Fig. 4

Reporter gene assay determined interactions of miR-142-5p and miR-365a-3p with cKIT 3′-UTR. Vectors containing cKIT 3′-UTR were co-transfected with pre-miR-142-5p and pre-miR-365a-3p, each in concentrations 10 nM and 25 nM. Measurement of reporter gene activities was performed 48 h after transfection. a Relative reporter gene activity of wild type cKIT 3′-UTR (WT) was repressed by miR-142-5p by 39% [10 nM] and 52% [25 nM]. b Predicted interactions of miR-142-5p within the binding region of cKIT 3′-UTR are illustrated here. c Co-transfection of cKIT 3′-UTR with miR-365a-3p resulted in repression of relative reporter gene activity by 24% [10 nM] and 24% [25 nM]. d The introduction of mutations in the binding region at positions 1143–1146 bp (MUT 1) slightly abolished effect on reporter gene activity. e Additional introduction of mutated bases at positions 749–754 bp (MUT 2) led to neutralization of the inhibitory effects of miR-365a-3p. f Predicted interactions of miR-365a-3p with cKIT 3′-UTR are depicted with mutated bases highlighted in gray. All reporter gene activities (n ≥ 12 in 3 independent experiments) were normalized to activities of cells transfected with respective 3′-UTR target sequence vectors and pre-miR negative control (median ± interquartile range). Activities are shown relative to empty control vector (c) identically transfected and normalized as 3′-UTR target sequence vectors. Mann–Whitney U-test; **p ≤ 0.01, ***p ≤ 0.001