FIG 3.

miR-195 associates with Igf2r mRNA. (A) Schematic representation of the Igf2r mRNA depicting the target site for miR-195 in its 3′ untranslated region (3′ UTR). Alignment of the Igf2r mRNA sequence with miR-195 is shown. Top strand, Igf2r mRNA; bottom strand, miR-195. (B) Levels of biotinylated miR-195 24 h after transfection. Values represent means ± SEM of results from three separate experiments. *, P < 0.05 (compared with cells transfected with control scramble oligomer). (C) Binding of biotinylated miR-195 to mRNAs encoding IGF2R, IGFBP5, and STIM1: (a) levels of mRNAs in the materials pulled down by biotin-miR-195; (b) levels of total input mRNAs. (D) Levels of reporter activities as measured by analysis of the Igf2r 5′-UTR, various fragments of CR, or 3′-UTR luciferase (Luc) reporters after ectopic overexpression of miR-195. (Left) Schematic of plasmids of different chimeric firefly Luc-Igf2r reporters. At 24 h after transfection with pre-miR-195, cells were transfected with different Igf2r luciferase reporter plasmids. The results were expressed as the mean ± SEM from three separate experiments. *, P < 0.05 (compared with cells transfected with control scrambled oligomer). (E) Effect of deletion of miR-195-binding site (schematic) on luciferase reporter activity after ectopic miR-195 overexpression. WT, wild type; Mut, mutant.