Regulation of Apelin Is Associated with Proliferation and Angiogenesis in Gastric Cancer

Background: Apelin is an emerging endogenous ligand, which is involved in proliferation and angiogenesis in certain cancers. However, few studies have reported its functions and underlying mechanisms in human gastric cancer (GC). Therefore, the present study aimed to investigate the effect of Apelin expression in human GC and the underlying mechanisms of Apelin in the promotion of proliferation both in vitro and in vivo. Methods: A total of 178 patients diagnosed with GC under postoperative care were enrolled for the study to investigate clinicopathological and immunohistochemical factors of Apelin expression. Survival of patients was analyzed using the Kaplan-Meier method and Cox regression model. We adopted quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), western blot and ELISA to analyze human GC specimens and cell lines. The role and mechanisms of Apelin were evaluated by performing in vitro and in vivo experiments to analyze exogenous Apelin and its overexpression in human GC cells. Results: The expression of Apelin was higher in human gastric cancer cells than in adjacent normal tissues. Apelin, which was overexpressed in vessel invasion (P <0.01), lymph node metastasis (P <0.01), late-staged tumor (T) status (P <0.05), pathological type (P <0.05) and nerve invasion (P <0.05), also exhibited a positive correlation with vascular endothelial growth factor (VEGF). Apelin overexpression or exogenous Apelin activated downstream of ERK/Cyclin D1/MMP-9 signaling pathway to promote MGC-803 cell proliferation and invasion in vitro. Apelin overexpression promoted angiogenesis aiming at accelerating growth of subcutaneous xenograft in vivo. Conclusions: This study has elucidated the relationship between Apelin and its clinicopathological features in human GC, and the role of Apelin in tumor cell proliferation in human GC cell lines. This is the rst study to elucidate underlying mechanisms of Apelin in the proliferation of GC. Apelin can be a potential therapeutic target for human GC.

consisting of 77 amino acids that may promote proteolytic maturation and subsequently generate different bioactive peptide fragments: Apelin-17, Apelin-36 and Apelin-13 that are the predominant types in the human plasma [8,9]. Apelin/APJ signaling pathway, which is widely expressed in the cardiovascular system, has been reported to be involved in various physiological and pathological processes such as angiogenesis, heart failure, energy metabolism and cancer progression [9,10]. The dysfunctional, abnormal vasculature morphology plays a signi cant role in the growth and development of tumor [10][11][12]. Previous studies have revealed that VEGF and its receptor are instrumental in angiogenesis, and have been used as a therapeutic target for over 30 years [11,12]. Apelin overexpression stimulates proliferation of endothelial cells, enhances tumor vascularization and formulates capillary tubes both in vivo and in vitro [13,14]. In addition, Apelin upregulation has been reported in previous studies, and strikingly, a correlation between the level of Apelin expression and clinical outcomes in speci c human cancers has been observed [15,16].
However, there are few studies on the role and molecular mechanisms of Apelin in GC. Therefore, the present study aimed to investigate the correlation between Apelin expression and clinical outcomes in human GC by performing a retrospective study, and to reveal the role and underlying molecular mechanisms of Apelin in GC cells in vitro and in mice.

Patients And Methods
Patients A retrospective study was conducted using surgical specimens by comparing tumor tissues and adjacent normal tissues obtained from 178 patients with histopathologically con rmed GC from Binzhou Medical College A liated Hospital. The clinicopathological data of patients covered the period between January 2009 and December 2011. The patients underwent radical surgical resection with D2 lymphadenectomy and were subjected to chemotherapy with or without radiotherapy. All patients were required to undergo computed tomography (CT) scanning for the neck, chest and abdomen, and anti-tumor therapies could not be administered before surgery. Postoperative chemotherapy began 1 month after surgery and included treatment with uoropyrimidine or capecitabine in combination with oxaliplatin or paclitaxel, repeated for at least 4 cycles. Eligible patients underwent radiotherapy by adopting intensive modulation radiotherapy (IMRT) and the prescribed dose was 45-50.4 Gy given as 1.8-2.0 Gy per fraction. The progression-free survival (PFS) time corresponded with the period from the beginning of operation to the occurrence of disease progression, death or the end of the study. Overall survival (OS) corresponded to the period from surgery to death or December 31, 2016.

