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

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.


Introduction
The incidence of liver cancer, which is the second most commonly diagnosed cancer overall, is higher among men than women. In developed countries, liver cancer is the fifth leading cause of cancer-related deaths among men. The incidence of liver cancer is also high in developing countries. Oftentimes, no symptoms are presented during early stage disease. Hence, liver cancer tends to be diagnosed at an advanced stage, especially in developing countries. The incidence of liver cancer is lowest in South Asia, Northern Europe, Central Europe, and Eastern Europe and highest in East Asia, Southeast Asia, North Africa, and West Africa. Globally, the most common primary liver cancer is hepatocellular carcinoma (HCC), which accounts for 75%-85% of all primary liver cancer cases [1][2][3].
Studies have shown that E2F mRNA expression levels are related to different cancer stages and pathological grades of HCC patients, and E2F4 protein is highly expressed in advanced liver cancer [11] . E2F4 is the most abundantly expressed E2F transcription factor. Additionally, E2F4, which accounts for the majority of E2F activity, is involved in various cellular functions. E2F4 has previously been reported to function as a transcriptional repressor. However, recent studies have reported transcriptional activation functions as well [12,13].
The role of E2F4 in HCC pathogenesis remains unclear. Hence, this study aimed to elucidate the role of E2F4 in HCC. The expression of E2F4 in clinical HCC tissues and adjacent non-tumorous tissues, as well as in HCC and normal liver cell lines, was examined using quantitative real-time polymerase chain reaction (qRT-PCR). The findings of this study indicated that E2F4 might promote HCC carcinogenesis and might function as a candidate biomarker for diagnosis and as a therapeutic target.

Clinical samples
HCC and adjacent tissues were collected from 40 patients undergoing thoracic surgery in the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Chongqing Medical University, from January 2018 to January 2019. The samples were collected from 33 male and seven female patients. The patients were randomly selected, all showed primary HCC, and had not been treated with radio-or chemotherapy before surgery. Based on the tumor, node, metastasis (TNM) staging, 11, 7, and 22 cases were classified as stage I, stage II, and stage III, respectively. The tissues were immediately placed in RNA preservation solution in liquid nitrogen after surgical removal. The study protocol was approved by the Research Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, and written informed consent was obtained from each patient.

Cell lines and cell culturing
Five human HCC cell lines (Huh7, Hep3B, HepG2, Bel-7402, and SK-HEP-1) and one modified normal liver cell line (LO2) were purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). The other cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS. All cells were cultured in a cell incubator at 37 °C and 5% CO2. The pcDNA3.1 plasmid and the adenoviral vector encoding control short interfering RNA (siRNA) and red fluorescence protein (RFP) were obtained from the Infectious Disease Laboratory of Chongqing Medical University. The pcDNA3.1-E2F4 overexpression plasmid and the adenoviral vector encoding si-E2F4 (Ad-siE2F4) were constructed by our group and verified by sequencing. Plasmid transfection was performed using Lipofectamine 3000, according to the manufacturer's instructions. The virus titers used for cell infection were >10× [11].

RNA extraction, reverse transcription, and qRT-PCR
Total RNA was extracted from tissues and cells using TRIzol following the manufacturer's instructions. cDNA was synthesized using 1 µg of the isolated RNA as a template. The synthesized cDNA was diluted using double distilled water (1:9) and stored at -20 °C until use. qRT-PCR analyses were performed in triplicate. The relative expression levels of E2F4 were calculated using the 2 −ΔΔCt method. The primers used for qRT-PCR were listed in Table S1.

Cell proliferation, migration, and invasion assays
Cells were transfected with pcDNA3.1-E2F4 or Ad-siE2F4 for 24 h. The cells were then resuspended and counted using a cow-abalone counting plate. The proliferation of cells (2 × 10 3 /well) seeded in a 96-well plate was examined. When the cells adhered, a long-term dynamic cell imaging system was used to analyze the number of cells in each well and set as an initial value. Cells were counted once every 24 h, and the growth rate was calculated. A colony formation assay was performed using a six-well plate. Cells (500 cells/well) were cultured for one week and stained with crystal violet, and the colonies were counted. To perform the wound-healing assay, 4 × 10 4 cells/well were plated in a 96-well plate. When the cells adhered to the wall and the density reached at least 90%, the monolayer was scratched to introduce a wound gap. A long-term dynamic cell imaging system was used to capture images of the cells once every 12 h to analyze wound healing. Cell invasion assays were performed using transwell chambers. The upper chamber was coated with Matrigel (1:10). Cells (4 × 10 4 ) were resuspended in serum-free DMEM and seeded into the upper chamber. DMEM supplemented with 10% FBS was added to the lower chambers and the cells were incubated for 24 h. Transwell inserts were then removed and the cells were washed three times with phosphate-buffered saline. The cells were fixed in 4% paraformaldehyde at room temperature for 30 min, stained with crystal violet for 10 min, and counted using an optical microscope.

