Expression and prognostic value of E2F family members in gastric cancer

E2F is a family of transcription factor proteins involved in multiple processes, including cell cycle regulation, cell differentiation, the DNA damage response and cell death. Studies have shown that E2Fs have prognostic signicance in many cancers, but the expression patterns and prognostic values of E2Fs in gastric cancer have not been systematically elucidated. In this study, we used the ONCOMINE database and UALCAN online analysis to compare the transcriptional levels and expression of eight E2F family members between gastric cancer and normal samples. UALCAN was also used to analyze the relationship between the expression of 8 E2F members and clinicopathological parameters. A protein-protein interaction (PPI) network was constructed using the STRING database. The functions and pathways of the E2F family and 50 frequently changed genes closely associated with the family members were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) software. The prognostic value of the E2Fs was determined by Kaplan-Meier Plotter and Cox regression. Western blot and qPCR were used to analyze the expression of E2F2 in gastric cancer. CCK8 and EdU analyses were conducted to assess cell proliferation, and wound-healing and Transwell assays were used to analyze cell migration and invasion. the role of E2F2 in gastric cancer in detail. We found that members of the E2F family show differences in expression between cancer and normal tissues and may be closely related to the prognosis of gastric cancer. Among them, E2F2 can serve as an independent prognostic factor for gastric cancer and promote the proliferation, invasion and metastasis of gastric cancer cells. These data indicate that E2F2 could be a potential biomarker for gastric cancer. K-M Plotter was used to analyze the survival according to each of the 8 evaluated E2F members. The results showed that the mRNA expression of E2F family members was closely related to the prognosis of gastric cancer patients. High expression of E2F1 (HR = 2, 95% CI: 1.68-2.38, p = 1.1e -15 ), E2F2 (HR = 1.3, 95% CI: 1.09-1.58, P = 0.0044), E2F3 (HR = 1.89, 95% CI: 1.57-2.27, P = 3.5e -12 ), E2F4 (HR = 1.97, 95% CI: 1.65-2.36, P = 5e -14) , E2F5 (HR = 1.62, 95% CI: 1.35-1.95, P = 2e -7 ) was correlated with poor OS, whereas high expression of E2F6 (HR = 0.77, 95% CI: 0.65-0.92, P = 0.0033), E2F7 (HR = 0.59, 95% CI: 0.47-0.75, P = 1.1e 5 ), and E2F8 (HR = 0.53, 95% CI: 0.44-0.65, P = 1.8e -10 ) was associated with better OS (Figure 7). This indicates that E2F family members can be used as effective biomarkers for predicting the survival of gastric cancer patients. was signicantly upregulated, and its overexpression promoted cell proliferation and tumorigenicity. Patients with higher E2F1 levels have larger tumor sizes, more advanced tumor stages, and worse survival than patients with lower E2F1 levels (34). This is similar to the results of our study. The results of the bioinformatics analysis show that E2F1 overexpression may indicate a poor prognosis and is associated with a more advanced clinical stage (stage 3). E2F2 plays contradictory roles in the development of tumors. On the one hand, E2F2 can inhibit tumorigenesis by inhibiting cell cycle regulators. On the other hand, E2F2 can act as an "activator" to increase target expression and cause cancer (35). Previous studies have shown that changes in E2F2 protein expression are closely related to the occurrence of different cancers It has been reported that knocking down E2F2 signicantly reduces the metastatic ability of breast cancer cells, and mutations in E2F2 are related to tumor proliferation and survival in breast cancer patients (37). E2F2 functions as an oncogene in liver cancer (38), while in prostate cancer, E2F2 inhibits tumor cell proliferation by targeting miRNAs (39). in the and of cancer cells and is related to cell invasion and migration It has been found to be amplied in a variety of human tumors, including lung (41), bladder (42), liver ovarian breast (45), and pancreatic cancer A study by Li et found that silencing E2F3 had an inhibitory effect on proliferation and an inducing effect on apoptosis in gastric cancer cells There are also reports in the literature indicating that E2F3 can function as a direct target of miRNA to play a carcinogenic role in gastric cancer 49). Our research shows that E2F3 overexpression is associated with a poor prognosis in patients and occurs in gastric cancer cells, suggesting that E2F3 may be a therapeutic candidate for gastric cancer patients. In gastric cancer, Li et al. discovered that downregulation of E2F6 expression inhibited the proliferation and invasion of gastric cancer cells, suggesting that E2F6 may play a carcinogenic role in gastric cancer. E2F7 has been found to be involved in the development of breast (65), gallbladder (66), pancreatic (67), and cervical cancer (68), among others. E2F8 has also been shown to be involved in the development of various cancers, including breast (69), lung (70), and liver cancer (71), but the roles of E2F7 and E2F8 in gastric cancer have not been reported. After analyzing the expression of E2F family members in gastric cancer, we focused on the prognostic value of the E2F family in gastric cancer. The results of the online website Kaplan-Meier Plotter show that high expression of E2F1/2/3/4/5 may be related to poor OS in gastric cancer patients, while the high expression of E2F6/7/8 is related to better OS. Using univariate and multivariate Cox regression analyses, it was found that among the eight E2F members, E2F2 can be used as an independent prognostic factor for gastric cancer; this piqued our research interest. Since the E2F family is also directly or indirectly involved in the processes of cell proliferation, invasion and metastasis, we hoped to elucidate the role of E2F2 in gastric cancer cells. The results showed that the downregulation of E2F2 inhibited not only the proliferation of gastric cancer cells but also their invasion and metastasis. Therefore, we believe that E2F2 plays a role in promoting cancer in gastric cancer cells. However, the downstream signaling pathways that regulate the biological function of these tumor cells and their underlying molecular mechanisms need further study in the future. In conclusion, our results indicate that overexpression of eight E2F members was signicantly correlated with the clinical cancer stage and pathological tumor grade in gastric cancer patients. A high mutation rate (51%) for E2Fs was observed in gastric cancer patients. In addition, E2F2 can be used as an independent prognostic factor for gastric cancer, and downregulation of E2F2 can inhibit the proliferation, invasion and migration of gastric cancer cells. These results indicate that E2F2 can be used as a potential prognostic marker and therapeutic target for gastric cancer, and further study on E2F2 may improve the survival and prognostic accuracy of gastric cancer patients. The focus of this study is to provide new ideas for the clinical diagnosis and prognostic assessment of gastric cancer via a combination of bioinformatics analysis and cell experiments. Our results provide an important bioinformatics foundation and related theoretical foundation for guiding follow-up research on gastric cancer.


