Surya Chinamuthevi

Introduction: Although it is known that excess alcohol consumption during pregnancy can lead to fetal alcohol spectrum disorders (FASD), the molecular mechanisms behind these clinical manifestations are still poorly understood. ¹ Previous studies have alluded to the role of neural crest cells (NCCs) in FASD. As the multipotent progenitor cell population, NCCs give rise to cell types involved in craniofacial development. ² Epithelial-to-mesenchymal transition (EMT), autophagy, and apoptosis occur in the generation of neural crest derivatives and are essential for NCC development. MicroRNAs (miRNAs) play roles in regulating gene expression related to these cellular processes. Previous studies have indicated that ethanol can affect miRNA levels and these various pathways. Methods: A luciferase reporter system was made by cloning a 3’-UTR of each gene into a pMIR-Report vector. The vector was fused both with and without the 3’-UTR of these genes and miRNA mimic or miRNA control mimic were then co-transfected into NCCs. ³ Ethanol interplay with these NCC samples was then observed. Results: Ethanol exposure caused a significant decrease in mRNA expression of Snail1 in NCCs and downregulation of miR-34a by using miR-34a inhibitor prevented the ethanol-induced reduction in the mRNA expression of Snail1. These findings demonstrate that ethanol-induced up-regulation of mi-34a can downregulate Snail1 in NCCs. ³ Co-transfection of the 3’-UTR of Atg9a mRNA and miR-34a mimic into NCCs caused a significant decrease in luciferase activity and shows that Atg9a is a direct target of miR-34a. ⁵ NCCs transfected with miR-34a inhibitor were treated with ethanol for 24 h and down-regulation of miR-34a by the inhibitor greatly increased expression of Atg9a in NCCs exposed to ethanol. ⁵ Co-transfection of the 3’-UTR of Siah1 and miR135a mimic into NCCs caused a significant decrease in luciferase activity and shows that Siah1 is a direct target of miR-135a. ² Excessive expression of miR-135a by transfection with miR-135a mimics decreased ethanol-induced upregulation of Siah1 and highlighted how downregulation of miR-135a leads to ethanol-induced upregulation of Siah1 in NCCs. ² Increased expression of miR-135a significantly decreased ethanol-induced expression of phosphor-p38 MAPK and indicates that miR-135a can inhibit p38 MAPK pathway through downregulating Siah1. ² Conclusion: Ethanol treatment can disrupt the production and development of cranial NCCs through impacting EMT, autophagy, and apoptotic related genes. These biological mechanisms may contribute to the etiology of craniofacial defects seen with FASD. ¹ The associated genes and miRNAs present therapeutic targets for the prevention of FASD.

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