Statistical analysis
All statistical analyses were performed using SPSS 22.0. Data were expressed as the mean ± standard deviation from three independent experiments. The means between two groups were analyzed using Student's t-test, while those between multiple groups were analyzed using one-way analysis of variance (ANOVA). The correlation between E2F4 expression and the clinicopathological features of patients was analyzed using the chi-square (χ 2 ) test. Differences were considered significant at P <0.05.

Correlation between E2F4 expression and clinicopathological factors
The mRNA levels of E2F4 in matched HCC and adjacent non-tumorous tissues from 40 HCC patients were analyzed using qRT-PCR. The clinical tissue samples were divided into E2F4-high (21 cases) and E2F4-low groups (19 cases) based on median E2F4 expression ( Table 1). Analysis of the correlation between E2F4 expression and clinicopathological features revealed that most patients with tumor sizes > 5 cm (16/24) exhibited upregulated E2F4 expression. This suggests that the expression of E2F4 is positively correlated with tumor size. The absolute expression of E2F4 in the adjacent non-tumorous tissues (0.062 ± 0.0125) was significantly lower than the tumorous tissues (0.183 ± 0.035) (Fig. 1A). Additionally, E2F4 mRNA levels were examined in HCC (Huh7, SK-HEP-1, Hep3B, HepG2, Bel-7402) and normal liver (LO2) cell lines. Among the HCC cell lines, Huh7 and Hep3B exhibited the lowest and highest E2F4 expression, respectively. E2F4 expression in all HCC cell lines was higher than LO2 cells (Fig. 1B). These results suggest that E2F4 functions as an oncogene in liver cancer tissues. Survival analysis using the gene expression profiling interaction analysis (GEPIA; http://gepia.cancerpku.cn/index.html) database revealed that the survival rate of HCC patients in the E2F4-high cohort was lower than that of patients in the E2F4-low cohort (Fig. 1C), which was consistent with the findings of Huang et al. [11,14]. This suggests that E2F4 is involved in HCC development.

Effect of E2F4 on the HCC cell proliferation, migration, and invasion
The proliferation, migration, and invasion of Ad-siE2F4-transfected and Ad-E2F4-transfected HCC cells were examined next. As shown in Fig. 2A, the proliferation of Ad-E2F4-transfected Huh7 cells was greater than the control cells. The proliferation of Hep3B cells was significantly decreased upon transfection with Ad-siE2F4. The results of the colony formation assay revealed that the Ad-E2F4transfected Huh7 cells exhibited an increased number of colonies (Fig. 2B), whereas the Ad-siE2F4transfected Hep3B cells exhibited a significantly decreased number of colonies. In the wound-healing assay, the migration rate of Ad-E2F4-transfected Huh7 cells significantly increased (Fig. 2C), while for Ad-siE2F4-transfected Hep3B cells, it significantly decreased. The results of the transwell assay (Fig. 2D) revealed that the invasion of Ad-E2F4-transfected Huh7 cells was markedly higher than the control cells. In contrast, the invasion of Ad-siE2F4-transfected Hep3B cells was markedly decreased. These findings further confirmed that E2F4 could enhance the proliferation, migration, and invasion of HCC cells [15].

E2F4 promotes the progression of HCC cell cycle
E2F family transcription factors mediated several cellular functions, including apoptosis and proliferation. E2F-mediated enhancement of cell proliferation could be caused by either inhibition of apoptosis or enhancement of proliferation [16].
Huh7 cells transfected with either pcDNA3.1 or pcDNA3.1-E2F4 and subjected to flow cytometry analysis to examine apoptosis and cell cycle markers at 48 h or 72 h post-transfection. There was no significant difference in the number of cells at the late apoptotic (upper right quadrant) stage between the pcDNA3.1-and pcDNA3.1-E2F4-transfected groups (Figs. 3A and 3B). This suggests that E2F4 does not promote apoptosis in HCC cells. The pcDNA3.1-E2F4transfected group exhibited an increased number of cells in the S-phase (Figs. 3C and 3D), indicating that E2F4 promotes the transition from G0/1 to S-phase and contributes to cell cycle progression.

