miR-375 mediates the CRF signaling pathway to regulate catecholamine biosynthesis by targeting <i>Sp1</i> in porcine adrenal gland

<p>Corticotropin-releasing-factor (CRF) is a key regulator of catecholamines (CATs) biosynthesis in the adrenal gland. Furthermore, miR-375 has been confirmed to be localized in the mouse adrenal gland. However, the relationships between miR-375 and CRF in regulating CATs biosynthesis remain to be established. This study was designed to investigate the relationship between CRF and miR-375 in the regulation of CATs biosynthesis in the porcine adrenal gland. Eight adult female pigs (four controls; four injected intracerebroventricularly with 50 μg of CRF) were used for the <i>in vivo</i> experiments in this study. The results showed that miR-375 was exclusively localized in porcine adrenal medullary cells. Functional studies showed that miR-375 negatively regulated CATs synthesis in primary cells by affecting the expression of the CATs synthetases tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine-<i>N</i>-methyltransferase (PNMT). CRF up-regulated the expression of CATs synthetase in primary adrenal medullary cells under basal conditions and upon endogenous miR-375 inhibition; the enhanced effects vanished when cellular miR-375 was overexpressed by transfecting miR-375-mic. CRF decreased the expression of miR-375 both <i>in vivo</i> and <i>in vitro</i>. Our <i>in vitro</i> results showed that CRF significantly decreased the expression of miR-375, perhaps by binding to CRFR1. miR-375 functions by directly binding to the 3′-UTR region of specificity protein 1 (<i>Sp1</i>), which is involved in regulating <i>Th</i> and <i>Dbh</i> expression. These data collectively indicate that miR-375 plays an important role in regulating CATs synthesis and mediates the CRF signaling pathway in porcine adrenal medullary cells.</p>