes mentioned above displayed enhanced mrna expression.fig. western blot analysis and validation of several key proteins from microarray results. a cells were collected and lysed after 1-min ntp treatment at different time points, namely, and 24 h. with sds-page gel electrophoresis, the expressions of caspase parp, mtor, phospho-mtor, akt, phospho-akt, nf-κb, phospho-iκbα, jnk, phospho-jnk, p mapk, phospho-p mapk, c-jun, junb, c-fos, and actb were measured. qpcr and western blot validation to further validate some of the microarray profiles, seven representative candidate genes were selected for analyzing the gene expression levels via the qpcr method. the regulation patterns for all selected genes were consistent with the microarray data and only had marginal differences in the relative fold changes, except the egr and fos genes which presented extremely high mrna expression levels by qpcr when compared to microarray analysis. moreover, among the microarray results, we focused on and selected several candidate proteins for multiple pathways, which included apoptosis, mapk signaling pathway, jnk, akt and nf-κb pathways, and some critical transcriptional factors. among the western blot results, the caspase- and parp degraded over the time points, which indicated the classical apoptosis cascade. mtor and phosphorylated mtor levels peaked at 8 h post ntp treatment and descended thereafter. mtor is a serine/threonine protein kinase which is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy and stress signals. it plays a critical role in the phosphorylation of akt, which has also been validated in our western blot result in that the expression of phospho-akt was increased till 8 h after exposure. after that, the akt regulated the nf-κb signaling pathway by inducing release of nf-κb through phosphorylation and degradation of iκbα. ntp stimuli activated jnk, and phosphorylation of jnk on the site of thr183/tyr was increased in the time course, which modified the activities of numerous proteins that resided at the mitochondria or acted in the nucleus, and were involved in apoptosis, cell differentiation and proliferation. moreover, the jnk activated the ap- transcriptional factors. only junb showed continued enhancement with in the time course after ntp treatment, while the expression levels of c-jun and c-fos remained stable. overall, from the translational level results, ntp treatment induced cell apoptosis and cell cycle arrest to assist processes for cell repair.table verification of gene expression changes in 1-min ntp treatment at 4 h after exposure note: the results were shown as mean ± sd in the 2nd and 3ed columns, and asterisks indicated significant differences between two methods found using t-test discussion the impact of ntp on cancer cells has not been investigated using the microarray approach in detail to date. the present work was the first to employ this high-throughput technique to elucidate the responses of nsclc tumor cells to a particular ntp treatment in vitro. the microarray technique was applied to determine the activities of thousands of genes at once to create a partial picture of cellular functions of a cells following ntp exposure. in a previous study, the microarray technique had been used and to examine the therapeutic potential of a plasma jet on cancer cell lines and tumors, with a focus on the capability of selective tumor cell eradication and on deregulation of signaling pathways. these selective effects of ntp on different cell types can find important applications in cancer treatment. besides the apoptotic effects induced by long-time ntp exposures to a cells, we also focused on the biological effects induced by short-time ntp exposures, which were below the levels that induced apoptosis in most of the cells. the time points of and 4 h post cell treatment were selected for detection of early effects in terms of ntp modulated gene activities, which were indeed confirmed by the microarray data. the expressions of more than genes were modulated by ntp exposures, with at least -fold induction or repression when compared to those of non-treated cells. our results showed that the affected genes differed significantly between the control and treated cells depending on the conditions. both sub-groups contained differentially expressed genes encoding for molecules known to participate in the apoptotic process, cellular response to stress, chromosome organization, cell cycle, dna damage response and mirna regulation. the signaling and network analysis by cluego/cluepedia revealed that certain significantly regulated genes were involved in pathways which led to the p signaling pathway, mapk signaling pathway, tgf and tnf signaling pathways, and il-4/il- signaling pathways. likewise, the majority of the biological processes that decreased the expressions were mainly related to chromosome organization/assembly, cell cycle and metabolic processes. the most striking