艾烟可吸入颗粒物对人肺腺癌A549细胞增殖及基因表达谱的影响
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摘要
目的观察艾烟PM10对人肺腺癌A549细胞增殖及基因图谱的影响,全面深入地探讨艾烟PM10对肺腺癌A549细胞的潜在药性作用或(和)毒性作用机制,为今后艾烟的研究寻找更明确的方向,同时为其安全性研究提供参考。
方法本研究采用体外实验方法,以A549细胞为研究对象,用MTT法观察了艾烟PM10对其活力的影响,对比了艾烟PM10、香烟烟气PM10及广谱抗癌药顺铂对A549细胞以及人支气管上皮细胞BEAS-2B活力的影响；采用流式细胞术观察了艾烟PM10对A549细胞周期的影响,检测了艾烟PN10对A549细胞内活性氧(ROS)水平的影响；采用Hoechest33258染色法用荧光显微镜观察艾烟PM10对A549细胞的凋亡形态学的影响,并对细胞内Ca2+水平以及Caspase9、Caspase3/7活性和NF-κB(p65)表达量进行检测；采用新一代基因测序技术——数字基因表达谱法(Digital Gene Expression profiling, DGE)观察了艾烟PM10对人肺腺癌A549细胞基因表达谱的影响,筛选出艾烟PM10处理后差异表达的基因,对其差异基因表达模式进行聚类分析、Gene Ontology功能显著性富集分析和Pathway显著性富集分析,筛选了艾烟PM10干预A549细胞的过程中差异表达的基因及它们参与的生物过程和最主要的信号转导途径,分析了差异基因与A549细胞周期、细胞凋亡之间的关系。
结果艾烟PM10对A549细胞增殖影响的实验结果如下：
(1)艾烟PM10对A549细胞活力的影响
24h干预组内,与溶剂对照组相比,经不同浓度艾烟PM10处理过的A549细胞活力均显著降低(p<0.05,p<0.01)；48h干预组内,与溶剂对照组相比,经不同浓度艾烟PM10处理过的A549细胞活力均显著降低(p<0.01)；各艾烟PM10处理组内比较,随浓度增加,细胞活力显著降低(p<0.01)；与24h干预组内同浓度艾烟处理组相比,48h干预组内艾烟PM10处理各组细胞存活率均显著降低(p<0.01)。
(2)艾烟PM10、香烟侧流烟气PM10及顺铂对A549细胞活力的影响
三种样品同一浓度组间比较,顺铂组A549细胞存活率均低于艾烟PM10组和香烟PM10组(p<0.01)：40μg/ml组间比较,艾烟PM10组与香烟PM10组内A549细胞存活率无显著差异(p>0.05),其余各浓度组间比较,艾烟PM10组A549细胞存活率均显著低于香烟PM10组(p<0.05)。
(3)艾烟PM10、香烟侧流烟气PM10及顺铂对支气管上皮细胞BEAS-2B活力的影响
三种样品同一浓度间细胞存活率比较,香烟干预组BEAS-2B细胞活力均显著低于艾烟干预组(p<0.05)；顺铂干预组的细胞活力,除100μg/ml组与艾烟干预组无差异(p>0.05)外,其余各浓度组细胞活力均显著低于艾烟干预组(p<0.05)；40μg/ml组间比较,顺铂干预组细胞活力显著低于香烟PM10干预组,然而80μg/ml和100μg/ml组间比较时,香烟PM10干预组细胞活力却显著低于顺铂组。
(4)艾烟PM10干预24h对A549细胞周期的影响
与对照组相比,艾烟PM10处理各组S期细胞比例显著增加(p<0.05)。
与160μg/ml组相比,其余两个艾烟PM10处理组内S期细胞显著降低(p<0.05)；400μg/ml时,G1期细胞比例显著上升,G2期细胞比例显著下降,差异均有统计学意义(p<0.01).320μg/ml时,G1期和G2期细胞比例均无显著变化(p>0.05)。
与320μ g/ml组相比,浓度为400μg/ml时,G1期细胞比例显著升高(p<0.05),G2期细胞比例无显著差异(p>0.05).
