叉头框蛋白A1基因敲除对苯并[a]芘恶性转化细胞THBEc1转录组的影响  

Effects of forkhead box protein A1 knockout on transcriptome of benzo[a]pyrene-transformed human bronchial epithelial 16HBE cells

作　　者：马雪 李璐迪 王裕 邢云昆 付娟玲[1] 姚碧云[1] 赵鹏[1] MA Xue;LI Lu-di;WANG Yu;XING Yun-kun;FU Juan-ling;YAO Bi-yun;ZHAO Peng(Department of Toxicology,Beijing Key Laboratory of Toxicology Research and Risk Assessment for Food Safety,School of Public Health,Peking University,Beijing 100191,China;National Institute of Environmental Health,Chinese Center for Disease Control and Prevention,Beijing 100021,China)

机构地区：[1]北京大学公共卫生学院毒理学系,食品安全毒理学研究与评价北京市重点实验室,北京100191 [2]中国疾病预防控制中心环境与健康相关产品安全所,北京100021

出　　处：《中国药理学与毒理学杂志》2022年第3期185-196,共12页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81370079);国家自然科学基金(81001253);北京市自然科学基金(7132122)。

摘　　要：目的 研究叉头框蛋白A1(FOXA1)基因敲除对苯并[a]芘(BaP)恶性转化细胞THBEc1转录组的影响,探讨FOXA1在BaP致癌作用中的可能机制。方法 以FOXA1敲除细胞THBEc1-ΔFOXA1-c34和对照细胞THBEc1-ctrl为研究模型,采用Western印迹法测定FOXA1蛋白表达水平,分别采用克隆形成实验和Transwell实验测定细胞增殖和迁移能力。采用二代测序技术检测THBEc1-ΔFOXA1-c34与THBEc1-ctrl细胞间差异表达基因,并通过实时荧光定量PCR(RT-qPCR)对转录组测序结果进行验证。采用DAVID数据库对差异表达基因进行基因本体论(GO)和京都基因与基因百科全书(KEGG)通路富集分析。采用STRING 11.5数据库和Cytoscape 3.8.2对差异基因编码蛋白进行网络分析。结果 THBEc1-ΔFOXA1-c34细胞未检出FOXA1表达,且增殖和迁移能力均低于THBEc1-ctrl(P<0.01)。转录组差异分析共检出1332个基因在THBEc1-ΔFOXA1-c34与THBEc1-ctrl细胞间的表达差异倍数>2或<0.5(错误发现率<0.05),其中691个基因在THBEc1-ΔFOXA1-c34中表达高于THBEc1-ctrl,641个基因在THBEc1-ΔFOXA1-c34中表达低于THBEc1-ctrl。RT-qPCR验证的47个基因的表达差异和统计学P值与转录组测序结果一致。差异表达基因涉及多种生物学过程,主要包括血管生成、细胞增殖、迁移、黏附、炎症反应、免疫应答、信号转导[包括肿瘤坏死因子(TNF)、丝裂原活化蛋白激酶(MAPK)、磷脂酰肌醇-3-激酶/蛋白激酶B和转化生长因子β(TGF-β)等通路]和代谢等。在349个差异表达基因所编码的蛋白质形成的相互作用网络中,共发现分别以C-C趋化因子配体3(CCL3)、O-聚糖合酶葡糖胺基(N-乙酰基)转移酶3(GCNT3)、基质金属蛋白酶3(MMP3)和卷曲蛋白8(FZD8)为种子节点的4个核心功能模块。核心功能模块主要参与免疫应答和炎症反应、O-聚糖加工、胶原分解代谢过程以及典型WNT通路等。结论 敲除FOXA1后BaP恶性转化细胞THBEc1转录组明显改变,增殖和迁移能�OBJECTIVE To explore the possible mechanism of forkhead box protein A1(FOXA1)in benzo[a]pyrene(BaP)-induced carcinogenesis by determining changes in transcriptomes of BaPtransformed human bronchial epithelial 16HBE cells(THBEc1) after FOXA1 gene knockout. METHODS FOXA1 knockout THBEc1 cells THBEc1-ΔFOXA1-c34 and control cells THBEc1-ctrl were selected as cell models. Western blotting was used to determine FOXA1 protein expression levels. Colony formation assay and Transwell assay were performed to determine the effects of FOXA1 knockout on THBEc1cell proliferation and migration, respectively. Transcriptome sequencing technology was used to analyze and compare transcriptomes between THBEc1-ΔFOXA1-c34 and THBEc1-ctrl cells. Real-time quantitative PCR(RT-qPCR) was employed to confirm the transcriptome sequencing findings. The biological information, including Gene Ontology(GO) and KEGG pathway, was retrieved from the DAVID database. STRING 11.5 database and Cytoscape 3.8.2 were used to construct the interaction network of the proteins encoded by differentially expressed genes. RESULTS THBEc1-ΔFOXA1-c34cells did not express full-length FOXA1 protein, and their proliferation and migration potential was lower than that of THBEc1-ctrl. A total of 1332 genes were identified as differentially expressed genes(fold change>2 or <0.5, false discovery rate<0.05) via transcriptome sequencing analysis between THBEc1-ΔFOXA1-c34 cells and THBEc1-ctrl cells, among which 691 genes were up-regulated and 641 genes were down-regulated in THBEc1-ΔFOXA1-c34 cells. The expression differences between 47 genes determined by RT-qPCR were consistent with those of transcriptome sequencing. Differentially expressed genes were involved in many biological processes, including angiogenesis, cell proliferation, migration,adhesion, inflammatory response, immune response, signal transduction[involving tumor necrosis factor(TNF), mitogen-activated protein kinase(MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B and TGF-β pat

关 键 词：苯并[A]芘 叉头框蛋白A1 THBEc1细胞 

分 类 号：R99[医药卫生—毒理学]