{A one-tailed Student's t test identified five miRNAs which significantly reduced expression of firefly luciferase by targeting the mouse Mct1 3‚Ä≤ UTR (Fig. 3A). Four of these also significantly targeted the human MCT1 3‚Ä≤ UTR (Fig. 3B). miR-124 exerted the largest effect, decreasing luciferase expression by at least 50% in assays of both mouse and human sequences (P < 0.00001). miR-29a reduced expression by ‚â•33% (P < 0.01), and miR-29b reduced expression by ‚â•27% (P < 0.01) in both cases. A smaller effect was produced by miR-29c which reduced expression by ‚â•7% (P < 0.05). Transfection with miR-7 produced a small but nonsignificant decrease in luciferase activity for both human and mouse Mct1 sequences. Thus, 4 out of the 10 miRNAs tested significantly target both human and mouse Mct1 3‚Ä≤ UTRs: miR-124 and the three isoforms of miR-29. Of these, miR-29a, miR-29b, and miR-124 were capable of reducing expression by over one-quarter. The predicted binding sites for both miR-29b and miR-124 are found in the first 150 bp of the 3‚Ä≤ UTRs (Fig. 3E). PicTar predicted that miR-29a and miR-29c may also potentially target the miR-29b site. The 6 to 8 nucleotides at the 3‚Ä≤ end of miRNA binding sites, termed the ‚Äúseed region‚Äù (10), are highly complementary to the miRNA sequence and critical for binding. In both of the putative miR-29 and miR-124 binding sites, the 8 bp of the seed region were replaced with a restriction enzyme site with minimal complementarity to the miRNA sequence (Fig. 3F). Mutation of the putative miR-124 site abolished the effect of miR-124 while leaving the action miR-29a unaffected (Fig. 3G). This confirmed that the former region is an miR-124 binding site.Mutation of the putative miR-29 binding site led to the unexpected finding that while this virtually abolished the action of miR-29b, miR-29a was still able to target the mutated 3‚Ä≤ UTR albeit with an efficacy reduced by ~25% (P < 0.05) (Fig. 3G). This finding confirmed that this region forms an miR-29b binding site, and while miR-29a does bind this site, it also acts through binding at other sites within the Mct1 3‚Ä≤ UTR.}