Abstract
Background E2F is a family of transcription factor proteins involved in multiple processes, including cell cycle regulation, cell differentiation, the DNA damage response and cell death. Studies have shown that E2Fs have prognostic signi cance in many cancers, but the expression patterns and prognostic values of E2Fs in gastric cancer have not been systematically elucidated.

Methods
In this study, we used the ONCOMINE database and UALCAN online analysis to compare the transcriptional levels and expression of eight E2F family members between gastric cancer and normal samples. UALCAN was also used to analyze the relationship between the expression of 8 E2F members and clinicopathological parameters. A protein-protein interaction (PPI) network was constructed using the STRING database. The functions and pathways of the E2F family and 50 frequently changed genes closely associated with the family members were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) software. The prognostic value of the E2Fs was determined by Kaplan-Meier Plotter and Cox regression. Western blot and qPCR were used to analyze the expression of E2F2 in gastric cancer. CCK8 and EdU analyses were conducted to assess cell proliferation, and wound-healing and Transwell assays were used to analyze cell migration and invasion.

Results
The expression patterns of 8 E2F members were signi cantly related to the clinical cancer stage and tumor grade of gastric cancer patients. A high mutation rate (51%) for E2Fs was observed in patients with gastric cancer. In addition, high mRNA expression of E2F1/2/3/4/5 was a prognostic factor for poor overall survival (OS) in gastric cancer patients, while high expression of E2F6/7/8 was associated with better OS. Cox regression analysis found that E2F2 can be used as an independent prognostic factor for gastric cancer, and compared with normal cells, the expression of E2F2 in gastric cancer cells was signi cantly increased. In addition, we found that E2F2 silencing inhibits the proliferation, invasion and metastasis of gastric cancer cells.

Conclusions
Our study evaluated the expression and prognosis of the E2F family in gastric cancer and discussed the role of E2F2 in gastric cancer in detail. We found that members of the E2F family show differences in expression between cancer and normal tissues and may be closely related to the prognosis of gastric cancer.
Among them, E2F2 can serve as an independent prognostic factor for gastric cancer and promote the proliferation, invasion and metastasis of gastric cancer cells. These data indicate that E2F2 could be a potential biomarker for gastric cancer.

