王慧, 吕懿, 李玲, 李朝菲, 郭璨璨, 田凤, 穆箭兵, 郑金平. 二羟环氧苯并芘短期暴露对人支气管上皮细胞基因表达谱的影响[J]. 环境与职业医学, 2021, 38(1): 51-57,63. DOI: 10.13213/j.cnki.jeom.2021.20337
引用本文: 王慧, 吕懿, 李玲, 李朝菲, 郭璨璨, 田凤, 穆箭兵, 郑金平. 二羟环氧苯并芘短期暴露对人支气管上皮细胞基因表达谱的影响[J]. 环境与职业医学, 2021, 38(1): 51-57,63. DOI: 10.13213/j.cnki.jeom.2021.20337
WANG Hui, LYU Yi, LI Ling, LI Zhaofei, GUO Cancan, TIAN Fengjie, MU Jianbing, ZHENG Jinping. Effects on gene expression profile in human bronchial epithelial cells exposed to benzo[a]pyrene diol epoxide[J]. Journal of Environmental and Occupational Medicine, 2021, 38(1): 51-57,63. DOI: 10.13213/j.cnki.jeom.2021.20337
Citation: WANG Hui, LYU Yi, LI Ling, LI Zhaofei, GUO Cancan, TIAN Fengjie, MU Jianbing, ZHENG Jinping. Effects on gene expression profile in human bronchial epithelial cells exposed to benzo[a]pyrene diol epoxide[J]. Journal of Environmental and Occupational Medicine, 2021, 38(1): 51-57,63. DOI: 10.13213/j.cnki.jeom.2021.20337

二羟环氧苯并芘短期暴露对人支气管上皮细胞基因表达谱的影响

Effects on gene expression profile in human bronchial epithelial cells exposed to benzoapyrene diol epoxide

  • 摘要: 背景

    苯并a芘(BaP)可引起暴露人群呼吸系统损伤。既往研究表明,BaP及其活性代谢物染毒细胞24h,即可引发细胞能量代谢异常、氧化损伤,导致细胞生存率降低。

    目的

    观察BaP活性代谢物二羟环氧苯并芘(BPDE)短期暴露导致的人支气管上皮细胞(16HBE)基因表达谱的变化,为探讨BPDE急性毒性机制提供依据。

    方法

    选取人支气管上皮细胞(16HBE),分别用终浓度0.5、1.0、2.0、4.0μmol·L-1的BPDE染毒,同时设溶剂对照组(加入等体积二甲基亚砜)和空白对照组(加入正常培养基),处理24 h后,CCK8法检测细胞存活率。根据细胞存活率,挑选一个剂量染毒组和溶剂对照组利用BGISEQ平台进行转录组测序(RNA-Seq)。使用R语言中的DEseq2包检测不同样本的差异表达基因,并用phyper函数对差异基因进行基因本体论(GO)功能注释和京都基因与基因组百科全书(KEGG)通路分析。

    结果

    随BPDE染毒剂量的增加,16HBE细胞存活率呈下降趋势(Z=-7.79,P趋势 < 0.01);0.5、1.0、2.0、4.0 μmol·L-1 BPDE染毒组细胞存活率分别为94.56%±1.74%、84.80%±3.19%、80.08%±1.72%、62.72%±1.95%,均明显低于溶剂对照组(98.61%±0.61%)和空白对照组(100.00%±0.00%)(P < 0.05)。选取2.0 μmol·L-1 BPDE染毒组和溶剂对照组细胞进行测序,结果显示,以基因变化倍数的对数绝对值|log2FC|>1,P < 0.05为筛选标准,暴露组共检测出191个2倍及以上差异表达的基因;其中上调的基因87个,下调的基因104个。基因共表达网络分析发现,趋化因子CXCL6CXCL1CXCL3CXCL8和集落刺激因子CSF2具有较强的关联性。GO分析显示,差异表达基因主要参与细胞增殖的生物学过程,并通过KEGG通路分析发现,这些基因主要富集于白介素(IL)-17信号通路和炎症趋化因子等通路。进一步分析差异表达的mRNA,发现有670个mRNA表达上调,878个mRNA表达下调;差异表达mRNA主要富集于凋亡执行期负调控、细胞外信号转导负调控和信号受体活动的调节等生物学过程,主要影响铁死亡,调控干细胞多能性,赖氨酸退化等信号通路。

    结论

    BPDE短期暴露可引起16HBE细胞基因表达谱的改变,生物信息学分析结果提示IL-17信号通路和铁死亡途径可能参与该细胞损伤的过程。

     

    Abstract: Background

    Benzoapyrene (BaP) can cause respiratory damage in people exposed to it. Previous studies indicate that a 24-hour exposure to BaP and its active metabolites could cause abnormal cell energy metabolism and oxidative damage, leading to reduced cell survival.

    Objective

    This experiment observes the changes of gene expression profile of human bronchial epithelial cells (16HBE) caused by short-term exposure to benzoapyrene diol epoxide (BPDE), an active metabolite of BaP, and to provide evidence for the potential mechanism of acute toxicity of BPDE.

    Methods

    This experiment included four BPDE exposure groups (0.5, 1.0, 2.0, and 4.0 μmol·L-1, respectively), a solvent control group (dimethyl sulfoxide), and a blank control group (normal saline). CCK8 method was used to measure cell survival rate after 24 h treatment. According to the cell survival rate, an exposure group and a solvent control group were selected for transcriptional sequencing (RNA-Seq) using the BGISEQ platform. The DEseq2 package in R software was used to detect the differentially expressed genes (DEGs) in different samples. The phyper function in R software was used to perform Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the selected DEGs.

    Results

    The survival rates of 16HBE cells decreased with the increase of BPDE concentration (Z=-7.79, Ptrend < 0.01). The cell survival rates of the 0.5, 1.0, 2.0, and 4.0 μmol·L-1 BPDE groups (94.56%±1.74%, 84.80%±3.19%, 80.08%±1.72%, and 62.72%±1.95%, respectively) were significantly reduced compared with the solvent control group (98.61%±0.61%) and the blank control group (100.00%±0.00%) (P < 0.05). The cells in the 2.0 μmol·L-1 BPDE group and the solvent control group were selected for sequencing, and 191 DEGs satisfying both|log2FC|>1 and P < 0.05 were detected in the exposure group, including 87 genes up-regulated and 104 genes down-regulated. The results of gene co-expression network found that chemokines CXCL6, CXCL1, CXCL3, and CXCL8 had strong correlations with colony stimulating factor CSF2. The GO analysis results showed that these DEGs were mainly involved in the biological process of cell proliferation, and the KEGG pathway analysis results showed that these genes were mainly enriched in the interleukin (IL)-17 signaling pathway and the inflammatory chemokine signaling pathway. Further mRNA analysis results revealed that 670 mRNAs were up-regulated and 878 mRNAs were downregulated. The differentially expressed mRNAs were mainly involved in negative regulation of execution phase of apoptosis, negative regulation of extracellular signal transduction, regulation of signal receptor activity, etc., affecting ferroptosis, regulation of stem cells' pluripotency, lysine degradation, and other signaling pathways.

    Conclusion

    Short-term exposure to BPDE can cause changes in gene expression profile of 16HBE cells, and bioinformatics analysis results suggest that IL-17 signaling pathway and ferroptosis pathway might be involved in the BPDE associated cell injury.

     

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