The CYP11B1 and CYP11B2 genes encode 11β-hydroxylase and aldosterone synthase, which catalyse the production of cortisol and aldosterone, respectively, and have been implicated in the development of hypertension. For this study, we wished to investigate the role of microRNAs (miRNAs), a novel class of post-transcriptional gene regulators. To that end, we generated a profile of human adrenal miRNAs to study, and then investigated the action of one adrenal miRNA, miR-24, on CYP11B1 and CYP11B2 genes in vitro.
miRNA expression was measured in four non-tumorous human adrenal glands using μParaflo technology microarray. Putative miRNA-binding sites in the CYP11B1 and CYP11B2 genes were identified using four bioinformatic databases. The human adrenocortical cell line, H295R, was transfected with pre-miR 24 (50 nM) and a control, scrambled pre-miR. 48 h Post-transfection mature miR-24, CYP11B1 and CYP11B2 mRNA was measured by qRT-PCR and steroid secretion was quantified by liquid chromatography with tandem mass spectrophotometry.
Cross-referencing of microarray expression and bioinformatic data identified 23 adrenal miRNAs predicted to bind putative sites in CYP11B1 and 16 predicted to bind CYP11B2; 13 of these miRNAs were common to both genes. Transfection of H295R cells with pre-miR-24 produced a significant increase in mature miR-24 expression (P<0.001). CYP11B1 mRNA levels were reduced by 36.1±5.6% (P<0.05) and CYP11B2 mRNA by 36.4±7.3% (P<0.05). The level of secreted cortisol was reduced by 19.6±4.7% (P=0.07) and aldosterone by 15.9±3.1% (P<0.05).
These microarray and bioinformatic data have identified several candidate miRNAs that may be involved in regulation of the CYP11B1 and CYP11B2 genes, including miR-24: our in vitro data confirm its regulation of these genes. CYP11B1 and CYP11B2 are therefore subject to regulation by miRNAs and, given the role of these genes in adrenal pathophysiology and hypertension, may prove to be an important therapeutic target.