Figure 4.

Confirmation of bioinformatic predictions of miR targets. Human immortalized myoblasts (iMyo) cultivated in growth medium (P) or differentiation medium for 3 days (D) were transfected with either control LNA (anti-miR-C) or LNA against miR-1 and miR-206, miR-133a and miR-133b (A). Human rhabdomyosarcoma cells (RD) were transfected with plasmids coding for miR-128 and miR-30 precursors or scrambled sequence (scr) (B). Then the expression of corresponding microRNA and their randomly selected target genes was tested using qRT-PCR, normalization was performed using ΔΔCt method using GAPDH as a control gene and proliferating sample #1 as a reference sample (expression level 1). Transcriptome-supported target genes of miR-1/206 (C) and miR-133a/b (D) were downregulated during normal myogenic differentiation but not when it was accompanied by the transfection with corresponding anti-miRs. Transcriptome-supported target genes of miR-128 and miR-30 were downregulated when human rhabdomyosarcoma cells were transfected with lentiviral constructs overexpressing miR-128 (E) and miR-30 (F). The average of three independent experiments is shown. (*) indicates p-value <0.05; (G): Diagram showing the proportion of supported predictions that were qRT-PCR validated in this study. Black: validated targets, gray: non-validated targets.