Tissue culture validation was performed using HeLa cells to demonstrate whether two of the putative MRE sequences predicted by multiple algorithms on BACE1 are recognized physiologically by miR-107. These results indicate that, of the two tested putative MREs, one (B1) is physiologically relevant but not the other (B2). For this experiment, six different MREs were subcloned into pRLTK expression plasmids, along with PGL-3 control plasmids, to understand whether the MREs (designated B1 and B2) are changed as a result of endogenous miRNAs. Furthermore, siRNAs were transfected that mimic miRNAs. B1 and B2 each were compared versus appropriate controls that were matched except for a small shifting of nucleotides at the predicted seed-pair region. In addition, known positive and negative controls were used, namely LIN-28 and M2, the former a very strong MRE recognized by endogenous let-7 miRNAs as described previously (Kiriakidou et al., 2004, 2005). The bar signals shown to the right are the result of six different experiments (n = 3 performed twice); the signals represent the Renilla luciferase signal that has been corrected using the Firefly luciferase as a transfection efficiency control. Note that miR-107 siRNAs cause a statistically significant additional drop in the reporter construct expression, but miR-320 actually caused an apparent lack of inhibition on the B1 MRE. For C, Student’s t tests were performed. ##p < 0.0001; #p < 0.01. For the siR-107 experiments, # refers to the statistical significance of the difference in the overall reporter decrease with, versus without, siR-107 transfection.