Session: MON 723-757-Renin-Angiotensin-Aldosterone System/Endocrine Hypertension
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Poster Board MON-753
H295R cells were stimulated with AngII, Bu2cAMP (a substitute for ACTH) or KCl. miRNA expression in these and in basal cells was then assessed by microarray. miR-24 was detected in all cell groups, although Bu2cAMP-stimulated cells had significantly reduced levels of miR-24 relative to basal (p<0.003).
Luciferase reporter constructs containing full-length CYP11B1 or CYP11B2 3’UTR sequences were specifically mutated at each predicted miR-24 binding site. HeLa cells were separately transfected with each of these constructs and all were found to yield significantly higher levels of luminescence relative to their non-mutated counterparts (p<0.001). Furthermore, a construct mutated at both of the predicted CYP11B1 miR-24 sites produced significantly greater luminescence than either of the CYP11B1 constructs mutated at just one of the sites (p<0.001).
Co-transfection of non-mutated CYP11B1 or CYP11B2 constructs with miR-24 inhibitor also significantly increased luminescence (p<0.05) but this effect was not observed when miR-24 inhibitor was co-transfected alongside mutant constructs (p=0.24).
In conclusion, we confirm that miR-24 is expressed in the H295R cell line and that it is differentially expressed in response to Bu2cAMP; this may reflect a mechanism by which miR-24 could achieve fine control of gene expression via ACTH. Our reporter construct experiments validate bioinformatic predictions and these results are consistent with canonical miRNA binding and repression, confirming that miR-24 is capable of repressing CYP11B1 and CYP11B2 expression through direct binding of sites within the mRNA 3’UTR. 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.
Nothing to Disclose: LD, SA, JL, SW, SM, JMC, ED
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