There is a miR-24 binding site in the 3′-UTR of hALK4 mRNA. (A) Sequence alignment between miR-24 and its 2 putative binding sites in the 3′-UTR of ALK4 mRNA from different species. One lies in 445-467 bp downstream of the stop codon of the 3′-UTR of hALK4 mRNA and the other in 985-1011 bp. (B) HEK293 cells were transfected with luciferase reporter containing the hALK4 3′-UTR with wild-type or mutated target sites (shown in A, M1, and M2), along with empty or miR-24 vector. Luciferase assay was performed 48 hours after transfection. Reporter assay was performed in triplicate and the data represent the mean plus or minus SD of 3 independent experiments after normalized to R reniformis activity. The asterisk indicates a significant difference between miR-24 and control vector (P < .05).