SMAP reveals the transcription factor NKX3.1 as a miR-155 target.

<p>Section A: Luciferase reporter assays after mutagenesis proved NKX3.1 as a miR-155 target. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-155 mimic compared with a nonsense miRNA control together with the mutated seed (pTKGhNKX3.1m) as well as wild type control (pTKGhNKX3.1) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (****: P < 0.001, unpaired t test). Section B: mRNA degradation assays by means of RT-qPCR experiments are shown detecting relative NKX3.1 levels (reference gene: GAPDH). U937 were transfected with miR-155, nonsense miRNA and NKX3.1 siRNA. RNA was isolated at 24 and 48 h post transfection. Columns show mean relative NKX3.1 transcript levels (± SD) of three biological replicates each measured in triplicates compared with nonsense transfected controls. Section C: Western Blots detecting NKX3.1 and GAPDH (reference protein) are shown using the monocytic U937 cells transfected with miR-155, nonsense miRNA and NKX3.1 siRNA. Intrinsic NKX3.1 levels are decreased after miR-155 transfection compared with nonsense controls and siRNA (48 h post transfection). The bar graph shows the luminescence-based relative quantification of protein (NKX3.1:GAPDH) of three individual biological replicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P < 0.05, unpaired t test). Transfection efficiency was evaluated by transfecting Cy3 labelled nonsense siRNA and fluorescence microscopy. While NKX3.1 translation is markedly inhibited by miR-155, cellular NKX3.1 mRNA levels remained stable after miR-155 at 24 h and decreased after 48 h suggesting slow cellular turnover rates of miR-155 directed NKX3.1 inhibition. Section D: Endogenous miR-155 expression leads to decreased luciferase activity of transfected reporters. The bar graph on the left hand side shows relative miR-155 expression in U937 cells at 5 and 24 h after LPS stimulation. The other bar graph shows that endogenous miR-155 expression results in clear repression of luciferase activity (nonsense transfected) while ectopically introduced miR-155 results in elevated repression and mutated controls remain unaffected. The columns show means of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (**: P < 0.01,***: P < 0.001, unpaired t test).