Fig. 2

Analysis of miRNA cooperativity by HiTmIR assays. The 3’UTR sequences of 15 putative cooperative targets of miR-21-5p and miR-155-5p were cloned into pMIR-RNL-TK reporter plasmid and analyzed by semi-automated HiTmIR dual luciferase reporter assays. MiRNA induced effects on firefly luciferase activity were determined upon overexpression of only miR-21-5p or only miR-155-5p or upon co-expression of both miRNAs. For representation, the analyzed genes were categorized into four groups regarding the distances between neighboring miR-21-5p and miR-155-5p binding sites (< 40 nt (A), 40-240 nt (B), > 240–600 nt (C), > 600 nt (D)). Relative luciferase units (RLU) were related to the activity of an empty reporter control (pMIR-RNL-TK; under the respective miRNA condition) and are shown as the mean result (± standard error of the mean (± SEM)) of four independent experiments that were conducted in technical duplicates. Statistical evaluation was performed in comparison to the empty reporter control (gray asterisks) or for the co-expressional conditions in comparison to the effects upon single miRNA overexpression of miR-21-5p and miR-155-5p (black asterisks), respectively. Significant p-values after FDR adjustment are denoted (*p ≤ 0.05, **p < 0.01, *** p < 0.001)