The roles of E2Fs in gastric cancer: the transcription factors E2F1/2/3/4/5 as potential therapeutic targets and E2F6/7/8 as new prognostic biomarkers in gastric cancer


 Background E2F is a family of transcription factor proteins with multiple functions; E2F proteins are involved in cell cycle regulation, cell differentiation, the DNA damage response and cell death. Studies have shown that E2Fs have prognostic significance 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 website 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. The prognostic value of the E2Fs were determined by Kaplan-Meier Plotter. A protein-protein interaction (PPI) network was constructed using the STRING database. The functions and pathways of E2F family and its neighboring 50 frequently changed genes were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) software. Finally, quantitative PCR was used to verify the expression of E2Fs in gastric cancer cells. Results The expression patterns of 8 E2F members were significantly related to the clinical cancer stage and tumor grade of gastric cancer patients. High mRNA expression of E2F1/2/3/4/5 was a prognostic factor for poor OS in gastric cancer patients, while high expression of E2F6/7/8 was associated with better OS. In addition, a high mutation rate (46%) for E2Fs was observed in patients with gastric cancer. The expression levels of E2F1/2/3/5/8 in AGS and HGC27 cell lines were higher than those in normal cells, while E2F7 showed the opposite trend. Conclusions In summary, these results indicate that E2F1/2/3/4/5 can be used as potential therapeutic targets in gastric cancer, and E2F6/7/8 can be used as new prognostic markers to improve the survival rate and prognostic accuracy in gastric cancer.


Abstract
Background E2F is a family of transcription factor proteins with multiple functions; E2F proteins are involved in 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 website 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. The prognostic value of the E2Fs were determined by Kaplan-Meier Plotter. A protein-protein interaction (PPI) network was constructed using the STRING database. The functions and pathways of E2F family and its neighboring 50 frequently changed genes were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) software. Finally, quantitative PCR was used to verify the expression of E2Fs in gastric cancer cells.

Results
The expression patterns of 8 E2F members were signi cantly related to the clinical cancer stage and tumor grade of gastric cancer patients. High mRNA expression of E2F1/2/3/4/5 was a prognostic factor for poor OS in gastric cancer patients, while high expression of E2F6/7/8 was associated with better OS.
In addition, a high mutation rate (46%) for E2Fs was observed in patients with gastric cancer. The expression levels of E2F1/2/3/5/8 in AGS and HGC27 cell lines were higher than those in normal cells, while E2F7 showed the opposite trend.

Conclusions
In summary, these results indicate that E2F1/2/3/4/5 can be used as potential therapeutic targets in gastric cancer, and E2F6/7/8 can be used as new prognostic markers to improve the survival rate and prognostic accuracy in gastric cancer.

Introduction:
Gastric cancer, the second leading cause of cancer-related deaths, is one of the most common malignant tumors in the world (1). Despite improvements in surgical techniques and chemotherapy regimens, patient treatment results 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, the identi cation of prognostic markers related to gastric cancer is crucial to developing the individualized treatment plans for gastric cancer patients and improving the clinical outcomes of patients.
E2F is a family of transcription factor proteins, and E2F proteins 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 cancer (5), ovarian cancer (6), bladder cancer (7), prostate cancer (8), lung cancer (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, and 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 the 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 method to explore the molecular mechanisms of tumorigenesis and development and 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 or copy number variations, we studied the expression and mutation of the E2F family in gastric cancer patients in detail to determine the expression patterns, potential functions and unique prognostic values of E2F proteins in gastric cancer. Materials And Methods:

ONCOMINE database
The ONCOMINE database (www.oncomine.org) is an integrated online cancer microarray database containing data from DNA-and RNA-sequencing (RNA-seq) analyses used for differential expression classi cation of common cancer types and the corresponding normal tissues, as well as clinical and pathological analyses (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 cut-off and fold change threshold were as follows: p-value: 0.01, fold change: 1.5, gene grade: 10%, data type: mRNA.

UALCAN
Page 4/25 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. It 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 or ovarian cancer. To analyze the overall survival (OS), progression-free survival (FP) and postprogression survival (PPS) of gastric cancer patients, patient samples were divided 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 the signi cant associations of PPIs. (20). In this study, the STRING database was used to analyze the E2F family and its neighboring 50 frequently changed genes. 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.

Cell culture
The AGS, HGC27 and GES-1 cell lines 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% CO2. FBS and RPMI-1640 medium were purchased from Gibco (New York, USA).

Real-time quantitative PCR (qPCR)
Total RNA (1 µg) was isolated using a TRIzol (Invitrogen, Carlsbad, California, USA) kit, and its 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) method for relative mRNA expression was analyzed by 2-ΔΔ. The primer sequences are shown in Table 1. The following cycling conditions were used: 95 minutes or 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. The primers are shown in Table 1. Table 1 The primer of hub genes.

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 Fig. 1 and Table 2 (Fig. 5). This indicates that E2F family members can be used as effective biomarkers for the survival of gastric cancer patients.
4. E2F family genomic changes in patients with gastric cancer and the prediction of 50 frequently changed neighboring genes We used the cBioPortal online analysis tool to perform mutational analysis of the E2F family. As shown in Fig. 6A, 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 6B shows that among 360 gastric cancer samples, E2Fs were mutated in 183 samples (51%). E2F1, E2F5, E2F3 and E2F6 ranked the highest four genes of genetic alterations, and their mutation rates were 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, CDKN2A, etc., were closely related to E2F changes (Fig. 6C).