effect of ntp in the gene expression profiling analysis was the modulation of a large set of genes that were involved in multiple signaling pathways, including pi3k/akt and nf-κb mediated tnf signaling, mapk, jak-stat, tgf-β and vegf pathways. to better understand the influence of plasma-induced reactive species and potentially beneficial mechanisms of ntp on a cells, we examined the protein expression of apoptosis and related transcriptional factors which were mostly up-regulated or down-regulated after ntp exposure. a number of transcripts were identified to be significantly modulated for transcriptional factors. in this regard, cluego predicted the involvement of fos/jun/p in intracellular signaling in our experimental setup. further analysis of our data suggested that ntp might activate a broader range of p53-relevant processes. it is known that p is a critical transcription factor which participates in transcription as well as signal transduction, including cell cycle arrest, apoptosis, dna repair and damage prevention, cellular senescence and p negative feedback. the western blot assay also confirmed the multiple pathways involved in these complicated processes. the mapk, jnk and nf-κb pathways were all involved, and played critical roles in the regulation of responses to external stresses. in summary, the large number of negatively regulated genes involved in biological or cellular processes reflected the cellular responses to ntp, and helped confirm specific pathways or stress responses to ntp. although the details regarding the activation of specific pathways remained to be elucidated, our data presented new evidence that ntp treatment of a cells modulated the transcription of a variety of genes, which might center on p and mapk and tnf signaling pathways, etc. our data further indicated that it was possible to modulate cellular signaling as well as metabolism and/or apoptosis processes in ntp treated tumor cells on a genetic level.  overall, non-thermal plasma treated cells exhibited a pattern typical of damage responses, thereby validating several critical pathways/regulators, presumably mediated through multiplex signaling pathways. further investigation of these genes in other cell lines may reveal comprehensive mechanisms of plasma induced effects. 
the present work laid the foundation for further studies to clarify the roles of multiple pathways in plasma-induced biological processes. finally, a significant modulation in expression of genes related to cellular proliferation and differentiation was observed. we analyzed relevant signaling cascades associated with the tumor protein p in particular the cell cycle arrest, dna damage as well as the underlying apoptosis pathways. based on our results, the major effect from plasma exposure was found to be the activation of mapk and p signaling pathways, resulting in changes in gene expression of mekk, gadd, fos and jun. recent technological advances in atmospheric plasmas have made the creation of non-thermal atmospheric pressure plasma possible for utilization in the medical field. further investigation of these genes in other cell lines may reveal comprehensive mechanisms of plasma induced effects. overall, the presented data of the tumor transcriptome helped identify the key players in modulated gene expression following exposure to plasma at the molecular level, and also helped interpret the downstream processes.
 recent technological advances in atmospheric plasmas have made the creation of non-thermal atmospheric pressure plasma possible for utilization in the medical field. although accumulated evidence suggests that ntp induces cell death in various cancer cell types thus offering a promising alternative treatment strategy, the mechanism underlying its therapeutic effect is not fully understood.  we analyzed relevant signaling cascades associated with the tumor protein p in particular the cell cycle arrest, dna damage as well as the underlying apoptosis pathways. based on our results, the major effect from plasma exposure was found to be the activation of mapk and p signaling pathways, resulting in changes in gene expression of mekk, gadd, fos and jun. finally, a significant modulation in expression of genes related to cellular proliferation and differentiation was observed.  overall, the presented data of the tumor transcriptome helped identify the key players in modulated gene expression following exposure to plasma at the molecular level, and also helped interpret the downstream processes. the present work laid the foundation for further studies to clarify the roles of multiple pathways in plasma-induced biological processes. further investigation of these genes in other cell lines may reveal comprehensive mechanisms of plasma induced effects. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords non-thermal plasmagene profilingnsclc a cell linemultiple signal pathwaysissue-copyright-statement© the author 2015 