Figure 8

The ADAR2-mediated anti-tumoral effect is reversed by miR-221, miR-222 and miR-21 expression. (a) U118 cells (8 × 10⁴; untreated, dark gray), U118 ADAR2 cells (ADAR2, medium gray), and U118 ADAR2 cells transiently transfected with either scramble mimic (ADAR2 + scr, light gray) or with a mix of miR-221- and miR-222-mimic (ADAR2 + miR-221/222, red) were seeded and proliferation was monitored over 3 days. U118 untransfected cells (untreated, dark gray) were used as control. Error bars indicate standard deviations of four independent experiments. Mean ± standard deviation (n = 4), **P < 0.01 when ADAR2 plus miR-221/222 cells (red) are compared with the ADAR2 (dark gray) and ADAR2 plus scramble (light gray). (b) Protein lysates were extracted from the cells shown in (a) and analyzed by immunoblotting for p27^Kip1, a target of miR-221 and miR-222. (c) PDCD4 protein analysis after immunoblotting of total protein extracts from U118 ADAR2 and ADAR2 E/A cell lines and the corresponding quantitative densitometric analysis are shown. Each sample was normalized to GAPDH and compared with the ADAR2 E/A cells arbitrarily set to 1. A representative sample of two independent experiments is shown. (d) Representative photographs of U118 ADAR2 cells transfected with scramble mimic (scr) and miR-21-mimic (miR-21) at 0, 15 and 21 h after scratching the surface of monolayers cells. Only the U118 ADAR2 plus miR-21 cells show an increase in motility when compared with the control cells (scr). The wound healing assay was performed in a time interval in which the cells do not divide (data not shown).