Figure 5.

Silencing of Validated Target Genes by Adenovirus-Encoded miRNAs

(A) Time course of mature miRNA expression in MDA-MB-231 cells infected with AdΔ24E3-U6.pri-miR-1 or AdΔ24E3-U6.pri-miR-26b. Data shown are the individual results and means of three independent experiments, each measured in duplicate, and are given relative to uninfected cells. (B) Analysis of mRNA knockdown of miR-1 targets FOXP1 and MET and of miR-26b target PTGS2 in MDA-MB-231 cells infected with pri-miRNA-expressing adenoviruses. Data are from 5 or 3 independent experiments, each measured in duplicate, and are normalized to the expression measured in irrelevant virus-infected cells. Paired t test was used to test for significance of mRNA knockdown. (C) Comparison of collateral inhibition of gene silencing in cells infected with miRNA-expressing adenovirus. MDA-MB-231 cells were infected with AdΔ24E3-U6, AdΔ24E3-U6.pri-miR-1, or AdΔ24E3-U6.pri-miR-26b, and expression of non-target genes was quantified. Data are from 3 independent experiments, each measured in duplicate, and are normalized to the expression measured in uninfected cells. (D) Analysis of miRNA-mediated silencing of FOXP1 by measuring luminescence in an HCT116 reporter cell line carrying a FOXP1-3′UTR-RenSP plasmid, at 24 and 32 h after infection with miR-1 precursor-expressing adenoviruses or transfection with miR-1 mimics. Data shown are means + SD from three independent experiments done in triplicate. Repeated-measures one-way ANOVA was used to test for differences between groups. The results shown are from the analysis done on the data at 32 h after treatment. (E) Analysis as in (D), measuring luminescence 24 h after infection with the pri-miR-1 or pri-miR-26b-expressing adenovirus or transfection with miR-1 mimics. Data shown are means + SD from a representative experiment done in triplicate.