苯并[a]芘恶性转化细胞THBEc1中miR-584-5p/miR-211-5p/叉头框蛋白A1轴参与IDH1和HSPB1转录调控  

MiR-584-5p/miR-211-5p/FOXA1 axis is involved in transcriptional regulation of IDH1 and HSPB1 in benzo[a]pyrene malignant transformed cells THBEc1

作　　者：马雪 刘芷毓 邢云昆 姚碧云[2] 傅娟玲[2] 赵鹏[2] MA Xue;LIU Zhiyu;XING Yunkun;YAO Biyun;FU Juaning;ZHAO Peng(Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou 310051,China;Department of Toxicology,School of Public Health,Peking University,Beijing Key Laboratory of Toxicology Research and Risk Assessment for Food Safety,Beijing 100191,China)

机构地区：[1]浙江省疾病预防控制中心,浙江杭州310051 [2]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

出　　处：《中国药理学与毒理学杂志》2023年第11期841-852,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：目的探索苯并[a]芘(BaP)恶性转化人支气管上皮细胞T-16HBE-C1(THBEc1)中微RNA(miRNA)调控叉头框蛋白A1(FOXA1)表达上调的机制,并筛选和验证FOXA1的潜在靶基因。方法利用TargetScan,ENCORI数据库和THBEc1细胞与非转化细胞16HBE间miRNA的二代测序(NGS)结果,综合预测靶向调控FOXA1的miRNA,并通过实时荧光定量PCR(RT-qPCR)进一步筛选预测的miRNA。采用miRNA模拟物(mimics)转染THBEc1细胞,Western印迹法测定FOXA1蛋白表达水平,对靶向调控FOXA1的miRNA进行鉴定。利用hTF,JASPAR和ENCODE数据库结合FOXA1敲除细胞THBEc1-ΔFOXA1-c34和对照细胞THBEc1-ctrl间mRNA的NGS结果,综合预测FOXA1的潜在靶基因。利用RT-qPCR进一步对预测的靶基因[跨膜蛋白98(TMEM98)、IKAROS家族锌指2(IKZF2)、异柠檬酸脱氢酶1(IDH1)、肿瘤坏死因子受体相关因子5(TRAF5)、骨形态发生蛋白2型受体(BMPR2)、热休克蛋白B1(HSPB1)、Runt相关转录因子2(RUNX2)、golgin A7家族成员B(GOLGA7B)和维甲酸相关孤核受体A(RORA)]进行筛选。通过转染过表达质粒构建稳定表达FOXA1的细胞模型THBEc1-ΔFOXA1-c34-oe,即FOXA1功能回补,并利用RT-qPCR和Western印迹法验证FOXA1的潜在靶基因。结果TargetScan和ENCORI数据库分别预测到213和145个可能靶向调控FOXA1的miRNA。NGS共发现351个miRNA在THBEc1细胞中表达下调(差异倍数<0.5,且错误发现率<0.05),其中hsa-miR-584-5p(miR-584-5p),hsa-miR-142-5p(miR-142-5p)和hsa-miR-211-5p(miR-211-5p)在上述2个数据库中均被预测可靶向调控FOXA1。RT-qPCR结果证实,THBEc1细胞中miR-584-5p,miR-142-5p和miR-211-5p表达水平显著低于16HBE细胞(P<0.01)。转染miR-584-5p模拟物或miR-211-5p模拟物均可显著下调THBEc1细胞FOXA1蛋白表达水平(P<0.01),而转染miR-142-5p模拟物对THBEc1细胞FOXA1蛋白表达水平无明显影响。THBEc1-ΔFOXA1-c34和THBEc1-ctrl细胞间的20个差异表达基因(差异倍数<0.5或>2,且错误发现率<0.05)被hTF,JASPAR和ENCODE数据库均预测�OBJECTIVE To explore the microRNA(miRNA)regulation mechanism of up-regulated forkhead box protein A1(FOXA1)expression in benzo[a]pyrene(BaP)malignant transformed human bron-chial epithelial cell T-16HBE-c1(THBEc1)and screen and identify the potential target genes of FOXA1.METHODS TargetScan,ENCORI database and next-generation sequencing(NGS)results of miRNA between THBEc1 and non-transformed cell 16 HBE were used to comprehensively predict the miRNAs targeting FOXA1,and the predicted miRNAs were further screened via real-time quantitative PCR(RT-qPCR).The miRNA mimics were transfected into THBEc1 cells and the expression level of FOXA1 protein was determined by Western blotting to identify the miRNA targeting FOXA1.The potential target genes of FOXA1 were predicted by hTF,JASPAR and ENCODE databases combined with the NGS results of mRNA between FOXA1 knockout cell THBEc1-ΔFOXA1-c34 and control cell THBEc1-ctrl.RT-qPCR was used to further screen the predicted target genes[transmembrane protein 98(TMEM98),IKAROS family zinc finger 2(IKZF2),isocitrate dehydrogenase 1(IDH1),TNF receptor associated factor 5(TRAF5),bone morphogenetic protein receptor type 2(BMPR2),heat shock protein B1(HSPB1),RUNX family transcription factor 2(RUNX2),golgin A7 family member B(GOLGA7B)and RAR related orphan receptor A(RORA)].A cell model THBEc1-ΔFOXA1-c34-oe with stable expressions of FOXA1 was constructed via transfection of overexpression plasmid,that is,FOXA1 function was complemented,and the potential target genes of FOXA1 were identified by RT-qPCR and Western blotting.RESULTS TargetScan and ENCORI databases predicted that 213 and 145 miRNAs might target FOXA1,respec⁃tively.A total of 351 miRNAs were found to be down-regulated in THBEc1 cells(fold change<0.5,and false discovery rate<0.05).Among them,hsa-miR-584-5p(miR-584-5p),hsa-miR-142-5p(miR-142-5p)and hsa-miR-211-5p(miR-211-5p)were predicted to target FOXA1 in the above two databases.Realtime quantitative PCR(RT-qPCR)results confirmed that the expression levels of miR-584-5

关 键 词：苯并[A]芘 叉头框蛋白A1 miR-584-5p miR-211-5p 异柠檬酸脱氢酶1 热休克蛋白B1 

分 类 号：R99[医药卫生—毒理学]