芳香烃受体抑制miR-101a介导PM2.5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 PM2.5 mediated by aryl hydrocarbon receptor-suppressed miR-101a
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摘要:
背景 研究表明芳香烃受体（AhR）介导大气PM2.5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，PM2.5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PM2.5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PM2.5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（0.05 μmol·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miR-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（0.05 μmol·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FoxO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，PM2.5中的EOM激活AhR后抑制miR-101a的表达，进而通过
gsk3β 及cdc42 等基因干扰心脏发育，引起心脏畸形。Abstract:Background Previous studies have shown that aryl hydrocarbon receptor (AhR) mediates ambient PM2.5-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, PM2.5 may disturb miRNA expression via AhR signal pathway, leading to aberrant heart development.
Objective This study aims to investigate the regulation of miR-101a by AhR, and the effects of aberrant expression of miR-101a on heart development in zebrafish embryos exposed to PM2.5 extractable organic matter (EOM).
Methods PM2.5 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 (0.05 μmol·L-1) within 2 h post-fertilization (hpf). AhR knockdown was achieved by using morpholino, and miR-101a overexpression by miRNA 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 mRNA and miRNA expression levels were detected using qPCR. The miRNA 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 miR-101a in the heart of zebrafish embryos (decreased about 60%,P < 0.05). Both AhR small molecule inhibitor CH223191 (0.05 μmol·L-1) and AhR gene knockdown attenuated the EOM-induced miR-101a expression changes (P < 0.05). miR-101a agomirs alleviated the PM2.5-caused heart defects (from 19% to 11%,P < 0.05). In addition, predicted miR-101a target genes were enriched in FoxO and Wnt signal pathways which are essential to heart development. Moreover, EOM significantly increased the expression levels of Wnt signaling genegsk3β and the Rho GTPase family genecdc42 in the heart of zebrafish embryos (1.9 fold upregulation,P < 0.001, and 1.7 fold upregulation,P < 0.01, respectively), which were further counteracted by miR-101a agomirs (P < 0.01).Conclusion In the heart of zebrafish embryos, AhR activated by PM2.5 EOM significantly represses miR-101a expression, which in turn disrupts heart development via genes including
gsk3β andcdc42 , resulting in cardiac malformations.
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