Journal: Journal of cancer research and clinical oncology

Article Title: Upregulation of the ErbB family by EZH2 in hepatocellular carcinoma confers resistance to FGFR inhibitor.

doi: 10.1007/s00432-021-03703-6

Figure Lengend Snippet: Fig. 6 Proposed mechanism of FGFR/ErbB regulation by EZH2. The initial inhibition of FGFR by infigratinib leads to the downregulation of its downstream signalling proteins and several stem cell markers, including EZH2. However, long-term treatment eventually reactivates EZH2, which leads to an increase in the expression of the ErbB family and FGFRs. The dual inhibition of FGFR and ErbB effectively blocks resistance and results in a longer antitumour effect

Article Snippet: Stable EZH2 overexpression and knockdown cells A full-length EZH2 gene was cloned into pLX302 (Addgene #25896). shRNA targeting EZH2 was cloned into pLKO.1 (Addgene #8453). shRNA targeting luciferase was cloned 1 3 into pLKO.1 and served as the control plasmid.

Techniques: Inhibition, Expressing

Journal: Frontiers in Oncology

Article Title: An E2F1/DDX11/EZH2 Positive Feedback Loop Promotes Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3389/fonc.2020.593293

Figure Lengend Snippet: 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.

Article Snippet: Stable cell lines were constructed by transfecting cells with pcDNA (3.1) overexpression vector encoding full-length DDX11 cDNA or DDX11 shRNA purchased from Santa-cruz company (sc-77104-SH), and then selected by G418 for two weeks. siRNAs targeting p21 (#6456, Cell Signaling Technology), E2F1 (sc-29297, Santa-cruz Biotechnology), E2F2 (sc-29298, Santa-cruz Biotechnology), E2F3 (sc-37817, Santa-cruz Biotechnology), and EZH2 (#6509, Cell Signaling Technology) were also transiently introduced into HCC cells.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Over Expression, Knockdown, Activity Assay, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: An E2F1/DDX11/EZH2 Positive Feedback Loop Promotes Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3389/fonc.2020.593293

Figure Lengend Snippet: 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.

Article Snippet: Stable cell lines were constructed by transfecting cells with pcDNA (3.1) overexpression vector encoding full-length DDX11 cDNA or DDX11 shRNA purchased from Santa-cruz company (sc-77104-SH), and then selected by G418 for two weeks. siRNAs targeting p21 (#6456, Cell Signaling Technology), E2F1 (sc-29297, Santa-cruz Biotechnology), E2F2 (sc-29298, Santa-cruz Biotechnology), E2F3 (sc-37817, Santa-cruz Biotechnology), and EZH2 (#6509, Cell Signaling Technology) were also transiently introduced into HCC cells.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Over Expression, Knockdown, Activity Assay, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: An E2F1/DDX11/EZH2 Positive Feedback Loop Promotes Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3389/fonc.2020.593293

Figure Lengend Snippet: 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.

Article Snippet: Stable cell lines were constructed by transfecting cells with pcDNA (3.1) overexpression vector encoding full-length DDX11 cDNA or DDX11 shRNA purchased from Santa-cruz company (sc-77104-SH), and then selected by G418 for two weeks. siRNAs targeting p21 (#6456, Cell Signaling Technology), E2F1 (sc-29297, Santa-cruz Biotechnology), E2F2 (sc-29298, Santa-cruz Biotechnology), E2F3 (sc-37817, Santa-cruz Biotechnology), and EZH2 (#6509, Cell Signaling Technology) were also transiently introduced into HCC cells.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Over Expression, Knockdown, Activity Assay, Plasmid Preparation