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. 2013 Jun 26;89(2):25. doi: 10.1095/biolreprod.113.110049

FIG. 3.

FIG. 3

MicroR-424 targets a discrete 3′UTR sequence in FGFR1 and MAP2K1 genes. A) Schematic representation of the predicted miR-424 MREs within the 3′UTR of the FGFR1 mRNA. Alignments between the miR-424 binding sites and miR-424 are shown. B) Deletion analysis of FGFR1 3′UTR. HTR8/SVneo cells were transfected with the wild-type FGFR1-3′UTR reporter (WT) or with a promoter that harbors a mutation in each of the two miR-424 binding sites, alone (Δ1 or Δ2) or in combination (Δ1,2). Each reporter was transfected along with an empty vector (pcDNA3) or a vector expressing miR-424. C) Schematic representation of a conserved miR-424 MRE within MAP2K1 (site 1) and of a putative miR-424 8-mer site (site 2) in the sequence of MAP2K1P1. Alignments between miR-424 and MAP2K1 and MAP2K1P1 are shown. Asterisks indicate the diverging nucleotides in the seed region between the sequences of MAP2K1 and MAP2K1P1. D) Luciferase reporter assay using MAP2K1-3′UTR and MAP2K1P1 reporter that harbors a mutation in each of putative binding sites (Δ1 or Δ2) or wild-type (WT) MRE. Relative luciferase unit (RLU) activity from each reporter, normalized to firefly luciferase activity, was determined 48 h later. Experiments using MAP2K1 (left panel) were performed seven times (n = 7), and statistical significance was determined using a linear mixed-effects model with the experiment batch as the random effect. Using this method, MAP2K1 WT was repressed in the presence of transfected miR-424 by about 17.45%, with P = 3.28 × 10−14 (**). Data derived from the MAP2K1P1 construct are geometric means of three independent experiments performed in duplicate. Statistical significance was determined by the Student t-test (SEM). *P < 0.01.