Figure 5.

MiR-195 inhibits translation of DCLK1. A: levels of miR-195 in HEK-293 cells transfected with biotinylated miR-195 for 24 h. Values are the means ± SE (n = 3 replicates). *P < 0.05 compared with cells transfected with scramble oligomer. B: binding of biotinylated miR-195 to mRNAs encoding DCLK1 and Myc: levels of mRNAs in the materials pulled down by biotin-miR-195 (a); and levels of total input mRNAs (b). *P < 0.05 compared with scramble (n = 3 replicates). C: levels of miR-195 in cells transfected with pre-miR-195 for 48 h. *P < 0.05 compared with control (n = 3 replicates). D and E: changes in the levels of DCLK1 protein and Dclk1 mRNA in cells treated as described in C. F: newly synthesized DCLK1 protein as measured by L-azidohomoalaine (AHA) incorporation assays in cells described in C. Three separate experments were performed and showed similar results. G: distribution of Dclk1 (a) and Gapdh (b) mRNAs in each gradient fraction of polysomal profiles prepared from cells described in C. H: Levels of the Luc reporter activity 48 h after transfection with pre-miR-195. Left, schematic of different chimeric firefly luciferase reporters bearing the wild-type Dclk1 3′-UTR or its deletion mutation. *P < 0.05 compared with control (n = 3 replicates). Statistical significance was analyzed using unpaired, two-tailed Student’s t test. DCLK1, double cortin-like kinase 1; miR195-Tg, miR-195 transgenic; miRNAs, microRNAs.