经艾烟PM10处理过的各组细胞在流式细胞图中出现了明显的“凋亡峰”
(5)艾烟PM10干预4h对A549细胞内ROS水平的影响
与对照组相比,艾烟PM10处理各组细胞内ROS水平均显著降低(p<0.05).
(6)艾烟PM10对A549细胞凋亡形态学的影响
艾烟PM10处理4h在400μg/ml时出现部分凋亡细胞。
(7)艾烟PM10对A549细胞内Ca2+水平的影响
艾烟PM10干预A549细胞4小时后,随浓度增加,细胞内Ca2+水平显著增加(p<0.01).
(8)艾烟PM10对A549细胞内Caspase9、Caspase3/7活性的影响
与对照组相比,经不同浓度艾烟PM10处理后,A549细胞内Caspase9活性无差异(p>0.05).当艾烟PM10浓度为280μg/ml时,细胞内Caspase3/7活性明显下降(p<0.01)；而浓度为140μg/ml时,Caspase3/7活性无明显变化(p>0.05)。
(9)艾烟PM10对A549细胞NF-κB(p65)表达的影响
与空白对照组比,艾烟干预4小时,各浓度组A549细胞内NF-κB(p65)表达量均降低(p<0.05)；干预20小时,70μg/ml组和280μg/ml组内p65表达量均降低(p<0.05),140μg/ml组内p65含量无明显变化(p>0.05)。相同浓度下,不同时间的艾烟干预各组间对比,p65表达量无明显变化(p>0.05).
艾烟PM10对A549细胞基因表达谱的影响实验结果如下：
(1)差异基因筛选
与对照组相比,艾烟干预4小时差异表达基因共1109条(上调602条,下调507条)；干预20小时差异表达基因共3565条(上调1394条,下调2171条)。与艾烟干预4小时相比,干预20小时后差异表达的基因有2149条(上调1010条,下调1139条).
(2)差异基因表达模式的聚类分析
在艾烟PM10干预的过程中(干预4h、干预20h及从4h到20h的过程中)均有差异表达的基因共有316条,这些基因在艾烟PM10干预4h后,以上调为主；在干预20小时后,则以下调为主.
在艾烟PM10干预的过程中差异表达的基因总共有4415条。对促进细胞周期进程的基因以下调为主。艾烟干预过程中,对促凋亡基因BAD在干预4h时上调,随时间延长又表现为明显下调；对Caspase家族基因和抑制凋亡的基因Bcl家族也时而促进,时而抑制。
(3)差异基因参与的生物功能显著性富集分析
与空白组相比,艾烟PM10干预4h引起差异表达的基因涉及1425个生物过程。其中,显著富集的生物功能共有39个,富集最为显著的为“细胞周期”。
与空白组相比,艾烟PM10干预20h引起差异表达的基因涉及2211个生物过程。其中,显著富集的生物功能共有77个,富集最为显著的为“细胞代谢”。
与干预4h相比,艾烟PM10干预20h引起差异表达的基因涉及1786个生物过程。其中,显著富集的生物功能共有22个,富集最为显著的生物过程为“细胞过程”。
(4) Pathway显著性富集分析
与空白组相比,艾烟PM10干预4h引起差异表达的基因涉及214条信号通路或生物代谢途径。其中,显著富集的有细胞周期、内质网中的蛋白处理、氧化磷酸化、幽门螺杆菌感染的上皮细胞信号、p53信号途径及亨廷顿氏病。
与空白组相比,艾烟PM10干预20h引起差异表达的基因涉及228条信号通路。其中,显著富集的信号通路由高到底排列如下：细胞周期、DNA复制、代谢通路、剪接酶、糖胺聚糖的生物合成-硫酸角质素、嘧啶代谢、卵母细胞减数分裂、幽门螺杆菌感染的上皮细胞信号、内质网的蛋白处理、泛素介导的蛋白水解、氧化磷酸化、黏附连接点、p53信号通路、孕激素介导的卵母细胞成熟、糖基磷脂酰肌醇(GPI)锚合成、叶酸参与的单碳池代谢、亨廷顿氏病以及Wnt信号通路。
与干预4小时相比,艾烟PM10干预2h引起差异表达的基因涉及221条信号通路。其中,显著富集的信号通路由高到底排列如下：细胞周期、嘧啶代谢、DNA复制、p53信号通路、剪接酶。
结论
1.艾烟PM10对具有抑制人肺腺癌A549细胞增殖的作用。艾烟PM10对支气管上皮细胞BEAS-2B的毒性低于香烟PM10,对A549细胞的增殖有抑制作用,而香烟在同等浓度时对A549细胞活力无影响,说明艾烟PM10中含有抗肿瘤的成分。
