| Objective:To observe the effect of PM10in moxa smoke on the growth and the gene expression profile of A549cells.Methods:In this study, the effect of PM10in moxa smoke on the vitality of the human lung adenocarcinoma cell line-A549and human bronchial epithelial cells-BEAS-2B were observed by MTT assay, compared with the PM10in cigarette smoke and the broad-spectrum anti-cancer drug cisplatin; to examine whether PM10in moxa smoke could influence the cell cycle progression, FACS was performed, the intracelluar reactive oxygen species (ROS) levels were detected as well; the morphological changes of apoptosis of A549cells were observed by the fluorescence microscopy using staining Hoechest33258and the intracellular Ca2+level, activity of Caspase9, Caspase3/7and the content of NF-kappa B (p65) were detected; the gene expression profile of A549cells after intervention by PM10in moxa smoke was analyized by the next-generation gene sequencing technology-digital gene expression method (Digital Gene Expression profiling, DGE), which would provide some information about the differentially expressed genes(DEGs), clustering analysis of DEGs pattern, gene ontology functional enrichment analysis for DEGs and pathway enrichment analysis for DEGs of A549cells after treated by PM10in moxa smoke.Results:Results of the research on the effect on the proliferation of A549cells cultured with PM10in moxa smoke are as follows:(1) Effect of PM10in moxa smoke on viability of A549cellsWithin24h and48h intervention groups, compared with the solvent control group, the viability A549cells treated with PM10in moxa smoke were significantly lower.The cell viability was significantly reduced (p<0.01) with increasing concentration of PM10in moxa smoke.Compared with the same concentration in24h intervention group, the cell viability was significantly lower (p<0.01) in the48h intervention group.(2) Effect on viability of A549cells of PM10in moxa smoke, PM10in sidestream cigarette smoke and cisplatinCompared with the two kinds of PM10groups, cell viability in the cisplatin group was were significant lower(p<0.01) in every the same concerntration. When compared in the concerntration of40μg/ml, there was no significant difference (p>0.05) between the two PM10groups, while the survival of A549cells were significantly lower (p<0.05) in the rest concentrations of moxa smoke PM10group than the cigarette moke groups.(3) Effect on viability of BEAS-2B cells of PM10in moxa smoke, PM10in sidestream cigarette smoke and cisplatinCompared with the moxa smoke PM10group, the cell viability in every the same concerntration of cigrarette smoke PM10was was significantly lower (p<0.05), as for the cisplatin group, the cell viability in every other the same concerntration except100μg/ml group(p>0.05) was significantly lower (p<0.05) as well.In comparision with the cisplatin group, the cell viability was significantly higher in cigarettes PM10intervention group under the concerntration of40μg/ml, however, when the concerntration were80μg/ml and100μg/ml, cell viability were showed significantly lower.(4) Effect of24h intervention of moxa smoke PM10on A549cell cycleCompared with the control group, the proportion of S-phase cells increased significantly (p<0.05) in moxa smoke PM10group.Compared with the160μg/ml group, cells in S-phase was significantly lower (p<0.05) in the other two group(320μg/ml,400μg/ml); cells in G1-phase increased significantly (p<0.01), while decreased significantly (p<0.01) in G2-phase when the concerntration of moxa smoke PM10was400μg/mL However, the proportions of G1and G2phase cells were not significantly changed change (p>0.05)in comparision to160μg/ml group.Compared with320μg/ml group, cells in G1phase was significantly higher (p<0.05) in400ng/ml group, and it showed no significant difference (p>0.05) in the proportion of G2phase cells."Apoptotic peak" occured in the flow cytometry diagrams of A549cells cultured with moxa smoke PM10.(5) Effect of4h intervention of moxa smoke PM10on ROS levels in A549cellsCompared with the control group, ROS levels were significantly lower (p<0.05) in A549cells after4h intervention of moxa smoke PM10.(6) Effect of moxa smoke PM10on the morphology of apoptosis of A549cellsSome apoptotic cells were observed after treated with moxa smoke PM10in the concerntration of400μg/mL(7) Effect of moxa smoke PM10on intracellular Ca2+levels in A549cellsThe intracellular Ca2+levels increased significantly (p<0.01) A549cells after4hours intervention by moxa smoke PM10with increasing concentration.(8) Effect of moxa somke PM10on activity of Caspase9and Caspase3/7in A549cellsCompared with the control group, the intracellular Caspase9activity was no difference (p>0.05) in each concentration group of moxa smoke PM10after4hours’intervention. As for the activity of Caspase3/7, in comparasion with the control group, it was decreased significantly (p<0.01) when the moxa smoke PM10concentration is280μg/ml, while the difference was not statistically significant (p>0.05) in the concentration of140μg/ml(9) Effect of moxa smoke PM10on the expression of NF-κB(p65) in A549cellsCompared with the control group, the expression of NF-κB (p65) were decreased significantly (p<0.05) after intervention of moxa smoke PM10with different concerntrations for4hours. When the A549cells were cultured with moxa smoke PM10for20hours, the expression of p65were decreased significantly (p<0.05) in the concerntration of70μ/ml and280μg/ml, while it showed no significant change in the concerntration of140μg/ml (p>0.05).Results of effect on gene expression profiles of A549cells cultured with PM10in moxa smoke are as follows:(1) Differentially Expressed Genes (DEGs)Compared with the control group, there were1109different expressed genes in A549cells after intervention of moxa smoke in the concerntration of280μg/ml for4hours, including602up-and507down-regulated genes.When A549cells were cultred for20hours, there were3565different expressed genes in A549cells after intervention of moxa smoke in the concerntration of280μg/ml, including1394up-and2171down-regulated genes in the comparision to the control group.Compared with the4hours’intervention group, there were2149different expressed genes, including1010up-and1139down-regulated genes.