Essential hypertension is known to have a large genetic component. Variation in the CYP11B2 gene, which encodes the aldosterone synthase enzyme, is associated with excess aldosterone production and hypertension but the causative mechanism remains elusive. miRNAs are a class of post-transcriptional regulatory molecules, implicated in cardiovascular disease, development and tumourogenesis. They act by targeting the 3′ untranslated region (UTR) of mRNAs, inhibiting translation through mRNA cleavage or destabilisation
We propose a role for miRNAs in the regulation of CYP11B2 expression and in the development of essential hypertension. To study this, we characterised the miRNA profile of four normal adrenal glands and four aldosterone-producing adenomas (APA), which excrete excess aldosterone and cause hypertension. We then investigated the effect of four miRNAs (miR-125a-5p, miR-125b, miR-134 and miR-495a) which are expressed in these tissues and using bioinformatics were predicted to bind the 3′UTR of CYP11B2 mRNA.
Comparison of miRNA expression levels showed that miR-125a-5p and miR-134 are significantly down-regulated in APAs. The interaction of all four miRNAs with the CYP11B2 3′UTR was tested using a pEZX-reporter plasmid containing the full-length CYP11B2 3′UTR sequence. This was co-transfected into HeLa cells alongside a synthetic miRNA (pre-miR) or miRNA inhibitor (anti-miR). Both miR-125a-5p and miR-125b pre-miRs significantly decreased luciferase activity when over-expressed. Conversely, their anti-miRs significantly increased luciferase activity. This is consistent with canonical miRNA binding and repression, confirming our bioinformatic predictions. miR-495a and miR-134 pre-miRs and anti-miRs did not alter luciferase activity significantly.
In summary, we have identified two miRNAs, miR-125a-5p and miR-134, with aberrant expression in APA samples and putative binding sites in the CYP11B2 gene. We also confirmed that miR-125a-5p and miR-125b, but not miR-134, can repress CYP11B2 by directly targeting the 3′UTR. The regulation of CYP11B2 mRNA by miRNAs and altered expression in adrenal adenomas may give rise to novel therapeutic targets for the treatment of essential hypertension and adrenal tumours.