Abstract 1374: MicroRNA-155 Regulates Human Angiotensin II Type 1 Receptor Expression in Human Vascular Smooth Muscle Cells
The human angiotensin II type 1 receptor (hAT1R) gene has been found to be highly polymorphic. In particular, a single nucleotide polymorphism (SNP) has been described in which there is an A/C transversion at position 1166 in the 3′-untranslated region (3′-UTR) of this gene. The silent +1166 A/C SNP has been associated with cardiovascular disease; however, the physiological significance of this polymorphism is uncertain because of its location in the untranslated region. MicroRNAs (miRNA, ~22 bp) have recently emerged as a new class of small, non-coding RNAs that silence gene expression by base-pairing to complementary sequences within the 3′-UTR of target mRNAs. A search of the miRBase Target database (http://microrna.sanger.ac.uk/) suggested that a miRNA, designated miR-155, may interact with a target site which encompasses the +1166 A/C SNP. Therefore, the aim of the following study was to determine whether miR-155 could regulate hAT1R expression and to determine whether this polymorphism reduced the affinity of miRNA interaction, therefore resulting in increased hAT1R levels. Importantly, we demonstrate for the first time, that transfection of miR-155 into a human fetal vascular smooth muscle cell line (FLTRs) reduced the endogenous expression of the hAT1R (47±4%, n=4, p<0.01 vs. mock control FLTRs). The reduction in hAT1R expression was not due to changes in mRNA stability. Real-time PCR experiments demonstrated that mature miR-155 was expressed in FLTR cells and in all human tissues investigated. To investigate whether the level of miR-155 expression in FLTRs is physiologically relevant, these cells were transfected with various anti-sense RNA oligonucleotides. Importantly, these experiments demonstrated that only FLTRs transfected with anti-miR-155 showed an increase in hAT1R levels (39.6±4.1%, n=4, p<0.01 vs. mock transfected FLTRs). Finally, luciferase reporter assays demonstrated that the +1166 A/C SNP attenuated the ability of miR-155 to modulate luciferase levels. In conclusion, our results suggest that miR-155 plays a physiological role in regulating the expression of hAT1R in vascular smooth muscle cells. Additionally, our study provides a feasible mechanism by which the +1166 A/C SNP could lead to cardiovascular disease.