2.艾烟PM10可通过诱导细胞周期阻滞引起A549细胞死亡。艾烟PM10会引起细胞周期各阶段的明显阻滞,但以晚期阻滞作用明显。且该作用涉及对细胞周期调控的许多通路,如cyclin-CDK依赖性通路、p53调节通路、氧化还原调控以及代谢相关途径。
3.艾烟PM10可通过诱导细胞自噬引起A549细胞死亡。艾烟PM10可通过下调细胞外基质蛋白(ECM)和整合素的表达,使A549细胞出现失巢凋亡,并通过自噬作用使失巢凋亡的细胞死亡,这可能是艾烟PM10抑制癌细胞增长,诱导其死亡的主要机制之一,而且艾烟PM10可能通过该途径发挥抑制肿瘤转移和浸润的作用。
4.艾烟PM10对A549细胞凋亡的作用呈双向性,可促进又可抑制A549细胞凋亡,两种作用处于动态变化中.
5.艾烟PM10具有多种生物活性.艾烟PM10干预A549细胞引起大量差异表达的基因,这些基因参与多种生物过程,对机体的作用有利有弊,值得深入研究.
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 A549cells
Within24h 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 cisplatin
Compared 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 cisplatin
Compared 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 cycle
Compared 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 A549cells
Compared 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 A549cells
Some apoptotic cells were observed after treated with moxa smoke PM10in the concerntration of400μg/mL
(7) Effect of moxa smoke PM10on intracellular Ca2+levels in A549cells
The 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 A549cells
Compared 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 A549cells
Compared 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 genes
During 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 PM10
After 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 smoke
The 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.
方法本研究采用体外实验方法,以A549细胞为研究对象,用MTT法观察了艾烟PM10对其活力的影响,对比了艾烟PM10、香烟烟气PM10及广谱抗癌药顺铂对A549细胞以及人支气管上皮细胞BEAS-2B活力的影响；采用流式细胞术观察了艾烟PM10对A549细胞周期的影响,检测了艾烟PN10对A549细胞内活性氧(ROS)水平的影响；采用Hoechest33258染色法用荧光显微镜观察艾烟PM10对A549细胞的凋亡形态学的影响,并对细胞内Ca2+水平以及Caspase9、Caspase3/7活性和NF-κB(p65)表达量进行检测；采用新一代基因测序技术——数字基因表达谱法(Digital Gene Expression profiling, DGE)观察了艾烟PM10对人肺腺癌A549细胞基因表达谱的影响,筛选出艾烟PM10处理后差异表达的基因,对其差异基因表达模式进行聚类分析、Gene Ontology功能显著性富集分析和Pathway显著性富集分析,筛选了艾烟PM10干预A549细胞的过程中差异表达的基因及它们参与的生物过程和最主要的信号转导途径,分析了差异基因与A549细胞周期、细胞凋亡之间的关系。
