Figure 3.

miR455 is part of functional miRISC in BeWo cells. (a) Schematic of the dual luciferase assay used in this study. The exact complementary sequences of miR455-3 P and -5P, or the 3′UTRs of eight potential target genes of miR455, were cloned 3′ to the renilla luciferase (RL) ORF (upper lanes). Positions of the potential binding sites of miR455-3P (red) or miR455-5P (blue) are indicated. BeWo cells were transfected and treated for 48 h with either FSK (high miR455 levels) or DMSO (low miR455 levels). (b) FSK has no effect on renilla luciferase expression. Vector with no target sequences cloned 3' of renilla was transfected into BeWo cells. After 48 h of treatment, cells were lysed and the RL and FL activities were measured. RL/FL ratios were calculated for DMSO- and FSK-treated cells. P-values were calculated by the non-parametric T-test. (c and d) Vectors with miR455-3P or -5P target sequences cloned 3' of renilla were transfected into BeWo cells. RL/FL ratios were calculated for DMSO- and FSK-treated cells. P-values were calculated by the non-parametric T-test. (e and f) The 3′UTRs of eight potential miR455 targets were cloned into the dual luciferase plasmid. One day after transfection, cells were treated for 48 h with FSK or DMSO. RL/FL ratios were calculated for DMSO- and FSK-treated cells. Percentage repression of the respective miRNA target reporters was calculated by normalizing the RL/FL ratios after FSK treatment to the RL/FL rations after control treatment. P-values were calculated by the non-parametric T-test