Figure 8. Aurora-A upregulates miR-21 transcription via NF-κB in HCC cells.
(A) Western blotting detection of nuclear or cytoplasmic IκBα protein expression in SMMC-7721/shAurora-A (or SMMC-7721/shcontrol) or HepG2/Aurora-A (or HepG2/control) cells. Topo I or GAPDH was used as an internal control, respectively. (B) The activity of NF-κB was measured in SMMC-7721/shAurora-A (or SMMC-7721/shcontrol) or HepG2/Aurora-A (or HepG2/control) cells using a luciferase reporter system. (C) The promoter of miR-21 was activated by Aurora-A. SMMC-7721/shAurora-A (or SMMC-7721/shcontrol) or HepG2/Aurora-A (or HepG2/control) cells were treated with pGL3/miR-21-promoter or pGL3-Basic, respectively. (D) ChIP assays with anti-NF-κB/p65 antibodies showed binding of NF-κB to the promoter of miR-21 in SMMC-7721/shAurora-A (or SMMC-7721/shcontrol) or HepG2/Aurora-A (or HepG2/control) cells. The relative occupancies of NF-κB/p65 are indicated as vertical bars. The bar graphs show the averages of three independent ChIP experiments. (E) qRT-PCR and Western blotting detection of p65 mRNA and protein expression in SMMC-7721 transiently transfected with control/siRNA, p65/siRNA1 or p65/siRNA2, respectively. (F) qRT-PCR detection of miR-21 expression in SMMC-7721 cells transiently transfected with control/siRNA or p65/siRNA1, or HepG2/Aurora-A (or HepG2/control) cells or co-transfection with control/siRNA or p65/siRNA1, respectively. U6 was used as an internal control. (G) MTT analysis of IC50 values of ADR or CDDP in HepG2/Aurora-A (or HepG2/control) or co-tranfection with control/siRNA or p65/siRNA1, respectively. (H) Western blotting detection of above apoptosis-related proteins in HepG2/Aurora-A cells or co-transfection with control/siRNA or p65/siRNA1, respectively. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. N.S, P>0.05; *P<0.05; **P<0.01.