EFNA5 suppresses cell proliferation and tumor metastasis in hepatoma via epithelial-to-mesenchymal transition

Abstract

Background

EphrinA5 belongs to a subclass of ephrin ligands. Abnormal signal transduction of EFNA5 shows a relationship to the development of various tumors. In this study, we explored the level of EFNA5 in hepatoma cells and the influence of up regulation of EFNA5 expression level on the proliferation, invasion, and migration of HepG2 and LM3 cells. Additionally, this work focused on examining its possible mechanism of action, and future impacts on clinical practice.

Methods

Immunohistochemistry was utilized to explore the connection between EFNA5 and hepatoma. Real-time quantitative polymerase chain reaction was used for determining the expression levels of EFNA5 in several hepatoma cell lines and normal hepatocytes. Cells were transfected with a pCMV3-EFNA5-flag plasmid and an EFNA5 plasmid. The expression efficiency of EFNA5 was identified through qRT-PCR. For the purpose of further identifying cell proliferation, the Cell Counting Kit-8 assay was applied. To identify changes of cell migration and invasion ability, Transwell and Boyden tests were utilized. Western blot was employed to identify the expressions mof EFNA5 and possible downstream molecules.

Results

Data acquired from The Cancer Genome Atlas demonstrated that the level of EFNA5 in hepatoma was significantly downregulated in relative to the normal hepatocytes (P < 0.05). Upregulation of EFNA5 expression in hepatoma cells hindered the proliferative, invasive, and migratory ability of cells (P < 0.05). Additionally, EFNA5 downregulated the level of epithelial–mesenchymal transition-related molecules and EGFR.

Conclusions

The expression of EFNA5 was low in hepatoma cells. An increase in EFNA5 levels hinders the proliferation, invasion, and migration of hepatoma cells. These effects may occur through inhibition of hepatoma epithelial–mesenchymal transition by EFNA5. Moreover, the study on the mechanisms of proliferation, invasion and metastasis of hepatoma provides a novel theoretical basis, and may influence the clinical practice of tumor treatment in the future.

Introduction

At present, hepatoma is the most vital pathological category of primary hepatocarcinoma, and around 700,000 people across the world die of hepatoma annually. Compared with other solid tumors, its overall morbidity and mortality are among the highest globally [1]. Genomic analysis offers a clear picture of the main drivers which are accountable for tumor initiation and progression. Each hepatoma possesses an average of 40 genomic aberrations and few can be regarded to be drivers. Common mutations influence telomere maintenance [mutations in telomere reverse transcriptase (TERT)], Wnt pathway activation [mutations in catenin beta 1 (CTNNB1)], inactivation of cellular tumor antigen p53 (p53, encoded by TP53), chromatin remodeling [mutations in AT-rich interaction domain 1 A (ARID1A)], Ras signaling, mammalian target of rapamycin (mTOR) signalling and oxidative stress pathway activation. At present, merely a handful of these drivers are found to be druggable targets, including amplification of fibroblast growth factor 19 (FGF19) [2].

EFNA5 belongs to EphrinA subclass [3]. EFNA5 regulates cell adhesion and cytoskeleton remodeling by activating EPHA3 receptor [4]. Recent related research results have proved that the level of EFNA5 in chondrosarcoma, glioblastoma, colon cancer, hepatoma and kidney cancer is significantly down-regulated [5–8]. EFNA5 have been found to develop a highly variable cataract phenotype regarding morphology, severity, progression, and penetrance [9]. However, by contrast, the level of EFNA5 in hepatoma cell lines is specifically up-regulated, but unfortunately, these studies are underexpored. Recently, it has been indicated that the level of EFNA5 was positively associated with the prognosis of ovarian cancer, but this study did not compare the expression level of EFNA5 with that of normal tissues [10]. The difference in the level of EFNA5 shows a relationship to the biological characteristics of the tumor itself, which supports the hypothesis that the regulation of EFNA5 expression might become an effective measure in tumorigenesis. The down-regulation of EFNA5 may be related to the decrease of cell adhesion, which may be the potential causation of tumor spread. At the same time, previous researchers found that the aggressive growth and tumor spread in high-grade traditional chondrosarcoma and dedifferentiated chondrosarcoma were significantlyly related to the down-regulation of EFNA5 in chondrosarcoma. From this point of view, EFNA5 may make a protective impact on helping tumor to progress. The reconstruction of EFNA5 expression in chondrosarcoma cells can clarify the existing problem and may shed light on the role of EFNA5 in this tumor model [11]. In addition to its role in reverse signal transduction, EFNA5 makes an impact on tumor initiation and development by activating its known receptors (including EphA8, EphA7, EphA5, EphA4, EphA3, EphA2 and EphB2).

