Fig. 3

In silico prediction of 3’UTR secondary structures and exemplary validation miRNA cooperative effects. A, C: RNA secondary structures were predicted for LEMD3 (A) and RECK (C) 3′UTR sequences that showed cooperative binding of miR-21-5p and miR-155-5p as determined by the HiTmIR dual luciferase assays. Corresponding 3′UTR sequences (as cloned for the underlying assays) are shown in a linearized (upper panel; 5′→3′) and in a folded representation (lower panel), respectively. The included miRNA binding sites are indicated. B, D For the validation of miRNA cooperative effects, miR-21-5p and miR-155-5p binding sites were mutated within pMIR-LEMD3 (B) and pMIR-RECK (D) 3′UTRs. The reporter constructs were tested by HiTmIR dual luciferase reporter assays. Comparative measurements were conducted with both, the wild type and mutated reporter constructs. MiRNA induced effects were determined upon overexpression of only miR-21-5p or only miR-155-5p or upon co-expression of both miRNAs. 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 three independent experiments that were conducted in technical duplicates. Statistical evaluation of the cooperative effects was performed in comparison to the effects upon single miRNA overexpression of miR-21-5p or miR-155-5p (black asterisks). Significant p-values after FDR adjustment are denoted (*p ≤ 0.05, **p < 0.01, ***p < 0.001)