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Keywords:

  • Fetal Alcohol Spectrum Disorders;
  • Prenatal Nicotine Exposure;
  • Neural Stem Cells;
  • miR-9;
  • miR-21;
  • miR-153;
  • miR-335;
  • miR-140-3p;
  • Nicotinic Acetylcholine Receptors

Background

Ethanol (EtOH) and nicotine are often co-abused. However, their combined effects on fetal neural development, particularly on fetal neural stem cells (NSCs), which generate most neurons of the adult brain during the second trimester of pregnancy, are poorly understood. We previously showed that EtOH influenced NSC maturation in part, by suppressing the expression of specific microRNAs (miRNAs). Here, we tested in fetal NSCs the extent to which EtOH and nicotine coregulated known EtOH-sensitive (miR-9, miR-21, miR-153, and miR-335), a nicotine-sensitive miRNA (miR-140-3p), and mRNAs for nicotinic acetylcholine receptor (nAChR) subunits. Additionally, we tested the extent to which these effects were nAChR dependent.

Methods

Gestational day 12.5 mouse fetal murine cerebral cortical–derived neurosphere cultures were exposed to EtOH, nicotine, and mecamylamine, a noncompetitive nAChR antagonist, individually or in combination, for short (24 hour) and long (5 day) periods, to mimic exposure during the in vivo period of neurogenesis. Levels of miRNAs, miRNA-regulated transcripts, and nAChR subunit mRNAs were assessed by quantitative reverse transcription polymerase chain reaction.

Results

EtOH suppressed the expression of known EtOH-sensitive miRNAs and miR-140-3p, while nicotine at concentrations attained by cigarette smokers induced a dose-related increase in these miRNAs. Nicotine's effect was blocked by EtOH and by mecamylamine. Finally, EtOH decreased the expression of nAChR subunit mRNAs and, like mecamylamine, prevented the nicotine-associated increase in α4 and β2 nAChR transcripts.

Conclusions

EtOH and nicotine exert mutually antagonistic, nAChR-mediated effects on teratogen-sensitive miRNAs in fetal NSCs. These data suggest that concurrent exposure to EtOH and nicotine disrupts miRNA regulatory networks that are important for NSC maturation.