miR-381 and miR-539 target the 3′-UTR of MITF. (A) Schematic representation of the MITF 3′-UTR with the miR-381 and miR-539 binding sites. Luciferase reporter constructs with truncated fragments of the 3′-UTR shown with the lengths depicted. (B) Overexpression of miR-381 and miR-539 (miR cluster) inhibits luciferase activity in 3T3 cells. *P < .05. **P < .005. (C) Knockdown of the miRNA endonuclease, Dicer, results in increased luciferase activity with constructs containing the MITF 3′-UTR. *P < .05. (D) Western blot shows knockdown of Dicer expression with the sh-RNA-expressing lentivirus. MITF protein is increased in Dicer knockdown cells. (E) miR-381 and miR-539 specifically target the miR binding sites in the MITF 3′-UTR. Luciferase reporters with miR-381 and miR-539 binding sites show decreased activity with overexpression of miRs (miR cluster). Luciferase reporters with either miR-381 (Δ381) or miR-539 (Δ539) singly deleted also show decreased activity with overexpression of miRs. Luciferase reporter with and miR-381 and miR-539 binding sites deleted (ΔΔ 381-539) are not repressed by miR cluster overexpression. *P < .05. **P < .005. (F) PCR analysis shows overexpression of miR-539 and miR-381 in miR cluster transduced 3T3 cells. miR-17 is not significantly affected. P values are calculated with Student t test; all experiments are performed in triplicate. Luciferase activity is normalized to Sea Pansey luciferase activity