(2) Cluster analysis of the expression patterns of different genesDuring the intervention of moxa smoke PM10, there were316different expressed genes in common could be detected in each of the phases, including processes of4h intervention,20h intervention, and the phase from4h and20h. After the intervention for4h, most of these genes were upregulated in comparision to the control group, while they were mostly down regulated after20hours compared with the control group, gene below the main tone. A total of4415differentially expressed genes were detected in the three phases. Genes with the function to promote cell cycle progression were mostly down-regulated. While, BAD gene, a kind of pro-apoptotic gene, showed upregulated after intervention for4hours, then it was significantly down-regulated over time; the Caspase family genes and Bel, the anti-apoptotic gene family expressed up and down without rules.(3) Gene ontology (Biological Process) functional enrichment analysis for DEGs in A549cells treated with moxa smoke PM10After intervention for4hours, all the different expressed genes compared with the control group involved in1425kinds of different biological processes, of which39kinds were significantly enriched, and "cell cycle" was the most significant one.After intervention for20hours, all the different expressed genes compared with the control group involved in2211kinds of different biological processes, of which77kinds were significantly enriched, and "cell metabolism" was the most significant one.The different expressed genes between interventions for20hours and4hours involved in1786kinds of biological processes, of which22kinds of were significantly enriched, and "cellular process" was the most significant one.(4) Pathway enrichment analysis for DEGs of A549cells after treated by PM10in moxa smokeThe different expressed genes in A549cells which were cultured with moxa somke PM10for4hours compared with the control group, involved in214kinds of signaling pathways, of which6kinds were significantly enriched as follows:Cell cycle, Protein processing in endoplasmic reticulum, Oxidative phosphorylation, Epithelial cell signaling in Helicobacter pylori-infection, p53signaling pathway and Huntington’s disease pathway.The different expressed genes intervention with moxa smoke PM10for20hours compared with the control group, involved in228kinds of signaling pathways, of which the significantly enriched ones were as follows:Cell cycle, DNA replication, Metabolic pathways, Spliceosome, Glycosaminoglycan biosynthesis-keratan, Pyrimidine metabolism, Oocyte meiosis, Epithelial cell signaling in Helicobacter pylori, Protein processing in endoplasmic reticulum, Ubiquitin mediated proteo lysis, Oxidative phosphorylation, Ad here ns junction, p53signaling pathway, Progesterone-mediated oocyte maturation, Glycosylphosphatidylinositol(GPI)-anchor, One carbon pool by folate, Huntington’s disease and Wnt signaling pathway.The different expressed genes caused by moxa smoke intervention for20hours compared with that for4hours, involved in221kinds of signaling pathways, which significantly enriched pathways were arranged as follows:Cell cycle, Pyrimidine metabolism, DNA replication, p53signaling pathway and Spliceosome pathway.Conclusions:1. The moxa smoke PM10can inhibit human lung adenocarcinoma A549cell proliferatioa It had less toxic on bronchial epithelial cells BEAS-2B than cigarettes PM10did, but can inhibit the proliferation of A549cells. However, the PM10of cigarette somke with the same concentration had no effect on A549cell viability, which indicated that moxa smoke PM10may contain anti-tumor ingredients.2. Moxa smoke PM10induced A549cell death by causing cell cycle arrest. It can induce cell cycle arrest in various stages, expecially in the late blockade. And the effect on cell cycle regulation involves many pathways, such as cyclin-CDK-dependent pathway, p53regulatory pathway, redox regulation and metabolic pathways.3. Moxa smoke PM10induced A549cell death by causing autophagy. It can reduce the expressions of extracellular matrix proteins (ECM) and integrin in A549cells, which induced anoikis of the cells, and then cells died through autophagy programmed cell death. This may be one of the main mechanism of cell death which the moxa smoke PM10caused. Furthermore, moxa smoke PM10may play a role in tumor metastasis and invasion through this pathway.4. Moxa smoke PM10showed two-way effect on the apoptosis of A549cells. It can bothe promote and inhibit apoptosis of A549cells, and the two roles in a dynamic change.5. Moxa smoke PM10has many kinds of biological activities. It can cause a large number of different expressed genes in A549cells involved in various biological processes. It was both positive and harmful to the body, so it is necessary to carry out further research on moxa smoke. |
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