Endocrine Abstracts

The CYP11B1 and CYP11B2 genes encode the enzymes responsible, respectively, for the terminal stages of cortisol and aldosterone biosynthesis, and have been implicated in the development of essential hypertension. Previously, we investigated the role of microRNAs in the regulation of these genes and showed in vitro that levels of the adrenally-expressed microRNA-24 (miR-24) inversely correlate with those of CYP11B1 and CYP11B2 mRNA, as well as cortisol and aldosterone production. Bioinformatic analysis predicts two putative binding sites for miR-24 in the 3′UTR of CYP11B1 mRNA and one in CYP11B2. The purpose of this study was to ascertain whether observed changes in CYP11B1 and CYP11B2 mRNA levels in vitro were due to the direct action of miR-24 at these sites.

Luciferase reporter constructs containing full-length CYP11B1 and CYP11B2 3′UTR sequences were specifically mutated by a single base at the predicted miR-24 binding sites using site-directed mutagenesis. These constructs were then transfected into HeLa cells, either alone or alongside miR-24 inhibitor; luciferase luminescence was measured 48 h post-transfection.

Cells transfected with mutated plasmids yielded significantly higher luminescence compared to non-mutated plasmids (P<0.01). Co-transfection of non-mutated plasmids with miR-24 inhibitor also significantly increased luminescence (P<0.05), although this effect was eliminated when inhibitor was co-transfected with mutated plasmids (P=0.24). Furthermore, combined mutation of both the predicted CYP11B1 miR-24 sites resulted in greater effect than single mutations at either site.

These results are consistent with canonical miRNA binding and repression, and confirm that miR-24 is capable of regulating CYP11B1 and CYP11B2 expression through direct binding of 3′UTR sites on their mRNA. This is the first study to demonstrate directly such regulation of these genes at specific sites, and may have important implications for corticosteroid biosynthesis and its role in the development of hypertension.

Declaration of funding: This work was supported by the British Heart Foundation (grant number PG/09/092).