芳香烃受体抑制miR-101a介导PM_(2.5)有机提取物所致斑马鱼胚胎心脏畸形  

Heart malformations of zebrafish embryos exposed to extractable organic matter from PM_(2.5) mediated by aryl hydrocarbon receptor-suppressed miR-101a

作　　者：蔡畅 黄玉洁 陶怡舟 陈涛[1] 姜岩[2] CAI Chang;HUANG Yujie;TAO Yizhou;CHEN Tao;JIANG Yan(School of Public Health,Medical College of Soochow University,Suzhou,Jiangsu 215123,China;School of Biology and Basic Medical Sciences,Medical College of Soochow University,Suzhou,Jiangsu 215123,China)

机构地区：[1]苏州大学医学部公共卫生学院,江苏苏州215123 [2]苏州大学医学部基础医学与生命科学学院,江苏苏州215123

出　　处：《环境与职业医学》2021年第4期336-341,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(81972999)。

摘　　要：[背景]研究表明芳香烃受体(AhR)介导大气PM_(2.5)引起的斑马鱼胚胎心脏畸形,但其具体分子机制有待明确。小分子核糖核酸(miRNAs)等表观遗传机制在心脏发育中起重要作用,而且有报道AhR能调控miRNAs的表达。因此,PM_(2.5)有可能通过AhR信号通路干扰miRNAs表达,从而导致心脏发育异常。[目的]探讨PM_(2.5)有机提取物(EOM)激活AhR后对miR-101a的调控,以及miR-101a异常表达影响斑马鱼胚胎心脏发育的作用机制。[方法]收集苏州市区PM_(2.5)并以索氏提取法提取EOM,在受精后2 h(hpf)内加入EOM(5 mg·L^(-1))及AhR小分子抑制剂CH223191(0.05μmol·L^(-1))进行斑马鱼胚胎染毒。以吗啉寡核苷酸进行AhR基因敲减,以miR-101a类似物注射进行miR-101a过表达。在受精后72 hpf,观察各组斑马鱼胚胎发育情况,统计心脏畸形率。剖取斑马鱼胚胎心脏,提取总RNA,以实时荧光定量PCR方法检测miR-101a和elfa、gsk3β、cdc42 mRNA的表达。以在线数据库Targetscan进行斑马鱼miRNA靶基因预测,并以DAVID数据库对靶基因进行京都基因与基因组百科全书(KEGG)通路富集分析。[结果]与DMSO对照组相比,5 mg·L^(-1) EOM引起72 hpf的斑马鱼胚胎心脏畸形率由5%升高至18%(P<0.05)。EOM引起斑马鱼胚胎心脏中mi R-101a表达降低至对照组的60%左右(P<0.05),而AhR小分子抑制剂CH223191(0.05μmol·L^(-1))及AhR基因敲减均对EOM所致miR-101a表达变化有拮抗作用(P<0.05)。miR-101a类似物可减轻EOM所致的斑马鱼胚胎心脏畸形(由19%降至11%,P<0.05)。miR-101a的预测靶基因集中于Fox O及Wnt等心脏发育相关的重要信号通路,并且Wnt通路基因gsk3β及Rho家族小分子鸟苷酸三磷酸酶cdc42的表达在EOM染毒斑马鱼胚胎心脏中升高(升高1.9倍,P<0.001;升高1.7倍,P<0.01),但加入miR-101a类似物后恢复正常水平(P<0.01)。[结论]斑马鱼胚胎心脏中,PM_(2.5)中的EOM激活AhR后抑制miR-101a的表达,进而通过gsk3β及cdc42等基因干扰�[Background]Previous studies have shown that aryl hydrocarbon receptor(AhR)mediates ambient PM_(2.5)-induced heart defects in zebrafish embryos,but the molecular mechanisms remain to be clarified.Epigenetics such as microRNAs(miRNAs)play an important role in the heart development,and AhR has been reported to regulated the expression of miRNAs.Thus,PM_(2.5) may disturb miRNA expression via AhR signal pathway,leading to aberrant heart development.[Objective]This study aims to investigate the regulation of miR-101 a by AhR,and the effects of aberrant expression of miR-101 a on heart development in zebrafish embryos exposed to PM_(2.5) extractable organic matter(EOM).[Methods]PM_(2.5) was collected in an urban area in Suzhou city,and the EOM were extracted by Soxhlet extraction.Zebrafish embryos were exposed to EOM(5 mg·L^(-1))and AhR small molecule inhibitor CH223191(0.05μmol·L^(-1))within 2 h post-fertilization(hpf).Ah R knockdown was achieved by using morpholino,and mi R-101 a overexpression by mi RNA agomir injection.Embryos at 72 hpf were observed under microscope,and the rate of heart malformation was calculated.Total RNA was extracted from isolated hearts,and m RNA and mi RNA expression levels were detected using q PCR.The mi RNA target genes were predicted using the Targetscan online tool,and a Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed using the DAVID online tool.[Results]The EOM at 5 mg·L^(-1) significantly increased cardiac malformation rates in the zebrafish embryos at 72 hpf compared with the DMSO control(from 5%to 18%,P<0.05).Furthermore,EOM caused a significant decrease in the expression of mi R-101 a in the heart of zebrafish embryos(decreased about 60%,P<0.05).Both Ah R small molecule inhibitor CH223191(0.05μmol·L^(-1))and Ah R gene knockdown attenuated the EOM-induced mi R-101 a expression changes(P<0.05).mi R-101 a agomirs alleviated the PM_(2.5)-caused heart defects(from 19% to 11%,P<0.05).In addition,predicted mi R-101 a target genes were enriched

关 键 词：PM2.5 有机提取物 芳香烃受体 miR-101a 心脏发育 斑马鱼胚胎 

分 类 号：R114[医药卫生—卫生毒理学]