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, biological process, cell composition and molecular function. As shown in Table 3, we found that in biological processes, target genes were mainly enriched in transcription, the G1/S transition of the mitotic cell cycle, and DNA replication initiation, and in cell components, target genes were enriched in the nucleus, nucleoplasm and cytoplasm. The molecular functions 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 (Fig. 7).

Expression of E2Fs in gastric cancer cells
We conducted qPCR experiment to con rm the mRNA expression levels of E2Fs in gastric cancer (Fig. 8).
The results of the qPCR analyses con rmed that the expression of E2F1/2/3/4/5/6 in AGS and HGC27 cell lines was higher than that in GES-1 cell lines, while that of E2F7 showed the opposite trend. However, in our current study, no differential expression of E2F8 in gastric cancer was found, which suggests that we may need to conduct more experiments to study the role of E2Fs.

Discussion:
As transcription factors that regulate the cell cycle, E2F family members are involved in the development of various cancers (12). Although the role of E2Fs in tumorigenesis and their prognostic value have been con rmed (6)(7)(8)(9), it is still necessary to clarify the different roles of E2F family members in gastric cancer.
In this study, we analyzed the expression, mutation and prognostic value of different E2F family members in gastric cancer.
Our results showed that mRNA expression of all eight E2F factors was found and that the mRNA expression of E2Fs was closely related to the cancer stage and tumor grade of gastric cancer patients.
The prognostic analysis results showed that high expression of E2F1/2/3/4/5 was associated with poor OS in gastric cancer patients, while high expression of E2F6/7/8 was associated with better OS. In addition, a high mutation rate (51%) for E2Fs was observed in gastric cancer patients. Adjacent genes closely related to E2Fs were predicted, and genes related to the cell cycle were found to include CCNE1, CCNE2, CDK2, CDK4, CDKN1B, and CDKN2A. Related enrichment pathways included the p53 signaling pathway and TGF-beta signaling pathway.
E2F1 is the most studied transcription factor in the E2F family (10). E2F1 can function as an oncogene or tumor suppressor gene to regulate tumorigenesis according to the cellular environment (22). 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 (23), breast cancer (24), esophageal cancer (25), hepatocellular carcinoma (26) and pancreatic cancer (27). According to previous studies, the functional role of E2F1 in gastric cancer is different. Studies have shown that E2F1 overexpression inhibits gastric cancer progression in vitro (28). However, in a study by Xu et al., 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 poorer survival rates than patients with lower levels (29). This is similar to the results of our study. Overexpression of E2F1 indicated a poor prognosis and was associated with a more advanced clinical stage (stage 3).
E2F2 plays dual 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 (30). Previous studies have shown that changes in E2F2 protein expression are closely related to the occurrence of different cancers (31). It has been reported that knocking down the expression of 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 (32). E2F2 functions as an oncogene in liver cancer (33), while in prostate cancer, E2F2 inhibits tumor cell proliferation by targeting miRNAs (34). A study by Wang et al. showed that E2F2 was overexpressed in gastric cancer tissue samples. High levels of E2F2 were positively correlated with poor tumor differentiation, lymph node metastasis, more advanced stages and poorer OS. Silencing E2F2 signi cantly reduced cell proliferation, invasion and migration (35). This suggests that E2F2 plays a carcinogenic role in gastric cancer. Our research also supports this nding, which shows that E2F2 can be used as an effective biomarker for the diagnosis and treatment of gastric cancer.
E2F3 is considered an oncogene involved in the apoptosis and proliferation of cancer cells and related to cell invasion and migration (36). It has been found to be ampli ed in a variety of human tumors, including lung cancer (37), bladder cancer (38), liver cancer (39), ovarian cancer (40), breast cancer (41), pancreatic cancer (42), etc. A study by Li et al. found that silencing E2F3 had an inhibitory effect on proliferation and inducing effect on apoptosis in gastric cancer cells (43). 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 (44,45). Our research shows that overexpression of E2F3 is associated with a poor prognosis in patients and occurs in gastric cancer cells, suggesting that E2F3 may be a candidate therapeutic target for gastric cancer patients.

E2F4 is abundant in nonproliferating and differentiated cells and plays an important role in inhibiting
proliferation-related genes (46). A recent study showed that overexpression of E2F4 in the breast cancer cell nucleus was associated with various advanced clinical pathological features and a poor clinical prognosis in breast cancer patients (47). Sun et al. found that high E2F4 expression was signi cantly associated with poor OS, FP and PPS in lung cancer patients (48), 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 (49)(50)(51)(52)(53). In a study by Li et

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.

Competing interests
The authors declare that they have no competing interests. Authors' contributions HL and SZ analyzed the data and wrote the manuscript. LS, PW and SL assisted in editing the manuscript. YM, ZL and GW contributed to the design of the study. WQ and JL are the corresponding authors of the paper. All authors read and approved the nal manuscript. Figure 1 Transcriptional expression of E2Fs in 20 different types of cancer diseases (ONCOMINE database). Note:

Figures
Difference of transcriptional expression was compared by students' t-test. Cut-off of p value and fold change were as following: p value: 0.01, fold change: 1.5, gene rank: 10%, data type: mRNA.  Relationship between mRNA expression of E2F family members and tumor grades of GC patients.  Pathways enrichment map of E2Fs and their 50 neighboring genes.