Figure 4

Daam1 is a direct target of miR-335. (A) Schematic representation of 3'-UTR of Daam1 mRNA with the putative miR-335 binding sequence. Mutation was introduced to the Daam1 3'-UTR sequence in the complementary site for the seed region of miR-335. (B, C) Effects of miR-335 on expression of endogenous DAAM1 protein. Western blot analysis was used to monitor DAAM1 protein expression in rat astrocytes (B) and C6 cells (C) 72 h after transfection with miR-335 mimics or negative control mimics (NC) at the indicated concentrations. (D, E) Analysis of luciferase activity. C6 cells were cotransfected with psiCHECK-2-wild type Daam1 3'-UTR/-mutant Daam1 3'-UTR (indicated as WT or MUT on the × axis), and (D) miR-335 mimics or (E) antagomir-335, luciferase activity was assayed 72 h after transfection. (F) Effects of antagomir-335 on expression of DAAM1 and phosphorylation of MLC (myosin light chain). C6 cells were transfected with 100 nM antagomir-335 or antagomir-NC, and applied for western blot analysis 72 h later. (G) DAAM1 protein is significantly downregulated in astrocytoma. Endogenous DAAM1 expression in C6 cells and human astrocytoma tissues were detected by western blot. GFAP expression was used as a biomarker of normal astrocytes. (H) Quantified protein levels of DAAM1 are shown as normalized by β-actin. Data represent the means ± SD of three independent experiments. Statistical differences compared with the controls are given as *, P < 0.05; **, P < 0.01; ***, P < 0.001.