MiR-24 inhibited reporter activity through the 3′-UTR of hALK4 mRNA. (A) Schematic representation of the luciferase reporter constructs. The CMV promoter and the full-length 3′-UTR of hALK4 mRNA were inserted to pGL3-basic vector as indicated. (B) HEK293 cells were cotransfected with the hALK4–3′-UTR luciferase reporter (0.3 μg) and miRNA (1 μg) as indicated. At 48 hours after transfection, cells were harvested for luciferase assay (top panel). The expression of the 4 miRNAs was detected by stem-loop RT-PCR with U6 as loading control. The asterisk indicates a significant difference between miR-24 and the control (P < .05). (C) HEK293 cells were cotransfected with the hALK4–3′-UTR luciferase reporter and increasing amounts of miR-24 or miR-205 (1 μg). (D) Sequence of human miR-24/miR-189 stem-loop. (E) Detection of the miR-189 and miR-24 expression by Northern blot analysis in HEK293 cells transfected with the miR-24/miR-189 precursor vector. Con-miR, a vector expressing a nonspecific small RNA molecule (5′-AGCGGACTAAGTCCATTGCTT-3′) as a negative control miRNA. 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.