MiRNA-383 AND MiRNA-384 SUPPRESS PROOPIOMELANOCORTIN GENE EXPRESSION IN THE HYPOTHALAMUS: EFFECTS OF EARLY LIFE ETHANOL EXPOSURE

Introduction: Early life ethanol exposure is known to program hypothalamic proopiomelanocortin (POMC) neurons to express a reduced level of POMC and its control of stress axis functions throughout the life span. In this study we tested whether miRNAs contribute to the ethanol-induced suppression of Pomc gene expression during the developmental period. Methods: In in vivo studies, POMC-EGFP male mice were fed with 2.5 g/kg ethanol using milk formula (AF) or pair-fed isocaloric milk formula (PF) or left in the litter (AD) during postnatal day (PND)-2-6. In in vitro studies, mHypoA-POMC/GFP cells were treated with ethanol (50 mM) for a 24 h period. Hypothalamic tissues or cell extracts were used for measurement of miRNAs and POMC mRNA. Results: Determination of genome-wide microRNA expression profile identified 40 miRNAs significantly altered in hypothalamic tissues of AF mice. In-silico analysis further identified miRNA-383, 384 and 488 have putative binding sites at the POMC 3’UTR. However, only miR-383 and miR-384 are identified to be responsive to ethanol. Administration of miR-383 or 384 inhibitor oligos suppressed ethanol-stimulated miR-383 or 384 expression and restored Pomc mRNA and protein expression in AF mice. mHypoA-POMC/GFP cells when treated with ethanol showed elevated levels of miR-383 and miR-384 and reduced level of Pomc mRNA. Treatment with miR-383 or 384 mimic oligos reduced the level of Pomc mRNA, while treatment with miR-383 or 384 inhibitor oligos increased the level of Pomc mRNA. Reporter assay further confirms the binding specificity of miR-383 and miR-384 to Pomc 3’UTR. Conclusion: These data suggest that miR-383 and miR-384 suppress Pomc gene expression and may contribute to the ethanol-induced alteration of the stress axis functions.