Figure 2.

miRNA-132 directly targets CACT for downregulation. A: Gene expression profiling in INS-1 832/3 cells following miRNA overexpression. Differential expression (DE) resulting from miRNA-132 is plotted against DE for miRNA-212 24 h after overexpression of the miRNAs using oligonucleotides (P < 0.05). Larger circles indicate greater statistical significance of DE. Axes show the log₁₀ of the fold-change (miRNA over negative control). Negative values indicate downregulation of expression. CACT is highlighted as the most downregulated gene in response to both miRNAs. B: miRNA-132 decreases CACT protein level. Western blot analysis was used to evaluate the effect of miRNA-132 on CACT protein levels in INS-1 832/3 cells. Ads were used to overexpress either miRNA-132 or GFP. Cells were harvested 48 h after infection, and 25 µg of protein was loaded in each lane; Vdac protein was used to normalize loading. Blot is representative of three independent experiments. C: CACT is the direct target of miRNA-132. INS-1 832/3 β-cells were transfected with a dual luciferase reporter construct that contained the 3′-UTR for the rat CACT gene, and one of three viruses (MOI 10): Ad-GFP, Ad-miR-375, or Ad-miR-132 (n = 3 experiments for each condition). After 48 h, luminescence in the cells was measured. *P < 0.05; **P < 0.01.