Control of Myc expression by miRNAs. (A) Potential target sites for mature miRNAs in the mouse Myc 3′-UTR. Only mature miRNAs produced by miRNA genes down-regulated in γ-irradiation-induced lymphomas are shown in this scheme. Gene nomenclature is also indicated when different from the mature form (eg, mir-125b-1 is the gene that generates miR-125–3p). (B) Luciferase activity of a reporter construct carrying the Myc 3′-UTR downstream of the luciferase gene. The construct was cotransfected with a vector expressing each of the indicated miRNA precursors. All data are normalized versus the luciferase levels generated by scramble sequences. (C) Effect of miRNA genes on Myc protein levels. Transfection with miRNA genes was performed as described earlier in the Figure 3 legend, but cells were processed for immunoblot analysis for Myc or 2 different Myc targets, Mcm4 and Cyclin B1. A vertical line has been inserted to indicate repositioned gel lanes. The relative levels of Myc proteins were normalized using α-tubulin (α-tub.) as a loading control. (D) Luciferase activity of wild-type (wt) or mutant (mut) Myc 3′-UTRs in the presence of scrambled sequences (S) or the corresponding miRNAs. wt, indicates wild-type Myc 3′-UTR sequence; mut, single mutants for the indicated miRNA; and mut2, double mutant for the miR-125b-3p target sites. (E) Luciferase assays of wild-type Myc 3′-UTRs in the presence of pools of group 1 (G1; mir-132, mir-125b-1, let-7e, let-7-a, and mir-154), group 2 (G2; mir-301a, mir-148a, and mir-134), or group 3 (G3; mir-26b, mir-150, mir-207, and mir-223) miRNAs. In these pools, the sum of all miRNA vectors also equals 10 μg as in the scramble vectors or the previous assays. (F) Effect of G1–3 pools on Myc protein levels. Transfection with miRNA genes was performed as described earlier in the Figure 3 legend, but cells were processed for immunoblot analysis for Myc. The relative levels of Myc proteins were normalized using α-tubulin (α-tub.) as a loading control.