Fig. 3.

miR-18a and miR-19a negatively regulate ESR1 expression via its 3′-UTR. (A) Schematic representation of the human 3′-UTR of ESR1 indicating potential miR-18a and miR-19a binding sites. Triangles indicate possible binding sites for other miRNAs. Asterisks below the ESR1 3′-UTR display predicted poly(A) sites using a support vector machine as described in (43). The evolutionary conservation, shown below the ESR1 3′-UTR, was generated using the UCSC Genome Browser (human genome May 2004 assembly) (B) Histogram indicating the levels of luciferase activity in HEK-293 cells transfected with the miRNAs indicated together with the wild-type 3′-UTR (3′-ESR1-wt) reporter or with a mutant derivative (3′-ESR1-mut). Data shown are means of quintuplicate experiments and error bars represent standard deviations. Asterisks denote significant differences between indicated samples (*P < 0.05; **P < 0.01; *** P < 0.001 Student’s t test for unpaired data). (C) Western blot showing downregulation of ESR1 in MCF-7 cells following transfection with miRNA precursors or scramble control as indicated. Mock denotes untransfected cells and β-actin was used as loading control.