Introduction:
Gastric cancer, the second leading cause of cancer-related death, is one of the most common malignant tumors in the world (1). Despite improvements in surgical techniques and chemotherapy regimens, patient outcomes are often disappointing. Most patients with gastric cancer are diagnosed at an advanced stage, and the ve-year survival rate is still low (2). Therefore, identifying prognostic markers related to gastric cancer is crucial to developing individualized treatment plans for gastric cancer patients and improving their clinical outcomes.
E2F is a family of transcription factor proteins that are considered to be the main regulators of cell growth and proliferation. The E2F family is usually divided into two categories according to function: transcriptional activators (E2F1, E2F2 and E2F3a) and transcriptional repressors (E2F3b and E2F4-8) (3). The main functions of E2F proteins are to regulate the cell cycle, cell differentiation, the DNA damage response and cell death (4). E2F proteins have been found in several human malignant tumors, including breast (5), ovarian (6), bladder (7), prostate (8), lung (9) and gastrointestinal cancer (10).
To date, eight E2F factors have been identi ed in mammals, and these proteins (E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, and E2F8) were numbered according to the order of their discovery (11). E2F family member activators may have carcinogenic effects, whereas E2F family repressors may be related to tumor suppression (12). In most human tumors, E2F transcription factors undergo transcriptional changes or deregulation through different molecular mechanisms that inactivate the Rb family, and their uncontrolled expression can induce inappropriate S-phase entry and apoptosis (13). Studies have shown that overexpression of E2F1 in gastric cancer promotes cell death through various mechanisms, demonstrating the role of E2F1 in suppressing gastric cancer tumors (14). However, the expression levels of E2F family members in gastric cancer are unregulated, and their relationships with clinicopathological characteristics and prognosis have not been systematically clari ed.
Bioinformatic analysis based on high-throughput sequencing is an important approach to explore the molecular mechanisms of tumorigenesis and development and to identify biomarkers that can be used for early diagnosis and treatment. With the development of microarray technology, RNA and DNA research has become an important part of biology and biomedicine. By analyzing thousands of published gene expression levels and copy number variations, we studied the expression and mutation of E2F family members in gastric cancer patients in detail to determine the expression patterns, potential functions and unique prognostic values of E2F proteins in gastric cancer.

ONCOMINE database
The ONCOMINE database (www.oncomine.org) is an integrated online cancer microarray database containing data from DNA-and RNA-sequencing (DNA-seq and RNA-seq, respectively) analyses used to classify the differential expression between common cancer types and the corresponding normal tissues, as well as clinical and pathological data (15). In our study, the transcriptional expression data for 8 different E2F members in different cancer tissue samples and their corresponding adjacent normal samples were obtained from the ONCOMINE database. Differences in transcriptional expression were compared by Student's t test. The p-value cutoff and fold change threshold were as follows: p-value, 0.01; fold change, 1.5; gene grade, 10%; and data type, mRNA.

UALCAN
UALCAN (http://ualcan.path.uab.edu) is an interactive web resource developed based on the grade 3 RNA sequences and clinical data of 31 cancer types in The Cancer Genome Atlas (TCGA) database. This resource can be used to analyze the relative transcript expression of genes between tumor and normal samples and the correlations between expression and clinicopathological parameters (16). In this study, UALCAN was used to analyze the mRNA expression of eight E2F family members in primary gastric cancer tissue samples and the relationships of these members with clinicopathological parameters.
Differences in transcriptional expression were compared by Student's t test (p < 0.01).

Kaplan-Meier (K-M) Plotter
The online database K-M Plotter (www.kmplot.com) (17) was used to assess the prognostic value of E2F mRNA expression. The database contains gene expression data and survival information for patients with breast, lung, gastric and ovarian cancer. To analyze the overall survival (OS), progression-free survival (PFS) and postprogression survival (PPS) of gastric cancer patients, patient samples were strati ed into two groups according to the median expression level (high expression and low expression) and validated by K-M survival curves. Information on the number of high-risk cases, mRNA expression levels, hazard ratios (HRs), 95% con dence intervals (CIs) and p-values can be found on the K-M Plotter webpage. A p-value < 0.05 was considered statistically signi cant.