结果艾烟PM10对A549细胞增殖影响的实验结果如下：
(1)艾烟PM10对A549细胞活力的影响
24h干预组内,与溶剂对照组相比,经不同浓度艾烟PM10处理过的A549细胞活力均显著降低(p<0.05,p<0.01)；48h干预组内,与溶剂对照组相比,经不同浓度艾烟PM10处理过的A549细胞活力均显著降低(p<0.01)；各艾烟PM10处理组内比较,随浓度增加,细胞活力显著降低(p<0.01)；与24h干预组内同浓度艾烟处理组相比,48h干预组内艾烟PM10处理各组细胞存活率均显著降低(p<0.01)。
(2)艾烟PM10、香烟侧流烟气PM10及顺铂对A549细胞活力的影响
三种样品同一浓度组间比较,顺铂组A549细胞存活率均低于艾烟PM10组和香烟PM10组(p<0.01)：40μg/ml组间比较,艾烟PM10组与香烟PM10组内A549细胞存活率无显著差异(p>0.05),其余各浓度组间比较,艾烟PM10组A549细胞存活率均显著低于香烟PM10组(p<0.05)。
(3)艾烟PM10、香烟侧流烟气PM10及顺铂对支气管上皮细胞BEAS-2B活力的影响
三种样品同一浓度间细胞存活率比较,香烟干预组BEAS-2B细胞活力均显著低于艾烟干预组(p<0.05)；顺铂干预组的细胞活力,除100μg/ml组与艾烟干预组无差异(p>0.05)外,其余各浓度组细胞活力均显著低于艾烟干预组(p<0.05)；40μg/ml组间比较,顺铂干预组细胞活力显著低于香烟PM10干预组,然而80μg/ml和100μg/ml组间比较时,香烟PM10干预组细胞活力却显著低于顺铂组。
(4)艾烟PM10干预24h对A549细胞周期的影响
与对照组相比,艾烟PM10处理各组S期细胞比例显著增加(p<0.05)。
与160μg/ml组相比,其余两个艾烟PM10处理组内S期细胞显著降低(p<0.05)；400μg/ml时,G1期细胞比例显著上升,G2期细胞比例显著下降,差异均有统计学意义(p<0.01).320μg/ml时,G1期和G2期细胞比例均无显著变化(p>0.05)。
与320μ g/ml组相比,浓度为400μg/ml时,G1期细胞比例显著升高(p<0.05),G2期细胞比例无显著差异(p>0.05).
经艾烟PM10处理过的各组细胞在流式细胞图中出现了明显的“凋亡峰”
(5)艾烟PM10干预4h对A549细胞内ROS水平的影响
与对照组相比,艾烟PM10处理各组细胞内ROS水平均显著降低(p<0.05).
(6)艾烟PM10对A549细胞凋亡形态学的影响
艾烟PM10处理4h在400μg/ml时出现部分凋亡细胞。
(7)艾烟PM10对A549细胞内Ca2+水平的影响
艾烟PM10干预A549细胞4小时后,随浓度增加,细胞内Ca2+水平显著增加(p<0.01).
(8)艾烟PM10对A549细胞内Caspase9、Caspase3/7活性的影响
与对照组相比,经不同浓度艾烟PM10处理后,A549细胞内Caspase9活性无差异(p>0.05).当艾烟PM10浓度为280μg/ml时,细胞内Caspase3/7活性明显下降(p<0.01)；而浓度为140μg/ml时,Caspase3/7活性无明显变化(p>0.05)。
(9)艾烟PM10对A549细胞NF-κB(p65)表达的影响
与空白对照组比,艾烟干预4小时,各浓度组A549细胞内NF-κB(p65)表达量均降低(p<0.05)；干预20小时,70μg/ml组和280μg/ml组内p65表达量均降低(p<0.05),140μg/ml组内p65含量无明显变化(p>0.05)。相同浓度下,不同时间的艾烟干预各组间对比,p65表达量无明显变化(p>0.05).
艾烟PM10对A549细胞基因表达谱的影响实验结果如下：
(1)差异基因筛选
与对照组相比,艾烟干预4小时差异表达基因共1109条(上调602条,下调507条)；干预20小时差异表达基因共3565条(上调1394条,下调2171条)。与艾烟干预4小时相比,干预20小时后差异表达的基因有2149条(上调1010条,下调1139条).
(2)差异基因表达模式的聚类分析
在艾烟PM10干预的过程中(干预4h、干预20h及从4h到20h的过程中)均有差异表达的基因共有316条,这些基因在艾烟PM10干预4h后,以上调为主；在干预20小时后,则以下调为主.
