E2F4 Promotes the Proliferation of Hepatocellular Carcinoma Cells through Upregulation of CDCA3

Abstract

Liver cancer, the second most commonly diagnosed cancer, is associated with high mortality rates. E2F4 is a member of the E2F transcription factor family. There are limited studies on the role of E2F4 in hepatocellular carcinoma (HCC). In this study, the expression of E2F4 in HCC tissue samples and cell lines was analyzed using quantitative real-time polymerase chain reaction. E2F4 expression positively correlated with tumor size in patients with HCC. Additionally, E2F4 expression was greater in HCC cells than in normal LO2 cells. Furthermore, overexpression of E2F4 significantly enhanced the proliferation, migration, and invasion of HCC cells. The results of a luciferase assay revealed that E2F4 upregulated the expression of CDCA3 by binding to its promoter region (1863'-ACGCGCGAGAATG-1875') and consequently promoted proliferation and cell cycle progression of HCC cells. Taken together, these results demonstrated that E2F4 might play a vital role in HCC progression and could serve as a potential biomarker for the diagnosis and as a therapeutic target of HCC.

Keywords: CDCA3; E2F4; hepatocellular carcinoma; promoter.

Figure 1

Expression of E2F4 in the hepatocellular carcinoma (HCC) samples and the correlation with clinicopathological features. A. Expression of E2F4 in HCC and adjacent non-tumorous tissues was examined using quantitative real-time polymerase chain reaction (qRT-PCR). B. Expression of E2F4 in Huh7, SK-HEP-1, Hep3B, HepG2, and Bel-7402 cells. The immortalized normal liver cell line (LO2) was used as a control. C. The gene expression profiling interaction analysis (GEPIA; http://gepia.cancer-pku.cn/index.html) database was used to evaluate the correlation between E2F4 expression and the survival rate of patients with HCC.

Figure 2

Effect of E2F4 overexpression or knockdown on hepatocellular carcinoma (HCC) proliferation, migration, and invasion. E2F4 was overexpressed in Huh7 and knocked down in Hep3B cells. A. Results of the cell proliferation assay. The transfected cells were seeded and imaged using a long-term dynamic cell imaging system once every 24 h and the growth rate was calculated. B. Results of the colony formation assay. The transfected cells were cultured for one week. The cells were stained to examine the growth of colonies. C. Results of the transwell assay. The cells that passed through the membrane were stained and counted using a microscope (Magnification: 200×). D. Results of the wound-healing assay. The cell monolayer was scratched, and the migration of cells into the scratch area was monitored once every 12 h using the long-term dynamic cell imaging system.

Figure 3

E2F4 can promote cell cycle progression of hepatocellular carcinoma (HCC) cells. Huh7 cells were transfected with pCDNA3.1 or pCDNA3.1-E2F4. At 48 h post-transfection, the cells were analyzed via flow cytometry. A. Effect of E2F4 overexpression on apoptosis. B. Quantification of the number of late apoptotic cells in the pCDNA3.1-E2F4- and pCDNA3.1-transfected groups (upper right quadrant). C. Effect of E2F4 overexpression on the cell cycle. D. Quantification of the proportion of cells in the G0/1, S, and G2 phases of the cell cycle in the pCDNA3.1-E2F4- and pCDNA3.1-transfected groups.

Figure 4

Identification of E2F4 target genes. A. E2F4 was overexpressed in Huh7 cells and knocked down in the Hep3B cells. The expression of CDCA3, CENPI, CDC7, and KIF2C was examined using quantitative real-time polymerase chain reaction (qRT-PCR). B-C. The proliferation of Huh7 or Hep3B cells exhibiting overexpression or knockdown of E2F4. D-F. Schematic representation of the full-length and truncated CDCA3 promoters. 1 to 11 represent the predicted E2F4 binding sites. Results of the dual-luciferase reporter assay. Huh7 cells were transfected with CDCA3 full-length promoter [pGL3-CDCA3 (WT)] or three truncated CDCA3 promoter (pGL3-CDCA3-Δ1, pGL3-CDCA3-Δ2, and pGL3-CDCA3-Δ3) constructs. Luciferase activity was examined at 48 h post-transfection.