Protein-protein interaction (PPI) network construction and gene enrichment analyses
The STRING database (http://string-db.org/) provides information regarding the signi cant associations of PPIs. (20). In this study, the STRING database was used to analyze the E2F family and 50 frequently changed genes closely associated with the family members. We used the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (http://www.DAVID.org) (21) to conduct agonistic gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of 58 genes, including E2F enrichment analysis. GO enrichment analysis can predict gene function based on biological processes (BPs), cell composition (CC) and molecular function (MF), and KEGG can be used to analyze gene enrichment pathways. 6. Cell culture AGS, HGC27 and GES-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (Gibco, NY, USA). The cells were placed in an incubator at 37 °C and 5% CO 2 (22). FBS and RPMI 1640 medium were purchased from Gibco (New York, USA).

Transfection with lentiviral particles
The cells were seeded at a density of 2 × 105 cells/well in six-well plates, and 2 ml of complete medium was added to each well. The cells were incubated for 24 h and infected with lentiviral particles, and 12 h after infection, the LV-containing medium was replaced with fresh complete medium. The infected cells were then selected with 4 µg/ml puromycin for 96 h. The lentiviral expression vectors LV-control, LV-shE2F2 were purchased from Shanghai Gene Pharma Company (China).Virus packaging involves three plasmids, which are the tool vector plasmid (GV115, GV118, GV365) carrying the target gene or target sequence, the virus packaging auxiliary plasmid Helper 1.0 and the virus packaging auxiliary plasmid Helper 2.0. The shRNA lentiviral sequence of E2F2 is as follows: TAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAA 8. Real-time quantitative PCR (qPCR) Total RNA (1 µg) was isolated using a TRIzol kit (Invitrogen, Carlsbad, California, USA), and the concentration and purity were quanti ed using an ultraviolet spectrophotometer. Thereafter, cDNA was generated from the isolated RNA by reverse transcription using the Transcriptor First Strand cDNA Synthesis Kit (Roche, USA), and real-time PCR was performed using LightCycler 480 SYBR Green Master Mix (Roche Diagnostics GmbH). The expression level of each E2F gene was normalized to that of GAPDH. The cycle threshold (CT) for relative mRNA expression was analyzed by the 2-ΔΔ method. The primer sequences are shown in Table 1. The following cycling conditions were used: 95 °C for ve minutes followed by 40 cycles of 95 °C for 20 seconds and 60 °C for 30 seconds. QPCR assays were conducted in triplicate in a 10-mL reaction volume for each sample. QPCR was performed as described previously (23). The primer sequences for qRT-PCR were as follows. E2F2 Forward: CGTCCCTGAGTTCCCAACC;E2F2 Reverse : GCGAAGTGTCATACCGAGTCTT. GAPDH Forward: GGAGCGAGATCCCTCCAAAAT;GAPDH Reverse: GGCTGTTGTCATACTTCTCATGG. 9. Western blot analysis Cells were lysed in RIPA buffer containing PMSF on ice, and then the lysate was centrifuged at 13,000 x g for 5 minutes at 4 °C to remove cell debris. After the supernatant was collected, a BCA protein assay kit was used to determine the total protein concentration. Approximately 20 µg of protein per lane was separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis through 15% gels and then transferred to a polyvinylidene uoride membrane. The membrane was rst blocked with 5% skim milk and then incubated with a horseradish-peroxidase (HRP)-conjugated primary antibody against β-actin (120,000, Sigma). An antibody targeting Tspan7 (1:1000, Abcam) was also added, and the membranes were incubated at 4 °C overnight. Subsequently, the membranes were submerged in a secondary antibody solution (120,000, Abcam) for 1 hour at room temperature. Chemiluminescence was used to observe the antibody staining. Western blotting was performed as described previously (24).

Cell viability assay
After cells were digested with 0.25% EDTA-trypsin, they were seeded into 96-well plates at a density of 5000/well, and cell proliferation was measured at 24, 48, and 72 hours using the Cell Counting Kit-8 assay. Brie y, 10 µl of CCK-8 solution was added to each well and incubated with the cells at 37 °C for 2 hours.
The optical density (OD) at 450 nm was then measured with a microplate spectrophotometer (25). Another set of cells was seeded into a six-well plate and incubated overnight, after which EdU detection reagent was added, and cell proliferation was assessed by observing the uorescence under an inverted microscope (Olympus, Japan).

Scratch assay
Cells were seeded into 6-well plates at 90% con uence. Then, when a monolayer formed, it was scratched with the tip of a 200 µl pipette to form a wound along the center of each well. Next, the wells were washed twice with PBS to remove oating cells and debris, and fresh medium was added. Images were captured at 0 and 24 hours after scratching (100x magni cation) to assess cell migration into the wound area (26).

