Figure 4

Effects of miR-29 on target genes CDK6 and PI3K. (A,B) relative mRNA and protein expression levels (REL) of miR-29 target genes CDK6 (dark grey) and PI3K (light grey), assessed 24h, 48h and 72h after mimic/inhibitor transfection compared to NC-mimic/NC-inhibitor controls; graphs show means of biological triplicates ± SD. (C) Down-regulation of miR-29 target proteins CDK6 and PI3K is observed after IFN-γ stimulation of melanoma cells. (D) Schematic overview of CDK6 and PI3KR1 luciferase constructs with positions of conserved miR-29a binding sites predicted by TargetScan (bold) in the CDK6-3'UTR (BS1-3) and PI3KR1-3'UTR (BS) and corresponding miR-29a sequences (italics). (E) Luciferase activity in A375 cells transfected with constructs containing the positive control miR-29a full complementary sequence (29a-FC), parts of CDK6- or PI3K1-3'UTRs or CDK6 single binding sites (BS1-BS3) and miR-29a mimic or NC for 48h and 72h. Relative luciferase activity of miR-29a-transfected samples was normalized to NC-mimic-transfected control samples (luciferase activity of NC-mimic transfected samples was set to 100%). Bars show means of biological triplicates ± SD for each construct. (F) A375 and (G) FM55P cells transfected with CDK6 siRNA (black) show reduced proliferation compared to cells transfected with siRNA NC (grey). Results were reproduced at least in biological duplicates. Inserted bar diagrams show the mean confluence of at least biological triplicates at 0h and 72h. Shown are confluence ratios of si-CDK6/si-NC ± SEM. Significance was assessed by one-way ANOVA followed by a Bonferroni Post-Hoc test (A,B,E) or by a two-tailed t-test (F,G). * p<0.05, ** p<0.01 and *** p<0.001.