Suppression and Epigenetic Regulation of MiR-9 Contributes to Ethanol Teratology: Evidence from Zebrafish and Murine Fetal Neural Stem Cell Models
ABSTRACT Fetal alcohol exposure produces multiorgan defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol (EtOH) sensitivity of the miRNA miR-9 suggests one mechanism, whereby EtOH broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of EtOH teratology.
Zebrafish embryos were exposed to EtOH during gastrulation, or injected with anti-miR-9 or nonsense control morpholinos during the 2-cell stage of development and collected between 24 and 72 hours postfertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the nontargeted transcript, MECP2. Methylation at CpG islands of mammalian miR-9 genes was assessed in fetal murine neural stem cells (mNSCs) by methylation-specific PCR, and miRNA processing assessed by qRT-PCR for pre-miR-9 transcripts.
EtOH treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, EtOH transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of EtOH-induced teratology. In mNSCs, EtOH increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3.
EtOH exerts regulatory control at multiple levels of miR-9 biogenesis. Moreover, early embryonic loss of miR-9 function recapitulated the severe range of teratology associated with developmental EtOH exposure. EtOH also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between EtOH and miRNA regulatory networks in the developing embryo. The implications of these data for the expression and function of mature miR-9 warrant further investigation.
Article: MicroRNAs and Ethanol Toxicity[Show abstract] [Hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are a class of small nonprotein-coding RNAs (ncRNAs) that have been shown to promote the degradation of target messenger RNAs and inhibit the translation of networks of protein-coding genes to control the development of cells and tissues, and facilitate their adaptation to environmental forces. In this chapter, we will discuss recent data that show that miRNAs are an important component of the epigenetic landscape that regulates the transcription as well as the translation of protein-coding gene networks. We will discuss the evidence that implicates miRNAs in both developmental and adult effects of alcohol consumption. Understanding the interactions of this novel class of ncRNAs with the epigenome will be important for understanding the etiology of alcohol teratology and addiction as well as potential new treatment strategies.International Review of Neurobiology 01/2014; 115:245-84. DOI:10.1016/B978-0-12-801311-3.00007-X · 2.46 Impact Factor
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ABSTRACT: Adolescent binge alcohol abuse induces long-term changes in gene expression, which impacts the physiological stress response and memory formation, two functions mediated in part by the ventral (VH) and dorsal (DH) hippocampus. microRNAs (miRs) are small RNAs that play an important role in gene regulation and are potential mediators of long-term changes in gene expression. Two genes important for regulating hippocampal functions include brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT1), which we identified as putative gene targets of miR-10a-5p, miR-26a, miR-103, miR-495. The purpose of this study was to quantify miR-10a-5p, miR-26a, miR-103, miR-495 expression levels in the dorsal and ventral hippocampus of male Wistar rats during normal pubertal development and then assess the effects of repeated binge-EtOH exposure. In addition, we measured the effects of binge EtOH-exposure on hippocampal Drosha and Dicer mRNA levels, as well as the putative miR target genes, BDNF and SIRT1. Overall, mid/peri-pubertal binge EtOH exposure altered the normal expression patterns of all miRs tested in an age- and brain region-dependent manner and this effect persisted for up to 30 days post-EtOH exposure. Moreover, our data revealed that mid/peri-pubertal binge EtOH exposure significantly affected miR biosynthetic processing enzymes, Drosha and Dicer. Finally, EtOH-induced significant changes in the expression of a subset of miRs, which correlated with changes in the expression of their predicted target genes. Taken together, these data demonstrate that EtOH exposure during pubertal development has long-term effects on miRNA expression in the rat hippocampus.PLoS ONE 01/2014; 9(1):e83166. DOI:10.1371/journal.pone.0083166 · 3.53 Impact Factor
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ABSTRACT: Plasma or circulating miRNAs (cir miRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol (EtOH) altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. Pregnant sheep were exposed to a binge model of EtOH consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a third-trimester-equivalent period from gestational day 4 (GD4) to GD132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte, and leukocytes obtained from nonpregnant ewes, and plasma from pregnant ewes 24 hours following the last binge EtOH episode, and from newborn lambs, at birth on ~GD147. Pregnant ewe and newborn lamb cir miRNA profiles were similar to each other and different from nonpregnant female plasma, erythrocyte, or leukocyte miRNAs. Significant changes in cir miRNA profiles were observed in the EtOH-exposed ewe and, at birth, in the in utero, EtOH-exposed lamb. Cir miRNAs including miR-9, -15b, -19b, and -20a were sensitive and specific measures of EtOH exposure in both pregnant ewe and newborn lamb. Additionally, EtOH exposure altered guide-to-passenger strand cir miRNA ratios in the pregnant ewe, but not in the lamb. Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. Cir miRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment.Alcoholism Clinical and Experimental Research 03/2014; DOI:10.1111/acer.12378 · 3.31 Impact Factor