D1 Receptor Expression Is Regulated by MicroRNA Figure 9. Inhibition of miR-142-3p enhances D1 receptor signaling in CAD cells. (A) Relative b-galactosidase activity, normalized to total protein, in non-differentiated CAD cells co-transfected with pCMV b-gal+D1 39UTR (1277 bp) and either the control empty miRZipTM plasmid (gray bar) or the miRZipTM anti-142-3p plasmid (gray hatched bar). The cells were harvested 48 hours post-transfection. The bars represent the mean values 6 s.e.m. (n = 3). *P,0.05, the enzyme activity was significantly increased in cells transfected with miRZipTM anti-142-3p plasmid when compared to cells transfected with control empty miRZipTM plasmid (two-tailed pair-wise Student’s t-test). (B) Relative cAMP levels in non-differentiated CAD cells transfected with the control empty miRZipTM plasmid (gray circles) or the miRZipTM anti-142-3p plasmid (black squares) that were treated with increasing concentration of D1 receptor agonist, A77636, and 0.3 mM IBMX for 10 minutes. The cells were treated 48 hours post-transfection. The levels of cAMP were normalized to total protein and plotted relative to control cells that were only treated with 0.3 mM IBMX. The bars represent the mean values 6 s.e.m. (n = 3). Representative western blot (C) and cumulative data (D) showing the levels of phosphorylated and total DARPP-32 protein in non-differentiated CAD cells transfected with the control empty miRZipTM plasmid (white and white hatched bars) or the miRZipTM anti- 142-3p plasmid (gray and gray hatched bars) that were treated with 10 mM A77636 and 0.3 mM IBMX for 30 minutes. The cells were treated 48 hours post-transfection. The bars represent the mean values 6 s.e.m. (n = 4). *, **P,0.05, One Way ANOVA, post hoc SNK test. doi:10.1371/journal.pone.0049288.g009 vector plasmids. The results in Figures 9C and 9D shows that the levels of D1 receptor-activated phospho DARPP-32 is significantly higher in non-differentiated CAD cells transfected with miRZipTM anti 142-3p (,102% increase) compared to empty miRZipTM vector plasmid (,29% increase). Together these results strongly suggest that the miR-142-3p mediated regulation of D1 receptor protein expression is biologically significant. Micro RNA 142-3p Levels are Inversely Correlated to D1 Receptor Expression during Mouse Brain Development The results in Figure 1B and 1D showed that there is a significant increase in D1 receptor protein expression in vivo between P7 and P14 during mouse brain development. Figure 10A shows the increasing expression of D1 receptor protein at P7, P14 and P30 in the striatum region of mouse brain. We measured the expression of microRNA 142-3p in striatal brain tissue punches from P7, P14 and P30 using real-time RT-PCR. The result in Figure 10B shows that there is a significant reduction in miR-142- 3p expression on P14 and P30 compared to P7 which inversely correlates with the increase in D1 receptor protein expression. These results suggest that miR-142-3p might be involved in the D1 receptor post-transcriptional regulation during postnatal mouse brain development. PLOS ONE | www.plosone.org 12 November 2012 | Volume 7 | Issue 11 | e49288