Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease
Figure 4
Reduced miR-150 also targets p53 in STHdhQ7/HdhQ7 and STHdhQ111/HdhQ111 cells.
(A) Relative luciferase activity of cloned p53-3′UTR with miR-150 binding site (denoted by p53-UTR2) in STHdhQ111/HdhQ111 cells compared to STHdhQ7/HdhQ7 cells. Normalization of protein levels between the cells was done as described earlier. Relative luciferase activity of p53-UTR2 was found significantly higher (n = 3, p = 0.031) in STHdhQ111/HdhQ111 cells compared to STHdhQ7/HdhQ7 cells; (B) Fold increase (n = 3, p = 0.0056) in mature miR-150 expression detected by real time PCR using stem loop specific primers in STHdhQ111/HdhQ111 cells transfected with cloned pre-miR-150 compared to STHdhQ111/HdhQ111 cells transfected with empty vector U61, at 24 hours post transfection. Expression of miR-17-5p was used as endogenous control; (C) Reduced luciferase activity of p53-UTR2 co-transfected with pre-miR-150 in STHdhQ7/HdhQ7 cells (n = 3, p = 0.021) and STHdhQ111/HdhQ111 cells (n = 3, p = 0.04) compared to those obtained in respective empty vector U61 transfected cells; (D) Typical Western Blot showing reduction in p53 protein level in STHdhQ111/HdhQ111 cells 72 hours following transfection with pre-miR-150 compared to STHdhQ111/HdhQ111 cells transfected with empty vector U61. Average IOD compared to β-actin (n = 3, p = 0.043) is shown in the adjacent bar diagram.