Figure 4. PPP2R3A is a direct target of MiR-652.
(A) (top panel) Location of the miR-652 binding site within exon 2 (PR72) or exon 3 (PR130) of PPP2R3A isoforms. Second and third panels show deletions of either 3 bp (Del3) or 5 bp (Del5), respectively) created within the miR-652 binding site. (B) HEK293T cells were transfected with miR-652-3p mimics or negative control (NC) mimic, together with either empty pMIR-REPORT vector (pmiRreport), or pMIR-REP-PPP2R3A-Wt (PPP2R3A wt(wild type)) or PPP2R3A mt (mutant) deletion 3 or deletion 5 reporter plasmids along with pRL-TK. Luciferase activity was measured using a dual-luciferase reporter assay. The relative luciferase activity was calculated by LUCfirefly/LUCrenilla. The fold change of 652 mimic compared to NC mimic is shown; NC mimic set to 1. (C) HEK293T cells were transfected with either miR-652-3p mimics or NC alone, or with the addition of miR-652 antagomiR, together with either pMIR-REP-PPP2R3A-Wt (PPP2R3A wt or PPP2R3A mt deletion 3 or deletion 5 reporter plasmids along with pRL-TK. Luciferase assays were then performed as in (4B). (D) WB of PC3 cells transiently transfected for 3 to 7 days with either control mimic, miR-652 mimic, or miR-652 mimic together with miR-652 antagomir. Expression of PPP2R3A is shown, as is β-tubulin for protein loading control. (E) Densitometry of 3 independent WB performed as described in (4D), where PPP2R3A expression levels are normalized to β tubulin, and expressed as a percentage relative to cells transfected with negative control mimic. Data are represented as a mean ± standard error of the mean of three independent experiments and determined by the unpaired t-test (*P < 0.05; **P < 0.01, ***P < 0.001).