Epithelial–mesenchymal transition or EMT refers to a biological process through which cuboidal, tightly packed, and non-motile epithelial cells use a loosely organized mesenchymal or fibroblast-like phenotype possessing characteristics including reduced intercellular adhesion, loss of apical–basal polarity, gain of motility and invasive ability, elevated resistance to apoptosis, as well as enhanced ability of ECM production [12, 13]. In the development of tumor, EMT means the transformation of epithelial cells to mesenchymal phenotype [14, 15]. After acquiring mesenchymal markers, the motility of tumor cells will increase, and there is a high risk of lymph node metastasis or distant metastasis. The EMT process under various contexts were shown to be activated by several signaling molecules. For instance, EMT related to gastrulation is activated through the canonical Wnt signaling pathway, the TGFβ superfamily proteins Nodal and Vg1 and growth factors including FGF, and EGF. In the process of neural crest formation, EMT is triggered by signaling molecules including Wnt, BMP, FGF, and Notch. Type II EMT is triggered by factors including VEGF and TGFβ, while type III EMT, or EMT related to metastasis is triggered by a large set of signaling molecules like Wnt, TGFβ, BMP, FGF, EGF, HGF, PDGF, VEGF, Estrogen, and SCF. These molecules can stimulate a variety of signaling pathways, which can thus activate a small set of transcription factors (TFs) or master regulators of EMT. These contain Snail Family proteins Snail1 (Snail), Snail2 (Slug), Zinc finger E-box binding (Zeb) homeobox family proteins Zeb1 and Zeb2, and TWIST family proteins Twist1 and Twist2. Collectively, these TFs can hinder level of epithelial markers including E-cadherin, Claudin, Occludin, Mucin-1, PTEN, and RKIP as well as activate mesenchymal markers like N-cadherin, Vimentin, Vitronectin, and Matrix Metalloproteases [12]. Although there are many related articles published in the literature every year, there are few EMT involving hepatoma cells, and the exact pathway of this process and the interaction with other specific events in tumor microenvironment (such as tumor angiogenesis), and the interaction between inflammatory reaction and liver fibrosis characteristics are still challenging to clarify [16, 17]. Therefore, the occurrence of hepatoma-related epithelial–mesenchymal transition should not only consider tissue and circulating biomarkers, but also take the background of surrounding liver parenchyma into account.

There are few reports focusing on the role of EFNA5 in primary hepatoma. In early reports, EFNA5 exerts an anti-cancer impact on the onset and development of various tumors, and its possible mechanism is to regulate cell adhesion and cytoskeleton remodeling. In TCGA database, it was discovered that there exist differences between the hepatoma and the adjacent tissues. However, there is no relevant evidence to demonstrate whether EFNA5 influences the incidence and development of human hepatoma, whether it may affect hepatoma invasion and migration through regulating the epithelial–mesenchymal transition of hepatoma cells, and whether it can also regulate the development of hepatoma through controlling EGF/EGFR signaling pathway or regulating the incidence and development of hepatoma via other signaling pathways [14]. Therefore, the current work was aimed at exploring the connection between the differential expression of EFNA5 and the onset as well as the development of hepatocytes and the possible regulatory mechanism.

Materials and methods

Data acquisition

Complying with The Cancer Genome Atlas (TCGA) database publication guidelines and access policies, gene expression profiles (RNA sequencing) with corresponding clinical information of hepatoma and normal samples were acquired from the TCGA database (https://portal.gdc.cancer.gov/).

EFNA5 expression and survival analysis of hepatoma patients

This study was based on the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) in hepatocellular cancer and paracancerous tissues [18]. Using the Kaplan–Meier plotter, the critical part of EFNA5 in the survival of mammary cancer sufferers was further discussed.