Transwell assay
Transwell cell culture chambers containing Matrigel were used to evaluate cell invasion. Log phase cells were digested with 0.25% EDTA-trypsin, and the cell suspension was treated with serum-free DMEM. Then, 200 µl of cells was added to the upper well of the Transwell chamber, and 600 µl of medium containing 20% serum was added to the lower part of the well. After 24 hours of incubation, the cells that invaded to the lower chamber were xed with methanol for 30 minutes and then stained with Giemsa for 20 minutes. The cells remaining in the upper chamber were gently removed with a wet cotton swab, and the membrane was placed under an inverted microscope so that the remaining cells could be counted (26).

Statistical analysis
All statistical tests were performed with SPSS 19.0 (SPSS, Inc., Illinois, USA). Data are expressed as the mean ± standard deviation and were compared by Student's t test or analysis of variance. P < 0.05 was considered signi cant. Results:

Expression of E2Fs in patients with gastric cancer
Eight E2F family members have been identi ed in mammals. By using the ONCOMINE database and UALCAN online analysis website, we compared the transcriptional levels and expression of E2Fs between gastric cancer and normal samples. As shown in Figure 1 and Table 1, E2F2/3/7 mRNA expression was signi cantly higher in gastric cancer tissue samples than in corresponding normal tissue samples in multiple data sets. The DErrico database showed that compared with normal tissue, gastric intestinal type adenocarcinoma had increased expression of E2F2, with a fold change was 3.234 (p = 3.39E -7 ), while Cho observed a 1.473-fold increase in E2F2 mRNA expression in diffuse gastric adenocarcinoma (p = 1.45E -5 ). Signi cant upregulation of E2F3 expression was also observed in gastric cancer tissue samples. In the DErrico dataset, the expression levels of GI and G Mix A were increased 2.862-fold (p = 4.79E -6 ) and 2.374-fold (p = 1.50E -10 ), respectively. In the Chen and Cho datasets, E2F3 expression also showed similar trends. Compared with that in normal tissue samples, the expression of E2F7 in gastric cancer tissue samples was increased by 3.234-fold in the DErrico dataset (p = 3.39E -7 ) and by 1.473-fold in the Cho dataset (p = 1.45E -5 ). Next, through UALCAN, we further explored the mRNA expression patterns of the 8 E2F family members. Unlike the ONCOMINE database, the resources in UALCAN are based on the level 3 RNA-seq and clinical data of 31 cancer types from the TCGA database. As shown in Figure 2, compared with that in normal samples, the mRNA expression of the 8 E2F members in primary gastric cancer tissue samples was signi cantly upregulated (all p <0.05).

Relationships between the mRNA expression of E2F family members and clinicopathological parameters in patients with gastric cancer
After discovering the mRNA expression patterns in gastric cancer patients, we analyzed the relationships between the mRNA expression of different E2F family members and clinicopathological parameters, including patient cancer stage and tumor grade, through UALCAN. As shown in Figure 3, the mRNA expression of eight E2F family members was signi cantly correlated with the stage of cancer development in patients, and patients with advanced cancer had higher E2F mRNA expression. Among the E2F family members, E2F3, E2F4 and E2F7 had the highest mRNA expression in patients with stage 4 disease.
Similarly, the mRNA expression of the eight E2F family members was also signi cantly correlated with tumor grade. As the tumor grade increased, the mRNA expression of E2Fs tended to be higher. The highest mRNA expression of most E2F factors appeared in grade 3 tumors (Figure 4). In conclusion, the above results indicate that the mRNA expression of eight E2F family members in gastric cancer patients is signi cantly correlated with clinicopathological parameters.
3. Genomic changes in E2F family members in patients with gastric cancer and the prediction of 50 frequently changed genes closely associated with the E2F family We used the cBioPortal online analysis tool to perform mutational analysis of the E2F family. As shown in Figure 5a, the E2F family members had mutations in all four types of gastric cancer, with the highest mutation rate found in tubular stomach adenocarcinoma (63.29%). The mutation rates in stomach adenocarcinoma, mucinous stomach adenocarcinoma, and diffuse-type stomach adenocarcinoma were 46.64%, 40.91% and 29.71%, respectively. Figure 5b shows that among 360 gastric cancer samples, 183 samples (51%) had E2F mutations. E2F1, E2F5, E2F3 and E2F6 ranked as the four genes with the highest genetic alterations, with mutation rates of 15%, 15%, 11% and 9%, respectively. To further study the potential connections among E2F family members, the STRING tool was used to mine 50 frequently changed genes in the vicinity of E2F family genes. We found that genes related to the cell cycle, including CCNE1, CCNE2, CDK2, CDK4, CDKN1B, and CDKN2A, were closely related to E2F changes (Figure 5c).

