Figure 3

miR-486 is essential for normal myoblast fusion, cell cycle kinetics and viability in human skeletal myoblasts. (A) and (B) Myoblast fusion assay reveals significantly lower fusion (shown as Fusion Index % of cells with 2 or more nuclei) in miR-486-inhibited (anti-miR-486) cells. Normal and DMD myoblasts were infected at 50% confluency with lentivirus expressing miR-486, miR-486 inhibitor (anti-miR) or a scrambled miRNA control virus and allowed to differentiate to 90% confluency. Note the decreased levels of fusion in both the normal and DMD myoblasts at two time points (day 2 (Figure 2A) and day 4 (Figure 2B) of differentiation) when infected with anti-miR-486. (C) Increased cellular apoptosis measured using a caspase 3/7 enzymatic assay in normal and DMD myoblasts overexpressing (miR-486) or inhibiting (anti-miR-486) miR-486. DMD myoblasts overall had significantly higher caspase 3/7 activity compared with normal human myoblasts across all samples. The y-axis indicates average RLUs (that is, caspase 3/7 activity), and the x-axis indicates the condition of the myoblasts. Mock and scrambled miRNA-GFP-infected myoblasts served as negative controls (n = 3 replicates/cohort). Cells treated with hydrogen peroxide (H₂O₂) for 12 hours served as positive controls. (D) Ki-67 levels measured by immunofluorescence in normal and DMD myoblasts which have increased miR-486 expression, reduced miR-486 expression or expression of scrambled miRNA (negative control). Note the decreased levels of Ki-67 in myoblasts in which miR-486 expression is knocked down. *P < 0.005 and **P < 0.05 for comparisons between scrambled miRNA (control) versus miR-486- and/or anti-miR-486-infected myoblasts.