Immunohistochemical analysis
Immunohistochemical analyses were performed on tumor tissues previously xed in formalin and embedded in para n. The tissue sections (4 µm) were depara nized with xylene and rehydrated in gradients of alcohol. Heat-induced antigen retrieval was performed in 0.0 l M citrate buffer (PH = 6) for 5 minutes and subsequently the slides were heated in a microwave oven for 15 minutes. Tissue sections were incubated in 3% H 2 O 2 -methanol solution to block endogenous peroxidase at 37.0 °C for 20 minutes.
After blocking with albumen for 10 minutes, the sections were incubated overnight at 4 °C with anti-Apelin, anti-VEGF or anti-CD34 antibodies at dilution ratios of 1:200, 1:100 or 1:200, respectively (Abcam, the USA). The EnVision-HRP detection system (Dako, Carpinteria, CA, the USA) was used to evaluate the tissues, with diaminobenzidine as the chromogen and Mayer's modi ed hematoxylin as the counterstain. Tissues were incubated with 80 µL goat anti-rabbit or rabbit anti-mouse immunoglobulin G (IgG) (Dako, Carpinteria, CA, the USA) labeled with horseradish peroxidase using a dilution ratio of 1:250 at 37 °C for 30 minutes. The tissues were evaluated by two investigators who were blinded to clinical data. The investigators initially disagreed on the evaluation of approximately 10% of the tissues but they reached a consensus after further consultations. Apelin or VEGF immunostaining score was calculated by multiplying the positive cell area score and the staining intensity score. An immunoexpression score > 3 was considered positive, whereas a score ≤ 3 was considered negative. The staining intensity score was classi ed into four levels: no staining (0), light yellow staining (1), yellow staining (2) and deep yellow staining (3). The positive cell area score was based on the percentage of positive cells and was classi ed as follows: no positivity (0), less than 10% positivity (1), 11-50% positivity (2), 51-75% positivity (3) and more than 76% positive cells (4) [15,16]. Microvessel densities (MVD) were established by labeling capillaries (0.02-0.10 mm) with CD34 (Dako, Carpinteria, CA, the USA). Morphometric analysis was performed on three sections per slide using computer-aided CUE-2 software (Olympus Vanox, Tokyo, Japan) as previously described [17].

Construction of stable transfected cell lines
Apelin complementary DNA was purchased from Thermo Fisher Scienti c (Shanghai, China). DNA encoding areas were ampli ed using the following primers: 5'-CGCGAATTCGGCATGAATCTGCGGCTCTG-3'and 5'-GCGCTCGAGTCAGAAAGGCATGGGTCC-3'. The ampli ed PCR products were subcloned with Apelin cDNA into the pcDNA 3.1 vector adopting EcoRI and XhoI restriction enzymes (Invitrogen, Carlsbad, CA, USA). Vector expression was veri ed through DNA sequencing. An empty vector or an Apelinencoding pcDNA 3.1 vector was transfected into the MGC-803 cell line, which has been demonstrated to express a relatively low level of Apelin using Lipofectamine 2000 transfection reagent (Thermo Fisher Scienti c, Shanghai, China) according to the manufacturer's protocol.
RNA isolation and quantitative real-time reverse transcription-polymerase chain reaction Total RNA was isolated from each human GC cell line with TRIzol reagent (Invitrogen, Carlsbad, CA, the USA) in accordance with the manufacturer's guidelines. It was reverse-transcribed into cDNA using the murine leukemia virus reverse transcriptase. PCR ampli cations were carried out under the following cyclic conditions: 42 cycles of incubation at 95 °C for 20 seconds and at 60 °C for 30 seconds. PCR products were visualized by electrophoresis on 2% agarose gels stained with ethidium bromide. The primers for PCR were: Apelin, 5'-GATGCCGCTTCCCGATG-3'(forward) and 5'-ATTCCTTGACCCTCTGGGCT-3'(reverse); β-actin, 5'-TGCTGTCCCTGTATGCCTCT-3' (forward) and 5'-AGGTCTTTACGGATGTCAACG-3' (reverse). The mRNA levels were calculated with the 2 −ΔΔCt method [18]. Gene expression level of Apelin mRNA in MGC-803 cell line was determined using the same method at 48 hours after transfection with control or Apelin-encoding pc DNA 3.1 vector.

Protein Extraction and Western Blot analysis
Cultured cells at logarithmic growth phase were resuspended in lysis buffer containing 5µL Protease Inhibitor Cocktail (cOmplete, Sigma, Germany) and then lysed as described previously [19]. The protein concentration in the supernatants was measured using the Bradford protein assay kit (Bio-Rad, Hercules, CA, the USA). Equivalent amounts of protein samples (80 µg/lane) were fractionated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto polyvinylidene uoride (PVDF) membranes at 0.8 mA/cm 2 for 20-30 minutes. The membranes were blocked with Tris-buffered saline (TBS) containing 10% nonfat milk at 37 °C for 2 hours, and incubated with primary antibodies against Apelin, β-actin, pERK, pAkt, Cyclin D1 and MMP-9 at 4 °C overnight. Membranes were washed four times with TBST, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at 37.0 °C. The blots were visualized using the ECL reagent and quanti ed using Quantity One software. The primary antibodies were used at the following dilutions