Pathway enrichment analysis of EFNA5 related genes

With the TCGA database, a cohort of hepatoma samples containing EFNA5 was screened [19]. The data text included totally 474 liver tissue samples (including 50 samples of normal liver tissue and 424 samples of hepatoma tissue). According to normal tissue samples and tumor samples, the samples were grouped, and differential gene expression was explored using Wilcox-test. A list of differentially expressed genes was acquired. To conduct KEGG and GO enrichment analysis on the significantly expressed genes, the clusterProfiler package in R language (version 4.1.3) was employed. Meanwhile, the genes were sequenced according to the significance of P value. The most significant biological characteristics and pathways were listed.

Construction of protein network of EFNA5 related genes

Using the STRING database, the interaction between genes or proteins is further analyzed. At first, EFNA5-related genes (ERGs) are imported into the STRING database(http://string-db.org) [20]. In addition, the protein network of EFNA5-related genes is further constructed to provide the interaction analysis of EFNA5-related proteins afterwards.

Cell culture and transfections

Four human hepatoma cell lines, including HepG2, PLC/PRF5, LM3, HuH7 and a normal human hepatocyte line LO2, were selected in the current experiment, all of which were provided by the Cancer Center Laboratory of Southern Medical University. Among them, HepG2 and HuH7 cells were negative for HBV. The expression of HBsAg could be detected by PLC/PRF5. LO2 cell line is a cell line with typical morphological characteristics of hepatocytes. Cell transfection was carried out with Lipofectamine 3000 (Thermo Scientific, Waltham, MA, USA). Quantitative real-time PCR was adopted for detecting the expression level of EFNA5.

Main reagents for the experiment

High glucose DMEM, fetal bovine serum and RPMI-1640 medium involved in the current experiment were purchased from Gibco Biotechnology Company. SYBR Premix Ex Taq fluorescent quantitative PCR kit and DNA molecular weight standard Marker were purchased from TaKaRa Biotechnology Company. EphrinA5 primer sequence was designed and synthesized by Invitrogen Company of the United States. Trizol kit, OptiMEN, Lipofectamine 3000 liposome and protein pre-dyed molecular weight standard Marker were all purchased from Invitrogen Biotechnology Company. RIPA protein lysate is a product of Biyuntian Biotechnology Company. Enhanced chemiluminescence detection kit is a product of Syme Biotechnology Company. In this experiment, the used antibodies are as follows: EFNA5 (Cat. No. ABP50058, Abbkine), GAPDH (Cat. No. AP0063, Bioworld Technology), B-actin (Cat. No. 8480, CST), EGFR (Cat. No. 51067-2-AP, PTG), E-cadherin (Cat. No. 20874-1-AP, PTG), N-cadherin (Cat. No. 13769-1-AP, PTG), c-Jun (Cat. No. 9165, CST), c-Myc (Cat. No. ab32072, Abcam), and vimentin (VIM) (Cat. No. 60330-1-Ig, PTG); HRP labeled anti-rabbit/mouse secondary antibody were purchased from Epitomics Biotechnology company.

Total RNA extraction and quantitative real-time PCR

Based on the experimental method provided in the instruction manual of Trizol kit of Takara Company, the extraction of total RNA was performed. In line with the reaction system and reaction conditions in the following table, the mRNA expression level of the target gene was quantitatively detected. Besides, the final result is analyzed using the software of LightCycle 480 II (Roche) system. Using the relative quantitative method, the difference of mRNA expression level was compared. The control group was set up in the experiment, with B-actin being the internal reference; ΔΔCt = [mean Ct value of target mRNA (experimental group) − mean Ct value of B-actin (experimental group)] − [mean CT value of target mRNA (control group) − mean CT value of B-actin]. The following primer sequences were involved: EFNA5 plasmid upstream sequences: 5′-CAGGTGTCCACTCCCAGGTCCAAG-3′; EFNA5 plasmid downstream sequence: 5′-GGCAACTAGAAGGCACAGTCGAGG-3′; EFNA5 upstream primer: 5′-CACGCACGCTTCTCCATCT-3′; and EFNA5 downstream primer: 5′-TTGGAAGTGTGGTCGCAGG-3′.

Immunohistochemistry

hepatoma tissue arrays (HLivH180Su17) were purchased from Xinchao Company. In line with the manufacturer’s instruction, the IHC test kit (PV-6000) for EFNA5 protein expression analysis was bought from ZsBio (Beijing, China) and used. The Average optical density value is calculated by dividing IntDen score by Area score.

