MicroRNA-135a Protects Against Ethanol-Induced Apoptosis in Neural Crest Cells and Craniofacial Defects in Zebrafish by Modulating the Siah1/p38/p53 Pathway

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in various biological processes, including apoptosis, by regulating gene expression. This study was designed to test the hypothesis that ethanol-induced downregulation of miR-135a contributes to ethanol-induced apoptosis in neural crest cells (NCCs) by upregulating Siah1 and activating the p38 mitogen-activated protein kinase (MAPK)/p53 pathway. We found that treatment with ethanol resulted in a significant decrease in miR-135a expression in both NCCs and zebrafish embryos. Ethanol-induced downregulation of miR-135a resulted in the upregulation of Siah1 and the activation of the p38 MAPK/p53 pathway and increased apoptosis in NCCs and zebrafish embryos. Ethanol exposure also resulted in growth retardation and developmental defects that are characteristic of fetal alcohol spectrum disorders (FASD) in zebrafish. Overexpression of miRNA-135a significantly reduced ethanol-induced upregulation of Siah1 and the activation of the p38 MAPK/p53 pathway and decreased ethanol-induced apoptosis in NCCs and zebrafish embryos. In addition, ethanol-induced growth retardation and craniofacial defects in zebrafish larvae were dramatically diminished by the microinjection of miRNA-135a mimics. These results demonstrated that ethanol-induced downregulation of miR-135a contributes to ethanol-induced apoptosis in NCCs by upregulating Siah1 and activating the p38 MAPK/p53 pathway and that the overexpression of miRNA-135a can protect against ethanol-induced apoptosis in NCCs and craniofacial defects in a zebrafish model of FASD.

Keywords: Siah1; apoptosis; craniofacial defects; ethanol; miRNA-135a; neural crest cells; zebrafish.

FIGURE 1

Ethanol exposure decreased the expression of miR-135a in neural crest cells (NCCs) exposed to 50 mM ethanol for 24 h (A) and zebrafish embryos treated with 1% ethanol and collected at 24 h post-fertilization (hpf; B). The expression of miR-135a was determined by qRT-PCR, as described in the section “Materials and Methods”. Data are expressed as fold change over control and represent the mean ± SD of three separate experiments. * p < 0.05 vs. control.

FIGURE 2

Siah1 is the direct target of miR-135a in neural crest cells (NCCs). (A) The predicted binding sites of miR-135a in the 3′-UTR of Siah1 mRNA. (B) Luciferase reporter assays for the binding of miR-135a to the 3′-UTR of Siah1 in NCCs. (C) Western bolt analysis of the protein expression of Siah1 in NCCs transfected with miR-135a mimics or inhibitors for 48 h. Data are expressed as fold change over control and represent the mean ± SD of three separate experiments. * p < 0.05 vs. control.

FIGURE 3

Overexpression of miR-135a significantly decreased the ethanol-induced upregulation of Siah1 in neural crest cells (NCCs) and zebrafish embryos. NCCs transfected with control mimics or miR-135a mimics were treated with 50 mM ethanol for 24 h. Zebrafish embryos microinjected with control or miR-135a mimics were treated with 1% ethanol and collected at 24 h post-fertilization (hpf). The protein expression of Siah1 was examined in NCCs (A) and zebrafish embryos (B) by western blot. Data are expressed as fold change over control and represent the mean ± SD of three separate experiments. * p < 0.05 vs. control; # p < 0.05 vs. ethanol.

FIGURE 4

Overexpression of miR-135a reduced the ethanol-induced activation of the p38 mitogen-activated protein kinase (MAPK)/p53 pathway in neural crest cells (NCCs). NCCs transfected with control mimics or miR-135a mimics were treated with 50 mM ethanol for 24 h. The expression of Siah1, total p38, and phosphorylated p38 (A), and the expression of Siah1, phosphorylated p53, p53, p53 upregulated modulator of apoptosis (PUMA), and Bcl-2 homologous antagonist/killer (Bak; B) were determined by western blot. Data are expressed as fold change over control and represent the mean ± SD of three separate experiments. * p < 0.05 vs. control; # p < 0.05 vs. ethanol.

FIGURE 5

Overexpression of miR-135a significantly decreased ethanol-induced caspase-3 activation and apoptosis in neural crest cells (NCCs) and zebrafish embryos. NCCs transfected with control mimics or miR-135a mimics were treated with 50 mM ethanol for 24 h. Zebrafish embryos microinjected with control or miR-135a mimics were treated with 1% ethanol and collected at 24 h post-fertilization (hpf). Ethanol-induced apoptosis in NCCs (A-C) and zebrafish embryos (D,E) was determined by analysis of caspase-3 cleavage (A), caspase-3 activity (B,D), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis (C,E). White arrows in (E) indicate brain (B) and eye (E) in zebrafish embryos. Data are expressed as fold change over control and represent the mean ± SD of three separate experiments. * p < 0.05 vs. control; # p < 0.05 vs. ethanol. Scale bar = 200 μm.

FIGURE 6

Microinjection of miR-135a mimics diminished ethanol-induced growth retardation and dysmorphology in zebrafish larvae. Embryos microinjected with control or miR-135a mimics were treated with 1.5% ethanol from 3 to 24 h post-fertilization (hpf). Zebrafish larvae were collected at 4 days post-fertilization (dpf) for morphological analysis. Analysis of ethanol-induced growth retardation and dysmorphology was performed by using a stereoscopic microscope. The black arrows indicate the heart and the arrowheads indicate eye. Data represent the mean ± SD of three separate experiments. * p < 0.05 vs. control; # p < 0.05 vs. ethanol.

FIGURE 7

Microinjection of miR-135a mimics attenuated ethanol-induced craniofacial cartilage defects in zebrafish larvae. Embryos microinjected with control or miR-135a mimics were treated with 1.5% ethanol from 3 h post-fertilization (hpf) to 24 hpf. Zebrafish larvae were collected at 5 days post-fertilization (dpf) for skeletal staining with Alcian blue. (A) Lateral and ventral view of Alcian blue-stained craniofacial cartilages in larvae from different treatment groups. (B) Morphometric analysis of the craniofacial cartilages in larvae from different treatment groups. The length of Meckel’s cartilage (m), palatoquadrate (pq), hyosymplectic (hs), ceratohyal (ch), and the distance between m and pq joint, and between the arch of m and basihyal (bh) were measured. (C) Comparisons of ceratohyal cartilage (ch) angle between different treatment groups. Data represent the mean ± SD of three separate experiments. * p < 0.05 vs. control; # p < 0.05 vs. ethanol. e, ethmoid plate; t, trabeculae cranii; cb, ceratobranchial; bh, basihyal. Scale bar = 200 μm.