Figure 4

Chromatin modifying genes may be targets of miRNA-mediated expression in DLBCL. miR-10393-3p is involved in miRNA:mRNA interactions with chromatin modifiers MLL2/KMT2D and EP300. (a, b) miRNA and mRNA display anti-correlated expression patterns and the mRNA has a predicted binding site for miR-10393-3p (M10393). Orange dots represent centroblast libraries, red dots represent DLBCL libraries with a somatic mutation in MLL2/KMT2D or EP300, respectively, and blue dots represent DLBCL patient samples without the mutation. The boxplots to the top and right of each scatter plot summarize miRNA and mRNA expression in DLBCL (‘D’) and Centroblasts (‘C’); (c, d) Top: Schematic representations of the putative miR-10393-3p binding sites on MLL2/KMT2D or EP300. Putative seed regions within each site are underlined and in red font. Bottom: Dose response of miR-10393-3p miRNA activity in HEK-293 cells was assessed using a psiCHECK2 dual luciferase reporter construct containing each of the putative MLL2/KMT2D or EP300 binding sites. Activity is measured as Renilla luciferase normalized to Firefly luciferase to control for transfection efficiencies. The data were shown as normalized relative luciferase units (RLU) with respect to the corresponding dose of the control mimic and are representative of three independent experiments (mean ± SEM). Statistically significant comparisons between the co-transfected M10393 miRNA and the NC2 control for the perfect binding reporter vector are noted over the solid colored bars. Statistically significant comparisons between perfect binding and mismatch constructs are indicated above double-headed arrows. *P <0.05. White bars, NC2 negative control mimics; Solid colored bars, M10393 mimics on perfect binding (PB) sites; Striped colored bars, M10393 mimics on mismatched (MM) sites.