Figure 6.

MiR-146a targets the Notch signaling inhibitor NUMB. A and B: miR-146a binds the 3’-UTR of NUMB transcript to inhibit NUMB translation. The miR-146a binding site in NUMB 3’-UTR was aligned with the miR-146a sequence (A). The wild type (WT) NUMB 3’-UTR or its mutant defective in miR-146a binding (MUT) was cloned into a luciferase reporter vector. The reporter plasmids were transfected into the MCF-10A cells stably expressing miR-146a or miR control (described in Figure 4C and 4D) for luciferase activity analysis (B). *P < 0.05 compared to column 1. C and D: NUMB expression is inversely correlated with miR-146a expression and invasiveness in breast cancer cell lines. Whole cell lysates were used for anti-NUMB blotting with β-actin as a loading control (C). The expression of the endogenous miR-146a (green dots) and NUMB protein (red) in the indicated breast cancer cell lines was determined by FISH-TSA as described in the Materials and Methods (D). The data represent the mean ± S.E. of at least three independent experiments in triplicate.