Session: FP40-Renin-Angiotensin-Aldosterone System/Endocrine Hypertension
Room 135 (Moscone Center)
Poster Board MON-728
Luciferase reporter constructs containing full-length CYP17A1 3’UTR sequence were co-transfected into HeLa cells alongside miR-320a inhibitor. Luciferase luminescence was measured 48 hours post-transfection and found not to differ significantly from that of control-transfected cells (relative expression 1.2 ± 0.3, p=0.58). This suggests that miR-320a does not bind the 3’UTR of CYP17A1 mRNA.
Other steroidogenic mRNA targets of miR-320a were therefore investigated using four bioinformatic databases (miRBASE, microRNA.org, TargetScan and miRanda). This identified CYP11A1, which encodes the side-chain cleavage enzyme, as a possible target.
To investigate the effect of miR-320a on CYP11A1 expression, H295R human adrenal cells were transfected with miR-320a mimic or inhibitor. Relative to control cells, levels of CYP11A1 mRNA 48 hours post-transfection were found to be significantly reduced in the presence of miR-320a mimic (0.8 ± 0.02, p<0.05), and significantly increased in the presence of miR-320a inhibitor (1.4 ± 0.03, p<0.01).
In summary, we have shown that modulation of CYP17A1 expression in H295R cells is not the result of miR-320a binding CYP17A1 transcripts. Further in vitro investigation has shown a possible direct modulatory effect of miR-320 on CYP11A1 mRNA, but this is yet to be confirmed. Future studies will investigate this, and also the alternative mechanisms by which miR-320 might modulate CYP17A1 expression with particular emphasis on its role in the aetiology of APA.
Nothing to Disclose: LD, SW, SM, JMC, ED
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