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. 2021 Feb 5;10:593293. doi: 10.3389/fonc.2020.593293

Figure 6.

Figure 6

E2F1/DDX11/EZH2 forms a positive feedback loop in HCC cells. (A) GSEA indicated that DDX11 may be a downstream target of E2F transcription factors. (B, C) HepG2 and PLC8024 cells were transfected with siRNAs for E2F family members, including E2F1, E2F2, and E2F3. The expression of E2Fs and DDX11 mRNA was determined by qRT-PCR (B) and western blot (C). (D) E2F1 was overexpressed in HCC cells. Expression of E2F1, DDX11, EZH2, and p21 was examined. (E) Dual luciferase reporter assays were performed in HepG2 cells with E2F1 overexpression or knockdown to indicate the effect of E2F1 on the activity of DDX11 promoter. **P < 0.01, ***P < 0.001. (F) ChIP assays were used to detect the enrichment of E2F1 on DDX11 promoter. *P < 0.05. (G) Correlation between DDX11 mRNA and E2F1 was determined in 24 HCC tissues (Pearson correlation analysis). (H) The positive correlation of E2F1 and DDX11 protein expression was confirmed in 303 paraffin-embedded HCC tissues. Patients with high expression of E2F1 were accompanied with more DDX11 expression. (I) Cells with E2F1 silence were transfected with DDX11 overexpression vector. Colony formation was performed to examine the role of DDX11 in shE1F1-mediated cell growth suppression. *P < 0.05. (J) Cells were transfected with E2F1 siRNA and DDX11 overexpression vector for 36 h. The mRNA expression of EZH2 was examined. ns, not significant. (K) According to the published data (GDS2445), DDX11 mRNA was downregulated in EZH2-/- cells. (L) The association of EZH2 and DDX11 was determined in TCGA cases. (M) Cells were overexpressed with EZH2 and/or knockdown of E2F1. The mRNA expression of DDX11 was examined. *P < 0.05.