Differential Allelic Expression of Dopamine D1 Receptor Gene (DRD1) Is Modulated by microRNA miR-504

Background: Previously, we reported that dopamine D1 receptor gene (DRD1) is associated with nicotine dependence (ND) and demonstrated that two alleles (A and G) of polymorphism rs686 in the 3'-untranslated region (3'UTR) of DRD1 are expressed differentially. However, the mechanism underlying the differential expression remains to be determined. We hypothesize that it is caused by miRNA targeting. Methods: We first used the MicroInspector algorithm to identify microRNAs (miRNAs) potentially targeting the rs686 polymorphism in the DRD1 3'UTR and then employed a luciferase reporter assay combined with site-directed mutagenesis to test the predicted miRNA targeting. We also examined the miRNA targeting of DRD1 with a gene expression assay. Results: Of two miRNAs predicted by computational analyses, we found that miR-504, not miR-296, upregulated reporter luciferase activity and increased DRD1 expression by targeting the DRD 3' UTR, whereas inhibition of miR-504, not miR-296, had the opposite effect. Furthermore, we showed that the direct binding of miR-504 to the DRD1 3'UTR, verified by site-directed mutagenesis, causes a significant expression difference between the two alleles. Conclusions: miR-504 up-regulates DRD1 expression by direct binding to the 3'UTR, which leads to differential allele-specific expression of DRD1.