Biological function and pathway enrichment analyses of E2Fs and their 50 neighboring genes
The functions of E2Fs and their 50 neighboring genes were analyzed by GO and KEGG analyses in DAVID. GO enrichment analysis predicts gene function from three aspects, namely, BPs, CCs and MFs. As shown in Table 2, we found that in BPs, target genes were mainly enriched in transcription, G1/S transition of the mitotic cell cycle, and DNA replication initiation; in CCs, target genes were enriched in the nucleus, nucleoplasm and cytoplasm; and the MFs were mainly zinc ion binding, DNA binding and transcription factor activity. KEGG enrichment analysis showed that 58 genes were enriched in cancer-related pathways, such as the p53 signaling pathway, TGF-beta signaling pathway, and cellular senescence ( Figure 6).  (Figure 7). This indicates that E2F family members can be used as effective biomarkers for predicting the survival of gastric cancer patients. 6. Independent prognostic value of E2F mRNA expression in terms of OS in gastric cancer patients After using KM Plotter and observing that the mRNA expression of E2F1/2/3/4/5/6/7/8 may be correlated with the prognosis of gastric cancer patients, we downloaded the clinical and E2F mRNA expression data of 407 gastric cancer patients from the TCGA database (Table 3). We used survival, glmnet and other software packages to integrate and standardize the downloaded raw data and then performed univariate and multivariate Cox survival regression analyses. In the univariate analysis, we found that age, regional lymph node involvement, distant metastasis, pathological stage, and E2F2 are closely related to patient survival (Table 4). In the multivariate analysis, we found that a higher pathological stage (p= 0.036) and higher E2F2 mRNA expression (p = 0.005) were related to shorter OS of gastric cancer patients (Table 5).

E2F2 downregulation inhibits the proliferation of gastric cancer cells
We used PCR and Western blot experiments to explore the expression of E2F2 in gastric cancer cells. We found that compared with GES-1 cells, AGS and HGC27 cells had signi cantly increased mRNA and protein expression of E2F2 (Figure 8a and b). We selected AGS cells for LV-shE2F2 transfection to better understand the effect of E2F2 on gastric cancer cell proliferation. Figure 8c shows the protein expression level of LV-shE2F2. When using CCK-8 analysis to evaluate cell viability over a 3-day period, we found that the viability of the LV-shE2F2 group was signi cantly lower than that of the NC group (Figure 8d). EdU experiments showed that when E2F2 was silenced, the number of cells in the proliferating state was signi cantly reduced compared to that of the NC group (Figure 8e). These results indicate that silencing E2F2 inhibits gastric cancer cell proliferation.

E2F2 downregulation inhibits the invasion and metastasis of gastric cancer cells
To assess the effect of E2F2 upregulation on cell movement, wound-healing and Transwell assays were performed. As shown in the wound-healing assay (Figure 9a), knockdown of E2F2 signi cantly inhibited cell migration compared to that of the NC group. In addition, in the Transwell analysis, the invasive ability of the LV-shE2F2 group was reduced (Figure 9b). These results indicate that knocking down E2F2 has an inhibitory effect on the invasion and metastasis of gastric cancer cells.