Cell counting Kit-8 assay

CCK8 detection is a widely used method which can be adopted for measuring cell viability and proliferation. The current experiment employs Nuo CCK-8 Cell Counting Kit (Dongren Chemical Tec) from Weizan Company. Cells were transfected with plasmids and 5000 cells per well were inoculated into 96-well plates. Then, CCK-8 reagent was supplemented. The cells were subject to incubation for 48 h. In addition, the absorbance of each group of cells was measured (λ = 450 nm) on the ultraviolet spectrophotometer respectively with the line graph of the obtained OD value being plotted with time as horizontal axis and 450 nm OD value as vertical axis.

Transwell

The migration ability of HepG2 and LM3 cells was compared between transfected plasmid and empty plasmid control. Cells (1.5 × 10⁵) were plated in a transwell chamber and cultured at a constant temperature for 48 h in triplicate. To take photomicrographs of migrating cells, five high-power fields were randomly selected under the light microscope. Using Image J software, the number of cells passing through the membrane was analyzed with the histogram being drawn.

Boyden invasion experiment in vitro

To compare the invasion ability of HepG2 and LM3 cells with transfection plasmid and empty plasmid control, cells (1.5 × 10⁵) were inoculated in a Transwell chamber covered with matrigel in advance, and cultured at a constant temperature for 48 h in triplicate. Micrographs of invasive cells were taken and histograms were drawn.

Western blotting

After the preparation of the protein gel (Epizyme Biotech, Shanghai, China), the protein sample (protein content is 25 µg–50 µg/hole) was sampled. Protein separation was carried out by gel electrophoresis. The protein was transferred to the membrane (0.45 μm nitrocellulose membrane or polyvinylidene fluoride membrane). The parameters of the membrane transfer instrument were the constant current of 350 mA, and the membrane was transferred in an ice water bath for 2 h. 5% skimmed milk powder was used for blocking, and the membrane was soaked in a suitable sealing solution and placed in a slow shaking table for 1 h. The antibody would be subjected to incubation with the specifically recognized protein primary antibody overnight on a slow shaking table at 4 °C for 16 h to bind the antibody to the target protein. The second antibody conjugated with enzyme was supplemented to the membrane and incubated for 1 h under the condition of room temperature. Using ECL chemiluminescence method to detect the linked enzyme or fluorescence, the signal of the membrane is detected and quantified by imaging system (Sage Creation Science, Beijing, China). The intensity of the signal generated by it is in direct proportion to the number of proteins in the sample, showing the depth of black and the thickness of stripes in the image. The quantification of protein expression level can be performed automatically through the measurement of the gray value with the software Image J.

Statistical analysis

Statistical analysis uses statistical software IBM SPSS20.0 for data statistics and data analysis. All the actual data results are represented by the mean soil standard deviation. All the used measurement data are from at least 3 independent repeated experiments. The functional experimental data of hepatoma cell line EFNA5 overexpression group and negative control group were confirmed by independent sample T test. The experimental data are all drawn up, with the difference being of statistical significance (P < 0.05).

Results

EFNA5 expression is related to good prognosis in hepatoma

Using the TCGA database, a cohort of hepatoma samples containing EFNA5 was screened and the data text included totally 474 samples (including 424 hepatoma tissue samples and 50 normal liver tissue samples). The results demonstrated that the level of EFNA5 in tumor tissues of hepatoma patients was lower in relative to with that in normal liver tissues, and the expression difference was statistically significant (P < 0.01, Fig. 1A). The K–M plotter database was used for further studying the vital role of EFNA5 in the survival of hepatoma patients (RNAseq ID: 1946, Fig. 1B). Survival prognosis analysis was proceeded on the dataset LIHC-EFNA5, and the result demonstrated that the expression level of EFNA5 influenced the prognosis of patients and showed a positive association, that is, high expression of EFNA5 indicated a good prognosis (high expression of EFNA5 significantly associated with OS, P = 0.0012). The protein interaction network of EFNA5-related genes was designed using STRING (Fig. 1C). It was found that EPHA8, EPHA7, EPHA5, NTRK2, EPHA4, EPHA3, EPHA2, EPHA1, RPTOR and EPHB2 were participated in the protein interaction network of EFNA5, and they were the key meeting points. Obtaining and constructing the interaction structure network of EFNA5 related genes through STRING database will provide enough theoretical support for further exploring the mechanism of EFNA5 in hepatoma. The genes were sequenced according to the significance of P value. The most significant biological characteristics and pathways were listed (Fig. 1D). The results indicated that the differential genes were mostly concentrated in the intrinsic component of plasma membrane in the cell composition and extracellular matrix. Molecular function is enriched in the signal pathway of glycosaminoglycan binding and extracellular matrix structural constituent. During biological processes, they are mainly concentrated in the signaling channels including locomotion, cell motility, and biological adhesion. The KEGG visualization demonstrated that various genes are mainly concentrated in ECM receptor interaction, retinol metabolism, arachidonic acid metabolism, drug metabolism cytochrome p450, PPAR signaling pathway, metabolism of xenobiotics by cytochrome p450, etc. (Fig. 1E). To further study the relationship between EFNA5 and hepatoma, we explored its expression in liver tumor arrays through immunohistochemistry and the results showed that EFNA5 expression in adjacent tissue was notably higher than that recorded in liver tumor tissue (p < 0.001) (Fig. 1F, G).

