ACTH-dependent regulation of microRNA as endogenous modulators of glucocorticoid receptor expression in the adrenal gland.

MicroRNA (miR) are a subset of small RNA molecules, which posttranscriptionally modulate target gene expression. Although miR have been demonstrated to impact a number of processes during development and tumorigenesis, little is known about the expression and the role of miR in the adrenal gland. Because tight regulation of steroid synthesis is crucial for maintaining homeostasis upon stressful stimuli, here, we determined the miR expression pattern in mouse adrenal glands under baseline conditions, as well as 10, 30, and 60 min upon ACTH stimulation, using miR microarray. Changes in miR expression levels detected by array analysis were confirmed by real-time PCR and further analyzed by bioinformatic tools to identify miR that putatively target genes involved in adrenal function. After selecting miR, with a significant change in their expression level upon ACTH stimulation, four of the predefined miR (miR-96, miR-101a, miR-142-3p, and miR-433) were found to putatively target the glucocorticoid receptor [nuclear receptor subfamily 3, group C, member 1 (Nr3c1)]. Nr3c1 expression levels were elevated 10 min after ACTH stimulation but decreased after 60 min in comparison with baseline conditions. Modified Nr3c1-3'-untranslated region constructs were further tested by in vitro luciferase assays. Thereby, we could confirm that miR96, miR101a, miR142-3p, and miR433 target the Nr3c1-3'-untranslated region and result in a 20-40% repression of it. Taken together, ACTH stimulation could be demonstrated to acutely influence adrenal miR expression pattern in vivo; thus, potentially modulating adrenal response to acute stressors.