Heart malformations of zebrafish embryos exposed to extractable organic matter from PM_2.5 mediated by aryl hydrocarbon receptor-suppressed miR-101a

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-101a by AhR, and the effects of aberrant expression of miR-101a 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 (0.05 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 PM_2.5-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 gene gsk3β and the Rho GTPase family gene cdc42 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 PM_2.5 EOM significantly represses miR-101a expression, which in turn disrupts heart development via genes including gsk3β and cdc42, resulting in cardiac malformations.