CDCA3 is a target gene for E2F4
E2F4 regulates the expression of various genes by binding to promoters [17]. As E2F4 promotes the progression of the HCC cell cycle, we hypothesized that it modulated the expression of cell cycle regulator genes. Previous studies have reported various target genes of E2F4 using chromatin immunoprecipitation (ChIP) analysis. Among these target genes, CDCA3, CENPI, CDC7, and KIF2C were involved in the regulation of the cell cycle [12,13]. The effect of E2F4 overexpression or knockdown on the expression of these four genes in HCC cells was examined using qRT-PCR. The expression of CDC7 was not detected in both Huh7 and Hep3B cells and expression of CENPI and KIF2C were unaffected by either overexpression of knockdown of E2F4. However, the expression of CDCA3 was positively correlated with that of E2F4 (Fig. 4A). CDCA3, a cell division cycle protein, promoted the proliferation and cell cycle progression of colorectal and gastric cancer cells [14]. The proliferation of CDCA3-transfected cells significantly increased and was significantly inhibited upon co-transfection with siE2F4 (Figs. 4B and 4C). This indicates that E2F4 regulates HCC cell cycle through CDCA3. 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.  The E2F4 binding sites located in the CDCA3 5'-untranslated region (UTR) comprise a 2000-bp sequence upstream of the transcription start site, which were analyzed using the UCSC database. According to the JASPAR database, 11 candidate sequences in the CDCA3 promoter region were predicted to serve as potential E2F4 binding sites (Figs. 4D and 4F). Therefore, we constructed a vector encoding the full-length CDCA3 promoter (pGL3-CDCA3; pGL3-Basic as the vector) as well as vectors encoding three CDCA3 truncated promoter constructs containing three central high-scoring sequences (with relatively strong binding potential) (pGL3-CDCA3-Δ1, pGL3-CDCA3-Δ2, and pGL3-CDCA3-Δ3) (Fig. 4D). A dual-luciferase reporter assay was performed by co-transfecting Huh7 cells with E2F4 and pGL3-CDCA3, pGL3-CDCA3-Δ1, pGL3-CDCA3-Δ2, or pGL3-CDCA3-Δ3. Huh7 cells cotransfected with E2F4 and PGL3-CDCA3-Δ3 exhibited the highest luciferase activity (Fig. 4E). Two candidate sequences in the pGL3-CDCA3-Δ3 construct, 1863-ACGCGCGAGAA-1873 and 1865-GCGCGAGAATG-1875, were located at the same site. This indicates that E2F4 binds to this 13-bp sequence (1863-ACGCGCGAGAATG-1875) in the CDCA3 promoter.

Discussion
The E2F transcription factor family is involved in various cellular functions [15,16]. Previous studies have reported that E2F1 mediates carcinogenesis in various cancers. However, recent studies have also demonstrated that E2F1 is positively correlated with the tumor cell apoptosis index in cell carcinoma. Thus, E2F1 is a pro-apoptotic regulator that can also activate the tumor suppressor p53 [17,18]. Some studies have reported that E2F4 can arrest the cell cycle at G0 [19,20]. E2F4 is involved in the carcinogenesis of skin tumors, gastrointestinal tumors, and prostate cancer [21,22]. This study reported, for the first time, that E2F4 couldpromote the carcinogenesis of HCC through the upregulation of CDCA3, a cell cyclerelated protein.
In the G0 phase, activator E2Fs often form a complex with RB family members (including RB, p107, and p130). The cyclin/CDK complex phosphorylates the RB protein upon stimulation, resulting in the release of E2F. Free E2F can induce the transcription of cell cycle-related proteins and consequently promote cell cycle progression [4,5,[23][24][25][26]. E2F4, a member of the E2F transcription factor family, may dissociate from phosphorylated RB and upregulate CDCA3 expression by binding to its promoter region (1863-ACGCGCGAGAATG-1875). The upregulated expression of CDCA3 promotes the proliferation and cell cycle progression of HCC cells. However, further studies are required to elucidate the underlying mechanisms.
In this study, we analyzed the expression of E2F4 in HCC tissue samples and cell lines. E2F4 expression positively correlated with tumor size. The expression of E2F4 in HCC cell lines was higher than in LO2 cells. E2F4 overexpression enhanced the proliferation, migration, and invasion of HCC cells. The results of the luciferase reporter assay revealed that E2F4 upregulates CDCA3 expression by binding to its promoter region (1863-ACGCGCGAGAATG-1875) and consequently promotes the proliferation and cell cycle progression of HCC cells.