Abstract 101: The Role of MicroRNAs in the Regulation of Human Aldosterone Synthase Gene Expression
The human aldosterone synthase (hCYP11B2) gene encodes a key enzyme for the biosynthesis of aldosterone which is part of the renin-angiotensin-aldosterone system. Molecular variants in this gene have been associated with essential hypertension. MicroRNAs (miRNAs) are a family of small, noncoding RNAs that bind to the 3’-untranslated region (3’UTR) of the gene and regulate its expression. Our main objective in this study was to investigate whether miRNAs bind at the polymorphic sites present in the 3’UTR of hCYP11B2 gene and modulate its expression.
Previous studies have suggested that -344T/C (rs1799998) polymorphism in the hCYP11B2 promoter is associated with hypertension. Here, we compared nucleotide sequences of the promoter and 3’UTR of hCYP11B2 gene from hypertensive and normotensive subjects and found that rs1799998 is in linkage disequilibrium with 735G/A (rs28491316). Thus, variant -344T almost always occurs with variant 735G; whereas variant -344C almost always occurs with variant 735A. Bioinformatic algorithms of the full-length 3’UTR for miRNAs revealed that hsa-miR-766 was possibly binding at the 735G allele. To examine if this miRNA regulates hCYP11B2 gene expression, reporter constructs were made where luciferase gene was attached to 1.4 kb of hCYP11B2 3’UTR containing either 735G, or 735A and co-transfected into HEK293 cells with either pre-miR-766 or anti-pre-miR-766. Our results showed that pre-miR-766 reduced luciferase activity in a dose dependent manner in the presence of 735G allele. There was a significant decrease (65+2%) in the luciferase activity with pre-miR-766 (50 nM) in the presence of 735G allele, whereas it was insignificant (4+5%) in the presence of 735A allele. In H295R cells that express hCYP11B2 gene, pre-miR-766 (50nM) reduced luciferase activity by 81+1% in the presence of 735G allele. We also showed that transfection of pre-mir-766 reduced hCYP11B2 mRNA level by about 23.6% in H295R cells. In corroboration with these results, anti-pre-miR-766 did not decrease luciferase activity in presence of either allele in both cell cultures.
Our results suggest that hCYP11B2 gene expression may be regulated by differential binding of miR-766 at 735G/A polymorphism present in its 3’UTR.
- © 2012 by American Heart Association, Inc.