Fig. 3.

BACE1 is a miRNA target gene. (A) Schematic representation (not to scale) of the BACE1 3′UTR luciferase construct. TK, thymidine kinase promotor. Luc, luciferase gene. (B) BACE1 3′UTR (wt or mutant, see C) luciferase and Renilla luciferase constructs were transfected into HeLa cells with the indicated miRNA oligonucleotides at a final concentration of 75 nM. Normalized (to Renilla) sensor luciferase activity is shown as a percentage of the control with a scrambled oligonucleotide. Error bars represent standard deviations derived from three or more independent experiments performed in duplicate. Statistical significance between control (scrambled miR-treated) and candidate miR-treated HeLa cells was determined by a Wilcoxon test (*, P < 0.05, **, P < 0.01). (C) The sequence and putative binding sites of the miR-29a/b-1 family are shown. The miR-29a/b-1 seed sequence is highlighted in gray. In the hBACE1 3′UTR MUT construct, the binding site for miR-29a/b-1 is mutated as indicated. (D) Western blot analysis of endogenous BACE1 (probed with C-terminal antibody), Nicastrin (used as additional control) and β-actin in SK-N-SH cells treated with 75 nM (final concentration) of miR-29a/b-1. Note that cell extracts were treated with PGNase F (Roche) overnight to remove N-linked sugars before immunoblot analysis explaining the lower molecular weight of BACE1 and Nicastrin. Densitometric quantifications of BACE1 (shown as fold difference) are shown.