Fig. 1 [Images not available. See PDF.]

EFNA5 expression is associated with good prognosis in hepatoma. A TCGA analysis of EFNA5 expression difference between liver tumor tissue and normal liver tissue. **P < 0.01. B KM-plotter analysis of connection between EFNA5 expression and prognosis in hepatoma. C STRING online analysis of EFNA5 and related genes. D GO enrichment analysis of differential genes in hepatoma. E KEGG enrichment analysis of differential genes in hepatoma. F, G Representative IHC images and average density scores of EFNA5 expression in hepatoma and adjacent tissues

EFNA5 hinders the proliferation of hepatoma cells

To further study the level of EFNA5 in hepatoma cells, qRT-PCR was made to detect the expression of EFNA5 in four hepatoma cells. Consistent with the results, the expression of EFNA5 in HepG2, LM3, Huh7 and PLC/PRF5 cells was significantly down-regulated (Fig. 2A). To improve the expression level of EFNA5, two hepatoma cell lines were transfected with EFNA5-flag plasmid. QRT-PCR was employed to identify the level of EFNA5 in transfected cells. Based on the findings, the RNA level of EFNA5 in overexpression group was significantly higher than that in control group (HepG2: 56 times higher; LM3: 47-fold increased; P < 0.005, Fig. 2B). A blank plasmid transfection control group was established. The two groups of hepatoma cell lines were transiently transfected. The next cell function experiment was performed 24 h after transfection, and the conventional plating was carried out. Subsequently, the proliferation ability of EFNA5 overexpressed hepatoma cells was measured by CCK-8 method relative to the control group. The obtained results indicated that the elevated expression level of EFNA5 hindered the proliferation of hepatoma cells (Fig. 2C, D).

Fig. 2 [Images not available. See PDF.]

EFNA5 inhibits the proliferation of hepatoma cells. A EFNA5 expression in hepatoma cell lines and normal hepatic epithelial cell line at the RNA levels by quantitative real-time PCR. ***P < 0.001. B Expression of EFNA5 in EFNA5-overexpressing HepG2 and LM3 cells, as detected by quantitative real-time PCR assays. C, D The proliferation ability of EFNA5 overexpressed hepatoma cells in LM3 and HepG2 cells was measured by CCK-8 method compared with the control group

EFNA5 hinders the migration and invasion of hepatoma cells

Transwell experiment was applied for further exploring the influence of the change of EFNA5 expression on the migration ability of hepatoma cells. Transient transfection of plasmid upregulated the expression level of EFNA5 in two hepatoma cells. The findings indicated that in relative to the control group (NC), the cell migration ability of EFNA5 overexpression group was notably lowered (Fig. 3A, B) (P < 0.01). Based on cell invasion experiment (Boyden experiment), the influence of up-regulation of EFNA5 expression in hepatoma cells on cell invasion ability was explored. The results demonstrated that (Fig. 3C, D), compared with NC group, the invasion ability of the two hepatoma cell lines in EFNA5 overexpression group was significantly decreased (P < 0.01). Based on the obtained findings, we determined that EFNA5 was an inhibitory factor for the invasion of hepatoma cells.

Fig. 3 [Images not available. See PDF.]

