Figure 5

Pairing of the 3′ and 5′ end of miR-503 is necessary for direct targeting by miR-503 of the proto-oncogene DDHD2. (A) Pairing by the 3′ and 5′ end of miR-503 to the 3′UTR of DDHD2 is necessary to repress expression of the luciferase reporter construct. The Y-axis denotes relative luciferase units from miR-503 transfected HEK293 cells normalized to control RNA transfected cells. The X-axis indicates the type of 3′UTR (either wild-type or deletion as indicated) Error bars denote ± SD, n = 3. In the diagram, vertical lines indicate base pairing, dashes show deleted base pairs, and numbers in parenthesis denote location in the 3′ UTR, not genomic coordinates. P-values were calculated by Student’s one tailed t-test comparing miRNA normalized to Control siRNA luciferase activity with intact 3′ UTRs, to normalized activity with the respective miRNA target site deleted 3′ UTRs. *P < 0.05. (B) miR-503 transfection decreased protein expression of DDHD2. DDHD2 protein levels following miR-503 transfection in fibroblasts and HeLa cells compared to Control siRNA transfection. Band intensities were quantified, normalized to GAPDH, and shown relative to the Control siRNA.