Fig 2: 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.

Fig 3: circPTN regulates E2F2 protein expression through sponge miR-432-5p. (a) The binding sites between miR-432-5p and E2F2 mRNA were predicted by TargetScan. (b) Dual-luciferase reporter assay in A549 and H1229 cells using WT reporter (with wildtype binding sites) or MUT reporter (with mutated binding sites) in the presence of miR-NC or miR-432-5p mimic. (c) The protein level of E2F2 in A549 and H1229 cells upon miR-432-5p mimic transfection was detected by WB. (d) E2F2 protein levels in A549 and H1229 cells transfected with sh-NC, sh-circTPN, or sh-circTPN and miR-432-5p inhibitor were determined by WB. (e) qRT-PCR was used to detect the expression level of E2F2 in 90 pairs of NSCLC tissues and adjacent normal tissues. (f) The protein levels of E2F2 protein in three pairs of NSCLC tissues and corresponding adjacent tissues were determined by WB. (g) The correlation between the expression levels of E2F2 and miR-432-5p in NSCLC tissues. (h) The correlation between the expression levels of E2F2 and circPTN in NSCLC tissues. *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig 4: E2F2 overexpression facilitates LC cell proliferation, invasion, and migration after transfection with si-NORAD. NORAD was silenced in A549 cells in parallel with the overexpression of E2F2. (a and b) E2F2 expression in LC cells was assessed by RT-qPCR (a) and western blot analysis (b). (c and d) LC cell proliferation was examined by the CCK-8 method (c) and EdU assay (d). (e) A549 cell invasion and migration were measured by Transwell assay. The independent cell experiments were repeated three times. The results were presented as mean value ± standard deviation. One-way ANOVA was used to analyze the data in panels a, b, d, and e, and two-way ANOVA was used to analyze the data in panel c. Tukey’s multiple comparisons test was applied for post hoc test in panels b, c, and d. ** p < 0.01.

Fig 5: E2F2 activates PRR34-AS1 transcription in HCC cells(A) Western blot analyzed the knockdown or overexpression efficiencies of E2F2 in HCC cells. (B and C) The levels of E2F2 and PRR34-AS1 in indicated HCC cells were examined via quantitative real-time RT-PCR. (D) ChIP experiments measured the connection between PRR34-AS1 promoter and E2F2 in HCC cells. (E) The binding motif of E2F2 were predicted by JASPAR. (F) Luciferase reporter experiments assessed the luciferase activities of PRR34-AS1-Pro-WT and PRR34-AS1-Pro-Mut in HCC cells after E2F2 expression was increased or silenced. **p < 0.01.