EFNA5 can inhibit the migration and invasion of hepatoma cells. A, B Representative images and quantitative analysis of cell migration based on Transwell assays. C, D The representative images and quantitative dat of the EdU assay in HepG2 and LM3

EFNA5 inhibits EMT in hepatoma cells

EMT is featured by elevated level of N-cadherin and Vimentin, and reduced level of epithelial markers (including E-cadherin). Many studies have suggested that EMT exerts a vital impact on the occurrence, invasion, and metastasis of tumors [15, 17]. According to the function of regulating cell adhesion and cytoskeleton remodeling, to further explore the possible mechanism of EFNA5 and whether it is associated to the onset of EMT in hepatoma, and to demonstrate whether the up-regulation of EFNA5 expression affects the onset and development of EMT in hepatoma cells, the levels of EMT-related obvious molecules in EFNA5 plasmid transfection group and control group were identified using western blotting (Fig. 4A–C). Overexpression of EFNA5 inhibits the protein levels of B-actin, GAPDH, c-Myc, c-Jun, E-cadherin, EGFR, N-cadherin, Vimentin and EFNA5 in HepG2 and LM3 cells, while the level of E-cadherin tends to increase. This indicates that EFNA5 hinders the proliferation, invasion, and metastasis of hepatoma cells through controlling the occurrence of EMT. EFNA5 inhibits the occurrence of EMT in hepatoma cells.

Fig. 4 [Images not available. See PDF.]

EFNA5 inhibits EMT in hepatoma cells. A–C Analysis of epithelial–mesenchymal transition markers by western blotting and quantitative analysis in EFNA5 overexpression cell lysates. Overexpression of EFNA5 suppressed the protein levels of GAPDH, c-Myc, c-Jun, E-cadherin, N-cadherin, VIM, EGFR, and EFNA5 in HepG2 and LM3 cells

Discussion

Hepatoma refers to a commonly seen gastrointestinal malignant tumor [3], which is featured by high morbidity and mortality. Because hepatoma cells have strong proliferative ability, invasion and metastasis, and most patients are difficult to diagnose early, the overall survival time of hepatoma patients is significantly shortened. The pathogenesis of hepatoma has not been clarified, and there are many high-risk factors. An emerging reason for hepatoma refers to the metabolic syndrome owing to diabetes and obesity, as well as the related liver disease nonalcoholic fatty liver disease (NAFLD) and NASH. NAFLD or NASH might be along with chronic HBV infection-the exception to the rule that hepatoma usually relates to advanced hepatic fibrosis or cirrhosis. In accordance with a recently performed research requiring cautious verification, around 40% of patients undergoing hepatoma and NAFLD or NASH might not have cirrhosis [21]. High-throughput genomic studies focusing on gene sequencing of large cohorts have established the primary oncogenic drivers of hepatoma. Nevertheless, most of these drivers, including the TERT promoter, TP53 and CTNNB1, have not been demonstrated to be druggable and the comprehension of their role in hepatoma has not therefore translated into enhancing the management of the disease. Drug discovery which targets these complex proteins and regulatory mechanisms indicates a main breakthrough in hepatoma research [22]. In addition, the identification of driver mutations or amplifications in associated genes—including FGF19, CCND1 and VEGF—has not yet translated into proof-of-concept early clinical trials on the basis biomarkers. Comprehending the targets of the microenvironment in tumour progression and response to therapies has become an area needing to be further explored [23, 24], given the clinical relevance of this compartment in the risk of tumour development, prognosis as well as immunomodulation [25–27]. Using RTK inhibitors or targeting metastatic cancer cells through the Ras pathway (e.g. using PDFG-R/Raf inhibitor sorafenib) may not produce significant clinical benefits in metastatic HCC, and mesenchymal and chemoresistant (M-HCC) cells that express ZEB1 are addicted to PKCα and can be selectively eliminated by PKC inhibitors [13].

