Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease
Figure 3
Endogenous expression of p53 in STHdhQ7/HdhQ7 and STHdhQ111/HdhQ111 cells: decreased miR-125b target p53.
(A) Representative Western Blot showing increased p53 protein level in STHdhQ111/HdhQ111 cells compared to STHdhQ7/HdhQ7 cells; (B) Average integrated optical density (IOD) of p53 protein bands in A, normalized to β-actin level (n = 3, p = 0.024) in these cell lines. (C) Relative luciferase activity of cloned p53-3′UTR with miR-125b binding site (denoted by p53-UTR1) in STHdhQ111/HdhQ111 cells compared to STHdhQ7/HdhQ7 cells. Normalization of protein level between STHdhQ111/HdhQ111 cells and STHdhQ7/HdhQ7 cells was done by taking the ratio of RLU of cloned construct i.e. p53-UTR1 and empty vector pmiR. Relative luciferase activity of p53-UTR1 was found significantly higher (n = 3, p = 0.026) in STHdhQ111/HdhQ111 cells compared to STHdhQ7/HdhQ7 cells; (D) Reduced luciferase activity of p53-UTR1 co-transfected with pre-miR-125b in STHdhQ7/HdhQ7 cells (n = 3, p = 0.024) and STHdhQ111/HdhQ111 cells (n = 3, p = 0.0086) compared to those obtained in respective empty vector U61 tansfected cells; (E) Representative Western Blot showing reduction in p53 protein level in STHdhQ111/HdhQ111 cells 72 hours following transfection with pre-miR-125b compared to STHdhQ111/HdhQ111 cells transfected with empty vector U61, average IOD compared to β-actin (n = 3, p = 0.039) is shown in the adjacent bar diagram.