Figure 5.

DDX11 suppresses p21 via enhance the protein stability of EZH2 in HCC cells. (A) GSEA indicated that EZH2 signaling was activated in patients with high expression of DDX11. (B) In TCGA cases, a positive correlation between DDX11 mRNA and EZH2 mRNA expression was found. (C) The protein expression of DDX11 was associated with EZH2 protein expression in 24 fresh HCC specimens in SYSUCC cohort. (D) HepG2 and PLC8024 cells were transfected with DDX11 shRNA or overexpression vectors. The mRNA expression of EZH2 was examined by qRT-PCR. (E) The expression of DDX11, EZH2 and p21 in stable cells with DDX11 knockdown or overexpression was examined by western blot. (F) DDX11-expressing cells were transfected with EZH2 siRNA for 36 h. The effect of EZH2 on DDX11-mediated p21 suppression was tested. (G) The protein binding of EZH2 and DDX11 was confirmed by co-IP experiments. (H) The EZH2 protein stability was measured by CHX treatment in cells with DDX11 knockdown. *P < 0.05, **P < 0.01. (I) Cells were cultured with 20 mg/L CHX for indicated time, with or without pretreatment of MG132 (20 μM). The protein degradation of EZH2 was determined by western blot. (J) HCC cells with or without DDX11 knockdown were transfected with Ub for 48 h. The ubiquitination of EZH2 protein was examined by co-IP mediated by Ub antibody. (K) The role of EZH2/p21 axis was assessed by rescue experiment, using colony formation. *P < 0.05.