Discussion:
As transcription factors regulating the cell cycle, members of the E2F family are involved in the development of various cancers (12). Although the role of E2F members in the development of certain tumors and the prognostic value of these proteins have been reported, previous studies have focused on the mechanism of individual members of the E2F family in speci c cancer types (6)(7)(8)(9). Therefore, systematic analysis of the E2F family helps us to clarify its role in tumorigenesis and development and serves as a guide to nd new tumor markers.
To achieve this goal, we analyzed the expression, mutation and prognostic value of 8 E2F factors in gastric cancer through the ONCOMINE and TCGA databases and studied the role of E2F2 in gastric cancer in detail. Bioinformatics analysis indicates that the mRNA expression of E2Fs may be related to the cancer stage and tumor grade of gastric cancer patients. In addition, a high mutation rate (51%) of E2Fs was observed in gastric cancer patients. Using STRING and PPI software to predict the genes closely related to E2Fs, we found that the cell cycle-related genes CCNE1, CCNE2, CDK2, CDK4, CDKN1B and CDKN2A are associated with this family. Related pathways that were enriched include the p53 and TGF-β signaling pathways. E2F1, the most studied transcription factor in the E2F family (10), can function as an oncogene or tumor suppressor gene to regulate tumorigenesis depending on the cellular environment (27). A large number of studies have shown that E2F1 overexpression is of great signi cance in the poor prognoses of various cancers, including lung cancer (28), breast cancer (29), esophageal cancer (30), hepatocellular carcinoma (31) and pancreatic cancer (32). According to the literature, the functional role of E2F1 in gastric cancer is different. Studies have shown that E2F1 overexpression inhibits gastric cancer progression in vitro (33). However, Xu et al. reported that compared with that in noncancerous tissue samples, the expression of E2F1 in gastric cancer tissue samples was signi cantly upregulated, and its overexpression promoted cell proliferation and tumorigenicity. Patients with higher E2F1 levels have larger tumor sizes, more advanced tumor stages, and worse survival than patients with lower E2F1 levels (34). This is similar to the results of our study. The results of the bioinformatics analysis show that E2F1 overexpression may indicate a poor prognosis and is associated with a more advanced clinical stage (stage 3).
E2F2 plays contradictory roles in the development of tumors. On the one hand, E2F2 can inhibit tumorigenesis by inhibiting cell cycle regulators. On the other hand, E2F2 can act as an "activator" to increase target expression and cause cancer (35). Previous studies have shown that changes in E2F2 protein expression are closely related to the occurrence of different cancers (36). It has been reported that knocking down E2F2 signi cantly reduces the metastatic ability of breast cancer cells, and mutations in E2F2 are related to tumor proliferation and survival in breast cancer patients (37). E2F2 functions as an oncogene in liver cancer (38), while in prostate cancer, E2F2 inhibits tumor cell proliferation by targeting miRNAs (39).
E2F3 is considered an oncogene involved in the apoptosis and proliferation of cancer cells and is related to cell invasion and migration (40). It has been found to be ampli ed in a variety of human tumors, including lung (41), bladder (42), liver (43), ovarian (44), breast (45), and pancreatic cancer (46). A study by Li et al. found that silencing E2F3 had an inhibitory effect on proliferation and an inducing effect on apoptosis in gastric cancer cells (47). There are also reports in the literature indicating that E2F3 can function as a direct target of miRNA to play a carcinogenic role in gastric cancer (48,49). Our research shows that E2F3 overexpression is associated with a poor prognosis in patients and occurs in gastric cancer cells, suggesting that E2F3 may be a therapeutic candidate for gastric cancer patients.
E2F4 is abundant in nonproliferating and differentiated cells and plays an important role in inhibiting proliferation-related genes (50). A recent study showed that E2F4 overexpression in the nuclei of breast cancer cells was associated with various advanced clinicopathological features and a poor clinical prognosis in breast cancer patients (51). Sun et al. reported that high E2F4 expression was signi cantly associated with poor OS, FP and PPS in lung cancer patients (52), while in digestive tract tumors, E2F4 was found to promote the development of liver cancer, colorectal cancer and gastric cancer (10). In our study, high expression of E2F4 was associated with a poor prognosis in gastric cancer patients.
Previous data have shown that E2F5 is overexpressed in various types of human cancer, including breast cancer, ovarian epithelial cancer, prostate cancer, hepatocellular carcinoma, and colorectal cancer, and is closely related to cancer progression and prognosis (53)(54)(55)(56)(57). In a study by Li et al., knocking out E2F5 had a signi cant inhibitory effect on the growth rate of gastric cancer cells, suggesting that E2F5 may be an oncogene in gastric cancer (58). Our results indicate that elevated E2F5 mRNA expression levels are found in gastric cancer and that high expression is associated with an advanced cancer stage and tumor grade and a poor survival rate.
E2F6-8 have similar functions as a repressor group, but they exhibit completely different molecular mechanisms (59). Compared with E2F1-5, E2F6-8 lack the transactivation domain and Rb binding domain, allowing them to function as independent protein transcriptional repressors (60). In addition, it has been shown that E2F6 plays a repressive role by interacting with the multicomb complex, while E2F7 and E2F8 can form homodimers or heterodimers to inhibit the transcription of target genes (61). It has been reported that E2F6 expression is related to the prognosis of malignant tumors such as pancreatic (62), breast (63) and nasopharyngeal cancer (64). In gastric cancer, Li et al. discovered that downregulation of E2F6 expression inhibited the proliferation and invasion of gastric cancer cells, suggesting that E2F6 may play a carcinogenic role in gastric cancer. E2F7 has been found to be involved in the development of breast (65), gallbladder (66), pancreatic (67), and cervical cancer (68), among others. E2F8 has also been shown to be involved in the development of various cancers, including breast (69), lung (70), and liver cancer (71), but the roles of E2F7 and E2F8 in gastric cancer have not been reported.
After analyzing the expression of E2F family members in gastric cancer, we focused on the prognostic value of the E2F family in gastric cancer. The results of the online website Kaplan-Meier Plotter show that high expression of E2F1/2/3/4/5 may be related to poor OS in gastric cancer patients, while the high expression of E2F6/7/8 is related to better OS. Using univariate and multivariate Cox regression analyses, it was found that among the eight E2F members, E2F2 can be used as an independent prognostic factor for gastric cancer; this piqued our research interest. Since the E2F family is also directly or indirectly involved in the processes of cell proliferation, invasion and metastasis, we hoped to elucidate the role of E2F2 in gastric cancer cells. The results showed that the downregulation of E2F2 inhibited not only the proliferation of gastric cancer cells but also their invasion and metastasis. Therefore, we believe that E2F2 plays a role in promoting cancer in gastric cancer cells. However, the downstream signaling pathways that regulate the biological function of these tumor cells and their underlying molecular mechanisms need further study in the future.