在艾烟PM10干预的过程中差异表达的基因总共有4415条。对促进细胞周期进程的基因以下调为主。艾烟干预过程中,对促凋亡基因BAD在干预4h时上调,随时间延长又表现为明显下调；对Caspase家族基因和抑制凋亡的基因Bcl家族也时而促进,时而抑制。
(3)差异基因参与的生物功能显著性富集分析
与空白组相比,艾烟PM10干预4h引起差异表达的基因涉及1425个生物过程。其中,显著富集的生物功能共有39个,富集最为显著的为“细胞周期”。
与空白组相比,艾烟PM10干预20h引起差异表达的基因涉及2211个生物过程。其中,显著富集的生物功能共有77个,富集最为显著的为“细胞代谢”。
与干预4h相比,艾烟PM10干预20h引起差异表达的基因涉及1786个生物过程。其中,显著富集的生物功能共有22个,富集最为显著的生物过程为“细胞过程”。
(4) Pathway显著性富集分析
与空白组相比,艾烟PM10干预4h引起差异表达的基因涉及214条信号通路或生物代谢途径。其中,显著富集的有细胞周期、内质网中的蛋白处理、氧化磷酸化、幽门螺杆菌感染的上皮细胞信号、p53信号途径及亨廷顿氏病。
与空白组相比,艾烟PM10干预20h引起差异表达的基因涉及228条信号通路。其中,显著富集的信号通路由高到底排列如下：细胞周期、DNA复制、代谢通路、剪接酶、糖胺聚糖的生物合成-硫酸角质素、嘧啶代谢、卵母细胞减数分裂、幽门螺杆菌感染的上皮细胞信号、内质网的蛋白处理、泛素介导的蛋白水解、氧化磷酸化、黏附连接点、p53信号通路、孕激素介导的卵母细胞成熟、糖基磷脂酰肌醇(GPI)锚合成、叶酸参与的单碳池代谢、亨廷顿氏病以及Wnt信号通路。
与干预4小时相比,艾烟PM10干预2h引起差异表达的基因涉及221条信号通路。其中,显著富集的信号通路由高到底排列如下：细胞周期、嘧啶代谢、DNA复制、p53信号通路、剪接酶。
结论
1.艾烟PM10对具有抑制人肺腺癌A549细胞增殖的作用。艾烟PM10对支气管上皮细胞BEAS-2B的毒性低于香烟PM10,对A549细胞的增殖有抑制作用,而香烟在同等浓度时对A549细胞活力无影响,说明艾烟PM10中含有抗肿瘤的成分。
2.艾烟PM10可通过诱导细胞周期阻滞引起A549细胞死亡。艾烟PM10会引起细胞周期各阶段的明显阻滞,但以晚期阻滞作用明显。且该作用涉及对细胞周期调控的许多通路,如cyclin-CDK依赖性通路、p53调节通路、氧化还原调控以及代谢相关途径。
3.艾烟PM10可通过诱导细胞自噬引起A549细胞死亡。艾烟PM10可通过下调细胞外基质蛋白(ECM)和整合素的表达,使A549细胞出现失巢凋亡,并通过自噬作用使失巢凋亡的细胞死亡,这可能是艾烟PM10抑制癌细胞增长,诱导其死亡的主要机制之一,而且艾烟PM10可能通过该途径发挥抑制肿瘤转移和浸润的作用。
4.艾烟PM10对A549细胞凋亡的作用呈双向性,可促进又可抑制A549细胞凋亡,两种作用处于动态变化中.
5.艾烟PM10具有多种生物活性.艾烟PM10干预A549细胞引起大量差异表达的基因,这些基因参与多种生物过程,对机体的作用有利有弊,值得深入研究.
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 A549cells
Within24h 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 cisplatin
Compared 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 cisplatin
Compared 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 cycle
Compared 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 A549cells
Compared 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 A549cells
Some apoptotic cells were observed after treated with moxa smoke PM10in the concerntration of400μg/mL
(7) Effect of moxa smoke PM10on intracellular Ca2+levels in A549cells
The 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 A549cells
Compared 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 A549cells
Compared 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 genes
During 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 PM10
After 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 smoke
The 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.
引文
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