FIGURE 4.

miR-144 regulates agrin expression by directly targeting the 3′ UTR of agrin mRNA. (A) Schematic representation of three putative miRNA-binding sites on the agrin 3′ UTR sequence. (B) The 3′ UTR fragment of agrin was cloned into the Xbal restriction site downstream of the firefly luciferase gene of the pGL3-Basic vector. Luciferase activity of each sample was measured 48h after transfection and normalized to Renilla luciferase activity. pGL3-Basic vector was used as control. Graph error bars indicate SD calculated from at least three independent experiments. *P < 0.05, compared with the control. (C) Co-transfection of pSuper-144 with the firefly luciferase reporter gene linked to the wild-type and mutant-type sequence of miR-144 binding site within the agrin 3′ UTR. Luciferase activity was measured and normalized to phRL-TK activity. Three independent experiments were performed and data are presented as mean ± SD. *P < 0.05, compared with the control. (D) Protein level of agrin was assayed by western blotting. GAPDH was used as an internal control. (E) Protein expression was normalized to GAPDH. *P < 0.05, compared to the control group.