Figure 3

MiR-146a*, miR-524-5p and miR-582-3p regulate the expression of NFL mRNA 3′UTR. Relative quantitative RT-PCR was performed after the co-transfection of HEK293T cells with pre-miRNAs whose function was correlated with the differential expression observed sALS and a reporter construct expressing firefly luciferase coupled to NFL 3′UTR-S (white bar), -M (grey bar) or -L (black bar). Firefly luciferase was normalized to Renilla luciferase, the impact of each miRNA on the luciferase mRNA without NFL 3′UTR (pmirGLO vector control), and the effect of miR-let-7a (miRNA control) on the luciferase mRNA to obtain the specific effect on the NFL mRNA 3′UTR. Data are expressed as relative mRNA level change and show up-regulation as positive values and down-regulation as negative values. Experiments were performed in triplicate. Result are shown as mean ± SEM (t-test: *** = p < 0.001; ** = p < 0.01, relative to the pmirGLO vector control). Note that only results for those miRNAs that showed RT-PCR results in agreement with the reporter gene assay data are presented.