Induction of endogenous RelA by miR-324-3p.

When PC12 cells were transfected with 150 nM of premiR-324-3p, there was a significant induction of RelA mRNA expression compared to control premiR treated group at 3 days after transfection (A). Ago2 siRNA treatment prevented the RelA induction by miR-324-3p. The house-keeping control 18S rRNA expression was not changed by premiR-324-3p transfection or Ago2 knockdown (A). PremiR-324-3p transfection also led to significant increase in the RelA protein levels compared to control miR treated or vehicle treated control groups (B). Treatment with Ago2 siRNA prevented the premiR-324-3p mediated increase in RelA protein levels (B). The levels of β-actin used as a loading control were not affected by premiR-324-3p or Ago2 siRNA (B). The efficiency of Ago2 siRNA to knockdown Ago2 protein levels in PC12 cells in the presence and absence of premiR-324-3p and control miR was confirmed by Western blotting. The Ago2 siRNA knocked-down Ago2 protein levels by >85% in all the 3 groups (treated with vehicle or control miR or premiR-324-3p) compared to vehicle treated group (C). PremiR-324-3p transfection induced significant activation of caspase-3 as measured by cleaved caspase-3 levels in PC12 cells which was prevented by Ago2 knockdown (D). Bars represent mean ± SD of n  =  4/group of triplicate determinations. * p<0.05 compared with the control siRNA group (Student’s t-test).