Conclusion
In conclusion, our results indicate that overexpression of eight E2F members was signi cantly correlated with the clinical cancer stage and pathological tumor grade in gastric cancer patients. A high mutation rate (51%) for E2Fs was observed in gastric cancer patients. In addition, E2F2 can be used as an independent prognostic factor for gastric cancer, and downregulation of E2F2 can inhibit the proliferation, invasion and migration of gastric cancer cells. These results indicate that E2F2 can be used as a potential prognostic marker and therapeutic target for gastric cancer, and further study on E2F2 may improve the survival and prognostic accuracy of gastric cancer patients. The focus of this study is to provide new ideas for the clinical diagnosis and prognostic assessment of gastric cancer via a combination of bioinformatics analysis and cell experiments. Our results provide an important bioinformatics foundation and related theoretical foundation for guiding follow-up research on gastric cancer.

Availability of data and materials
The data that support the ndings of this study come from the public free-charged database, and some or all data, models, or code generated or used during the study are available from the corresponding author by request.

Ethics approval and consent to participate
This article does not contain any studies with human participants performed by any of the authors.

Consent for publication
Not applicable.  Figure 1 Transcriptional expression of E2Fs in 20 different types of cancer diseases (ONCOMINE database). Note: Differences in transcriptional expression were compared by Student's t test. The cut-off p-value and fold change were as follows: p-value, 0.01; fold change, 1.5; gene rank, 10%; and data type, mRNA.  Association of E2F mRNA expression with individual cancer stages in GC patients.

Figure 4
Relationship between mRNA expression of E2F family members and tumor grades of GC patients. Genomic changes in E2F family members in patients with gastric cancer and the prediction of 50 frequently changed genes closely associated with the E2F family. (a) Genome-speci c changes in E2F family members in 5 gastric cancer data sets. (b) A high mutation rate (51%) of E2F family members was observed in GC patients. (c) A network of E2F mutations and their 50 frequently altered genes closely associated with the E2F family was constructed.

Figure 6
Pathway enrichment map of E2F family members and 50 closely associated genes.

Figure 7
The prognostic value of the mRNA level of E2F factors in GC patients (Kaplan-Meier Plotter).  E2F2 downregulation inhibits the invasion and metastasis of gastric cancer cells. (a) In the wound-healing assay, the residual wound was much wider in the LV-shE2F2 group than in the NC group at 48 hours after scratching. (b) Transwell assays revealed markedly decreased migration and invasion abilities of the LV-shE2F2 group compared with those of the NC group. Data represent the means ± SD. **p<0.01, as assessed by Student's t test.