Figure 6. Resveratrol inhibition of Akt phosphorylation contributes to its regulation of miR-21 expression and function. A,

Resveratrol inhibits Akt phosphorylation in a dose-dependent manner. PC-3M-MM2 cells were exposed to vehicle or different doses of resveratrol (5-100 µM) for 24 h and whole cell lysates prepared from these cells were used for Western blotting. B, Inhibition of Akt phosphorylation by LY294002. PC-3M-MM2 cells were treated with LY294002 (10 µM), a PI3 kinase inhibitor, for 24 h and used for Western blotting to assess Akt activation. C, Akt inhibition reduced miR-21 levels as determined by microRNA array analysis. PC-3M-MM2 cells were treated with vehicle or LY294002 (10 µM) for 24 h, after which total RNA was isolated. The RNA samples were then subjected to microarray analysis of miRs. The miRs which are known to be regulated in prostate cancer [19] are shown using Affymetrix gene chip. Panels compare miRNA profile of vehicle and LY294002-treated PC-3M-MM2 cells. The intensity of red and green color indicates the extent of up- and down-regulation of miRNAs, respectively. Similar studies were performed on three independent samples for each treatment groups. D, LY294002 increased PDCD4 levels. PC-3M-MM2 cells were exposed to LY294002 (10 µM) for 24 h and used for Western blotting. E, LY294002 increased PDCD4-luciferase activity. Plasmid containing PDCD4 3′-UTR downstream of the luciferase gene coding sequence was transfected into the PC-3M-MM2 cells for 48 h. The cells were then treated with LY294002 (10 µM) for 24 h, after which cell lysates were prepared and subjected to luciferase assay. Data in bar graph are presented as the mean±SEM of at least 3 independent experiments. Asterisk (*) indicate statistically significant difference (p<0.05) from vehicle-treated cells.