Figure 9

Par-4 overexpression modulates translation of apoptosis-associated genes and sensitizes HT29 cells to 5-FU-mediated apoptosis via miR-34a regulation of BCL2 expression. (A) Post-translational regulation of apoptosis and anti-apoptosis gene expression by microRNAs upon Par-4 overexpression. Western analysis was performed to assess levels of anti-apoptotic proteins (BCL2, MCL1 and SGK1) and apoptotic proteins (Bim, p27 and VDAC1) targeted by the corresponding differentially expressed microRNAs in empty vector- and Par-4-transfected cells. Note that both miR-221 and miR-222 are predicted to target BCL2L11 (encoding Bim) and CDKN1B (p27). Heat map represents the relative microRNA expression levels between empty vector- and Par-4-transfected cells. (B) Western of BCL2 in empty vector- and Par-4-transfected cells treated with or without miR-34a antagomir. Par-4-overexpressing and empty vector-expressing cells were transfected with scrambled or miR-34a antagomir. Transfected cells were harvested at 0, 24, and 72 h to assess the effect of inhibiting miR-34a on BCL2 protein levels. Quantification of relative BCL2 protein levels were calculated from BCL2-to-actin ratios. Results are the mean ± S.D. of 3-4 independent experiments. *Significantly different from 0 h following miR-34a antagomir treatment (P < 0.05). (C) Inhibition of miR-34a in Par-4-overexpressing HT29 cells reverses apoptotic sensitization to 5-FU treatment. Empty vector-expressing cells transfected with scrambled antagomir (Vector with Scrambled), Par-4-overexpressing cells transfected with scrambled antagomir (Par-4 with Scrambled) and Par-4-overexpressing cells transfected with miR34a antagomir (Par-4 with anti-miR-34a) for 24 h were treated with 100 μM 5-FU for an additional 24 h and assayed for apoptosis. Results are the mean ± S.D. of 4-5 independent experiments. ^#Significantly different from Par-4 with Scrambled (P < 0.05).