Fig. 3

Validation of direct targeting by 3’ UTR luciferase assay and Taqman qRT-PCR. a Venn diagram containing genes that were found to be significantly downregulated in both MCF10A and MDA-MB-231 cells upon overexpression of has-miR-140-3p or 5’isomiR-140-3p to at least 65 % of the expression in control transfected cells. b and c MCF7 cells were transfected with the miRNA mimics and wildtype (b) or mutated (c) psiCHECK2 3’ UTR reporter plasmids as indicated. 72 h later, cells were lysed and the activity of renilla (480 nm) and firefly (560 nm) luciferase were measured. Renilla measurements were normalized to firefly and values were normalized to the negative control (mimic-ctrl2). Afterwards, values were normalized to the empty psiCHECK2 transfected with 5’ isomiR-140-3p or hsa-miR-140-3p. Bars represent the average of 6 biological replicates ± standard deviation indicated as error bars. d MCF10A and MDA-MB-231 cells were transfected with hsa-miR-140-3p, 5’ isomiR-140-3p or miRNA mimic negative control (mimic-ctrl2). 72 h later, cells were lysed and total mRNA was isolated and purified using RNeasy kit (Qiagen). The mRNA expression levels of the candidate genes were then assessed by Taqman qRT-PCR. Gene expression was normalized to HPRT and GAPDH housekeeping genes. Normalized gene expression is depicted as relative expression to cells transfected with mimic-ctrl2. Values represent the mean of three biological replicates (*** P ≤ 0.001, ** P ≤ 0.01, ns = non-significant compared to control, unpaired t test)