Eph receptor contains the largest receptor tyrosine kinase family, which interacts with Ephrin ligand to form a bidirectional cell signal transduction system, and studies have indicated that it is associated to the onset and development of various tumors [28]. EFNA5 belongs to a subclass of ephrin ligands, and it has been reported that it plays an anti-cancer part in many tumors, such as glioma, chondrosarcoma and leukemia [5–8, 29]. However, in breast cancer and ovarian cancer, it shows the role of promoting the appearance and development of tumors [30]. In early researches, both ephrinA5 isoforms hindered neurite outgrowth of dorsal root ganglia; nevertheless, ephrinA5S made a less inhibitory impact on the brain in the process of development [31]. Therefore, it appears that the biological effect of EFNA5 will be different owing to the change of cancer species. This discovery aroused our interest. Through the TCGA database, we found that there are differences in the level of EFNA5 between hepatoma tissues and normal liver tissues, and this difference in expression can predict different clinical prognosis. However, there are few reports on the specific biological effects and pathogenesis of EFNA5 in hepatoma. The current research studied the biological effects of EFNA5 on hepatoma at the molecular level. The qRT-PCR results suggested that in relative to normal hepatocytes, the level of EFNA5 in hepatoma cells was significantly down-regulated, suggesting that EFNA5 may be associated to the appearance and development of hepatoma. To further investigate how EFNA5 influences on the biological function of hepatoma cells, plasmid transfection technology was employed to specifically overexpress EFNA5 in HepG2 and LM3 cell lines. The results of CCK-8 demonstrated that the OD value of EFNA5-flag plasmid transfection group was lower than that of control group at 24, 48 and 72 h. It is indicated that up-regulating the level of EFNA5 can hinder the proliferation of hepatoma cells. Transwell and Boyden experiments further proved that up-regulating the expression of EFNA5 can effectively hinder the migration and invasion of hepatoma cells. In order to further study the possible molecular mechanism of EFNA5 in hepatoma [32], this study aimed to detect the expression level of EFNA5 and its related proteins through western blotting. The findings demonstrated that the expression levels of c-Myc, c-Jun, Vimentin and N-cadherin in EFNA5 plasmid transfection group decreased, while the expression level of E-cadherin tended to increase. Based on these results, EFNA5 can inhibit the proliferation, invasion, and metastasis of hepatoma cells through negatively regulating the occurrence and development of EMT [33]. The occurrence of EMT in tumor cells is resulted from diverse complex factors, and the abnormal activation of intracellular molecular signaling pathway and tumor microenvironment are vital initiating factors [16, 17, 34]. Notably, it has been reported that HepG2 cells are epithelial cells, sensitive to treatment, and may undergo EMT spontaneously, which needs to be further verified in subsequent experiments [35–38].

In the tumor microenvironment, tumor cells, immune cells, stromal cells, endothelial cells, and adjacent normal cells can actively communicate with each other through the microenvironment. Therefore, there are many factors affecting the tumor and its surrounding tissues [39]. As a commonly seen malignant tumor in digestive system, high recurrence rate and intrahepatic metastasis rate are important factors for poor clinical prognosis. Several features of cancer malignancy, such as tumour invasion, metastatic progression and treatment resistance, are associated with epithelial–mesenchymal transition [40]. EMT is a key process in cancer metastasis and certain proteins or miRNAs involved in it can regulate the EMT process [41]. For example, miR-520f-3p overexpression controls the expression of EMT biomarkers, up-regulates E-calmodulin and down-regulates waveform protein expression. YAP1 overexpression also promotes EMT in breast cancer, pancreatic cancer and hepatocellular carcinoma [40]. In this study, we found that in relative to normal liver cells, the expression of EFNA5 in hepatoma cells was notably down-regulated, whereas the increase of EFNA5 expression inhibited the proliferation, invasion, and metastasis of hepatoma cells. As a result, we hypothesized that the high level of EFNA5 can inhibit the recurrence and metastasis of hepatoma, therefore improving the disease-free survival rate and overall survival rate. Firstly, we confirmed that EFNA5 is an effective tumor suppressor, which can inhibit the development of hepatoma cells. Secondly, numerous studies indicate that EFNA5 interacts with EFNA receptors (such as EPHB2, A2, A3, and A5), and then mediates the signal cascade reaction. The inhibitory effect of EFNA5 subtype on the occurrence and development of hepatoma can be mediated by the downstream cascade of receptor-activated “positive signal”. Therefore, the overexpression of EFNA5 can inhibit the recurrence and metastasis of hepatoma, and can be applied as a biological index to judge the prognosis of hepatoma. Clinically, the treatment of hepatoma is still a difficult problem. Because most patients with hepatoma are in the advanced stage or even in the late stage when they are firstly diagnosed, they lose the chance of receiving early radical surgery. Systemic treatment is the main treatment, and the combination with local treatment has become a vital treatment model for hepatoma. Treating early and advanced hepatoma is single. Platinum-based combination chemotherapy has not achieved good survival benefits [42]. With the continuous development of targeted therapy and biotherapy, targeted therapy of hepatoma, which is mainly based on small molecule TKIs, has exhibited good anti-tumor activity, and its safety and tolerance are significantly better than systemic chemotherapy, bringing better choices for treating hepatoma. Currently, targeted therapy in combination with other systemic therapy and local therapy has become a novel direction of hepatoma treatment, and it has continuously enhanced the overall clinical prognosis of hepatoma. Targeted therapeutic drugs for hepatoma have been successfully approved. From the early sorafenib to today’s Ranvartinib, Regofinib, Capotinib and Apatinib, TKIs with small molecules and multiple targets have continuously exhibited its therapeutic advantages in the vicinity of hepatoma, which has also caused the climax of the exploration of molecular targeted therapeutic targets for hepatoma [43].

