D1 Receptor Expression Is Regulated by MicroRNA Figure 6. D1 receptor post-transcriptional regulation is mediated by microRNA miR-142-3p. (A) Levels of Dicer mRNA (normalized to b- actin) in CAD cells transiently-transfected with the pCMV- b-gal+D1 39UTR construct and either the negative control siRNA (-ve siRNA) or two different siRNAs (Dicer ‘‘a’’ and Dicer ‘‘b’’) targeting the dicer mRNA. The Dicer siRNAs were transfected individually or together (a+b) into non-differentiated CAD cells. The final concentration of the siRNA in the transfection was 10 nM. The cells were harvested 48 hours post transfection and mRNA levels measured using real-time RT-PCR with TaqManTM probes for dicer and b-actin. The bars represent the mean values 6 s.e.m. (n = 3 to 6). *P,0.001, the level of dicer mRNA was significantly reduced in cells transfected with the dicer siRNAs when compared to cells transfected with negative control siRNA, One Way ANOVA, post hoc SNK test. (B) Part of the harvested cells from the experiments in panel A, was lysed and the b-galactosidase reporter activity measured and normalized to total protein. The bars represent the mean values 6 s.e.m. (n = 3 to 6). *P,0.001, the level of b-galactosidase reporter activity was significantly increased in cells transfected with the dicer siRNAs when compared to cells transfected with negative control siRNA, One Way ANOVA, post hoc SNK test. Wild type pCMV- b-gal+D1 39UTR construct or pCMV- b-gal+D1 39UTR constructs with mutations in the binding site for miR-10a*, miR-142-3p or miR-294/295 were transfected into non-differentiated (C) or differentiated (D) CAD cells and 48 hours later b- galactosidase reporter activity was measured and normalized to total protein. The bars represent the mean values 6 s.e.m. (n = 6). *P,0.001, the level of b-galactosidase reporter activity was significantly increased in cells transfected with the pCMV- b-gal+D1 39UTR construct with the miR-142-3p binding site mutated when compared to other transfected cells, One Way ANOVA, post hoc SNK test. doi:10.1371/journal.pone.0049288.g006 (1277 bp) into non-differentiated CAD cells significantly increased b-galactosidase reporter activity compared to cells co-transfected with empty miRZipTM vector plasmid and pCMV b-gal+D1 39UTR (1277 bp). This result is consistent with above studies and suggested that the miRZipTM anti 142-3p plasmid also inhibits endogenous miR-142-3p and relieves D1 receptor post-transcrip- tional regulation in non-differentiated CAD cells. Using the D1 receptor selective agonist, A77636, we have previously shown that D1 receptors in CAD cells couple to adenylate cyclases and increase intracellular cAMP levels upon agonist stimulation [8–10]. To determine the effect of inhibiting miR-142-3p on D1 receptor signaling function, we transfected non-differentiated CAD cells with miRZipTM anti 142-3p or empty miRZipTM vector plasmids and compared D1 receptor stimulated increase in intracellular cAMP levels. The results in Figure 9B shows that non-differentiated CAD cells transfected with miRZipTM anti 142-3p had significantly higher D1 receptor- agonist induced cAMP levels compared to cells transfected with the empty miRZipTM vector (,85% increase in Emax). The EC50 values for D1 receptor agonist, A77636, were similar (0.136 nM PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49288