Abstract 239: MicroRNAs hsa-miR-584 and hsa-miR-31 Regulate Expression of Human Angiotensnogen Gene
Background: Hypertension is a risk factor for stroke, myocardial infarction, and congestive heart failure. A single nucleotide polymorphism (SNP) (rs7079) in 3’UTR of human angiotensinogen (hAGT) gene is associated with elevated blood pressure. We have used TargetScan V6.0, miRanda miRNA prediction algorithms and found that two microRNAs hsa-miR-584 and hsa-miR-31 may bind differentially to rs7079 and alter the expression of hAGT gene.
METHODS: The effect of hsa-miR-584 and hsa-miR-31 miRNAs on endogenous AGT expression levels in Hep3B cells were studied by transfection of individual miRNA mimics followed by quantitative real time PCR (Q-RT-PCR) of the hAGT gene. In addition, the 600 bp 3’UTR of hAGT gene containing either rs7079C or rs7079A was PCR amplified and cloned in to the multiple cloning site of pMIR-REPORT™ miRNA Expression Vector containing luciferase gene. These reporter constructs were then co-transfected with either miRNA mimics or inhibitors to study the effect of miRNAs on luciferase activity in Hep3B cells since these cells express hAGT gene. These experiments were also performed in HEK293 cells which do not express the hAGT gene.
Results: Q-RT-PCR showed that hsa-miR-584 and hsa-miR-31 mimics at 50nM concentration reduced endogenous hAGT mRNA levels by 38% and 30% respectively compared to the mock (without any microRNA) or negative control (which does not have any binding site for eukaryotic 3’UTRs) in Hep3B cells. Furthermore hsa-miR-584 and hsa-miR-31 showed 40% and 25% reduced luciferase activity of the construct containing rs7079 C allele. On the other hand these miRNAs did not affect the luciferase activity in the presence of rs7079 A. When dose dependent anti miRNA inhibitors were transfected along with miRNA mimics, these mimics abolished the microRNA induced down-regulation of luciferase activity.
Conclusion: hsa-miR-584 and hsa-miR-31 miRNAs bind strongly to the hAGT 3’UTR containing rs7079 C allele as compared to rs7079 A allele and may down regulate the expression of AGT gene containing rs7079 C allele. This may be one of the possible mechanism involved in association of rs7079 A allele with human hypertension.
- © 2012 by American Heart Association, Inc.