Figure 7. MicroRNA-130a mediates translational repression via the Cx43 3’UTR.

In panel (A), schematic of luciferase reporter constructs used for in vitro transfection assays. Firefly luciferase (F. luciferase) coding region is fused to the control 3’UTR vs. Cx43 3’UTR. Binding of miR-130a is predicted to inhibit translation of F. luciferase. Addition of a specific miR-130a inhibitor is predicted to allow translation of F. luciferase. As shown in (B), administration of a miR-130a inhibitor (300 picomoles) reduced miR-130a levels in 3T3 fibroblasts approximately 83%. Reporter constructs were transfected into NIH 3T3 fibroblasts (known to endogenously express miR-130a). In (C), luciferase assay with luciferase reporter constructs and miR-130a inhibitor vs. scramble. Inhibition of miR-130a had no effect on the translational efficiency of the control reporter. In contrast, translational repression of the reporter construct was relieved in a dose-dependent fashion. Co-transfection with a scramble inhibitor was performed as a control. Luciferase experiments were also carried out in HL-1 cardiomyocytes (also known to endogenously express miR-130a). Using quantitative PCR in (D), HL-1 cells contained 52.4% of the miR-130a compared to 3T3 cells. Administration of the miR-130a inhibitor resulted in a 73.2% reduction in miR-130a (E). Similar to 3T3 cells, luciferase assay demonstrated reduced luciferase activity in the presence of the Cx43 3’UTR reporter construct. Translational repression of the reporter construct was relieved with administration of a miR-130a inhibitor. Transfection with a scramble inhibitor was performed as a control. Experiments performed in triplicate × three independent experiments. * indicates p<0.01. 3’UTR: 3’ untranslated region; LNA: locked nucleic acid.