Figure 4.

Direct regulation of phosphatase and tensin homologue in cardiac fibroblasts by miR-21. After isolation, cardiac fibroblasts were cultured for 5 days. The cells were transfected with miRIDIAN mmu-miR-21 inhibitor or miRIDIAN mmu-miR-21 mimic to down-regulate or increase miR-21 levels. (A and B) miR-21 expression levels in cardiac fibroblasts 72 h post-transfection were determined using real-time PCR. miRIDIAN miRNA inhibitor/mimic negative controls were used for control transfections. Mean ± SD (n = 4), *P < 0.01 compared with control-transfected cardiac fibroblasts. (C) Phosphatase and tensin homologue protein levels in cardiac fibroblasts following knockdown of miR-21 in cardiac fibroblasts. (D) Quantification of phosphatase and tensin homologue level was performed using densitometry. Data were normalized to GAPDH. Data shown represent mean ± SD (n = 3). *P < 0.01 compared with control-transfected cells. (E and F) To demonstrate that phosphatase and tensin homologue is a direct target for miR-21 in cardiac fibroblasts, the cells were transfected with a pGL3-PTEN-3′-UTR firefly luciferase expression construct and co-transfected with pRL-TK Renilla luciferase expression construct along with either miR-21 inhibitor or mimic. An increase in relative firefly luciferase activity in the presence of miR-21 inhibitor or a decrease in the presence of miR-21 mimic indicates that the 3′-UTR of phosphatase and tensin homologue contains a target that is modulated by miR-21. Data represent mean ± SD (n = 3). *P < 0.05 compared with control-transfected cells.