Recently, abnormal signal transduction of tyrosine kinase receptors has become a research hotspot, such as EGFR, VEGFR, FGFR and PDGFR, and it is correlated with the occurrence and development of various malignant tumors, showing the role of promoting tumor proliferation, invasion, metastasis and tumor angiogenesis [44]. Among them, the relationship between EGFR and hepatoma has attracted our attention. It is indicated in recent studies that EGFR is abnormally expressed in human hepatoma cells, and its ligand EGF, as one of the mitogens required for the growth of hepatoma cells, can promote the occurrence and development of hepatoma. Meanwhile, in vitro experiments have proved that EGFR-TKIs represented by gefitinib can effectively hinder the migration and invasion of hepatoma cells [45, 46]. Therefore, EGFR may become a promising target for the therapy of hepatoma. In this study, it was indicated that the up-regulation of EFNA5 leads to the decrease of EGFR expression, indicating that EFNA5 may exert a synergistic role with EGFR- tyrosine kinase inhibitors in treating hepatoma. However, the clarification of the specific regulation mechanism involved in this process requires to be studied.

To sum up, the expression of EFNA5 gene is low in hepatoma. Up-regulating the expression level of EFNA5 in hepatoma cells can hinder the proliferation, invasion, and metastasis of hepatoma cells through negatively regulating the occurrence of EMT. Therefore, EFNA5 may be a potential biological index to judge the prognosis of hepatoma. Meanwhile, we found that up-regulating the level of EFNA5 in hepatoma cells can promote the degradation of EGFR, while the correlation and regulation mechanism between them need to be further studied and confirmed. EGFR is usually expressed in human hepatoma cells, and targeted inhibition of EGFR can effectively reduce the proliferation, invasion, and migration of hepatoma cells. Therefore, the negative regulation of EFNA5 and EGFR is a potential research direction, and EFNA5 may become a potential target in clinical application in the future, which may exert a synergistic role in anti-EGFR therapy by up-regulating the expression level of EFNA5, and may also become a promising therapeutic strategy for hepatoma.

Conclusion

To conclude, in this study, we found that there is a difference in the expression of EFNA5 between hepatoma cells and normal hepatocytes, revealing that this difference in expression can predict different clinical prognosis. Furthermore, the cellular function level and molecular level of two hepatoma cells (HepG2 and LM3) were confirmed, and it was demonstrated that the up-regulation of EFNA5 expression could hinder the proliferation, migration, and invasion of hepatoma cells. More importantly, it is indicated that EFNA5 may inhibit the proliferation, invasion, and migration of hepatoma through negatively regulating the occurrence of EMT of hepatoma cells.

Acknowledgements

Not applicable.

Author contributions

Z.Z. and S.H. designed the study, performed the experiments, collated the data, and contributed to editing the manuscript. J.L. interpreted data and edited the manuscript. J.L. and F.C. designed the study, X.W., J.L. And F.C. provided research funds. Z.Z., S.H., X.Z. and Y.Z. and X.W. participated in the course of the experiment. All authors read and approved the final manuscript.

Funding

The support of this study was provided by the National Natural Science Foundation of China (Grant No. 81872251); Natural Science Foundation of Guangdong Province (Grant Nos. 2020A1515010093 and 2021A1515012104); President Foundation of Integrated Hospital of Traditional Chinese Medicine, Southern Medical University (Grant No. 1202102002); Project of Administration of Traditional Chinese Medicine of Guangdong Province of China (20211267) and Beijing Xisike Clinical Oncology Research Foundation (Grant No. Y-2019Genecast-021).

Data availability

Publicly available datasets were explored in the current work. Additionally, the data can be found here: https://tcga.xenahubs.net.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.

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References

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