MicroRNA-9 regulates fetal alcohol-induced changes in D2 receptor to promote prolactin production

Abstract

Fetal alcohol exposure (FAE) is known to increase prolactin (PRL) secretion from the pituitary lactotropes. In this study, we determined whether microRNAs (miRs) are involved in FAE-induced alteration in PRL release. We employed a rat animal model of FAE involving feeding pregnant Fisher 344 rats with a liquid diet containing 6.7% alcohol between gestational days 7-21 (AF). Both cyclic and estradiol-implanted FAE females showed increased levels of plasma PRL and pituitary <i>Prl</i> mRNA but reduced levels of pituitary dopamine D2 receptor (<i>D2r</i>) and its short spliced form (<i>D2s</i>). FAE increased the expression levels of miR-9 and miR-326 and did not produce any significant changes in miR-153 or miR-200a levels in the pituitary. Effects of FAE on miR-9 and miR-326 were associated with reduced levels of <i>D2r</i> and <i>D2s</i>, increased levels of <i>Prl</i> in the pituitary, and in plasma. These effects of FAE on <i>D2r</i>, <i>D2s</i> and <i>Prl</i> were enhanced following estradiol treatment. In PRL-producing MMQ cells, ethanol increased miR-9 but not miR-326, reduced levels of <i>D2r</i> and <i>D2s</i> and increased levels of <i>Prl</i> Treatment of MMQ cells with an anti-miR-9 oligo reduced ethanol effects on miR-9, <i>D2r</i>, <i>D2s</i> and <i>Prl</i> miR-9 mimic oligos reduced the luciferase activity of reporter vector containing <i>D2r</i> 3'UTR, but failed to reduce the mutant luciferase activity. These data suggest that FAE programs the pituitary to produce increased amounts of miR-9 expression that represses the <i>D2r</i> gene and its spliced variant <i>D2s</i> by targeting its 3'UTR leading to an increase in PRL production and secretion.