Cell Proliferation Studies
To assess cell proliferation, 1 × 10 4 cells per well were seeded in triplicate onto at-bottomed 96-well microplates and cultured for 24 hours. The cells were synchronized for 12 hours in DMEM containing 0.5% FBS. After addition of different concentrations of Apelin-13 (0.02 µmol/L, 0.1 µmol/L, 0.5 µmol/L, 2.5 µmol/L 12.5 µmol/L, 62.5 µmol/L) for 24 hours, the cells were treated with MTT (5 mg/mL) at 37.0 °C for 4 hours. The 150µL dimethyl sulfoxide (DMSO) was added to each well to remove cells that were not reduced by MTT reagent. The absorbance of cell supernatants was measured at 570 nm using a microplate reader (Thermo Multiskan Ascent, the USA). We would also measure the absorbance at 570 nm using the optimal concentration obtained from the previous experiment at different time points (6 h, 12 h, 24 h, 36 h, 48 h). For certi cation the change of cell proliferation after transfection with pcDNA 3.1 or pcDNA 3.1-Apelin vector into MGC-803 cells, we also measured the absorbance at 570 nm at different time points as mentioned.

Cell Migration and Invasion Analysis
Transwell chambers (BD, the USA) were used to study cell migration and invasion. MGC-803 cells were divided into 4 groups and prepared in triplicate: cells without treatment, cells treated with Apelin-13, cells transfected with pcDNA 3.1, and cells transfected with pcDNA 3.1-Apelin. The 5 × 10 4 cells were seeded in the upper chamber, which was in advanced scribbled with Matrigel (BD, the USA), containing a medium without serum. The 600µL 10% DMEM were added in the lower chamber. The Transwell chambers were incubated for 24 hours. Next, cells that did not invade the membrane were removed. Those that invaded were stained with 4% polyoxymethylene for 15 minutes and then with 0.2% crystal violet, and counted under an inverted microscope at 200 × magni cation (Olympus, Japan).

Construction of Apelin-stable-transfection of MGC-803 cell
To determine the optimal concentration of puromycin (Beijing Inovogen Tech.Co. Ltd, China), cells growing at a logarithmic phase were cultured in 24-well microplates at a density if 2 × 10 4 cells/well overnight and then mixed with different concentrations of puromycin (0, 1, 2.5, 5, 7.5, 10, 15 µg/mL ). Puromycin at 7.5 µg/mL (the lowest concentration) killed all MGC-803 cells after 72 hours of treatment. This concentration, regarded as optimal concentration, was chosen for subsequent experiments. MGC-803 cells were seeded and cultured in 24-well plates with DMEM without FBS or antibiotic until the plate became 80% con uent. The 2 mL polybrene (Beijing Inovogen Tech.Co.Ltd. China) ( nal concentration 6 µg/mL) and viral supernatant 0.5 mL were added to each well and cultured at 37 °C for 4 hours. Fresh DMEM was added into each well for incubation at 37 °C for 24 hours. Three days after transfection, a medium containing puromycin (7.5 µg/mL) was added to each well and cultured at 37 °C. The medium was changed every 2-3 days until colonies were formed. Negative colonies were removed, and the remaining puromycin-resistant colonies were transferred to a 24-well microplate to cover an area nearly more than half of the dish. mRNA and protein expression of target genes were determined with RT-PCR and Western blot assays.

Subcutaneous xenografts in vivo
Fourteen nude mice (BALB/c nu/nu, female, 5-6 weeks old, obtained from the Laboratory Animal Resources of Chinese Academy of Sciences, Shanghai, China) were raised in pathogen-free conditions.
The xenograft tumors were established by subcutaneous injection of 0.2 mL MGC-803 cells (1 × 10 7 /mL) stably expressing Apelin or control into the mice. The long diameter (a), short diameter (b), and weight of tumor were measured twice a week by the same investigator. The tumor volumes (mm 3 ) was calculated using the following formula: V = ab 2 /2. Values obtained were used to plot a tumor growth curve. Mice were euthanized after 35 days of xenograft tumors formation for further examination. The tumor proliferation rate was calculated as: (the averaged weight of xenograft tumors in the experiment -the averaged weight of control group)/ the averaged weight of xenograft tumors × 100%.

Immunohistochemistry of MVD in Vivo
Immunohistochemical staining was performed for MVD in murine tumors as described previously in this study.

Statistical Analysis
All statistical analyses were performed using SPSS version 16.0 (IBM Corp., Armonk, NY, the USA). The chi-square test was employed to analyze the correlation of clinicopathologic factors with Apelin and VEGF expression. Continuous variables such as MVD were expressed as mean ± S.E.M. Differences between the two groups were determined by Student t-test or nonparametric tests. The association between Apelin-encoding protein and VEGF-encoding protein expression was analyzed by Spearman's rank correlation test. Kaplan-Meier analysis and Cox proportional hazards were used to draw survival curves, and log-rank test to assess survival rates. P < 0.05 was regarded statistically signi cant.

Results
The results revealed that there were 141 males and 37 females, with a median age of 62 years (range 34-38 years). The number of patients with the primary lesion site localized to the cardiac region, gastric fundus, gastric body and gastric antrum was 61, 14, 49 and 54, respectively. The histological types of tumor cells were divided into 4 categories based on the degree of tumor differentiation: well or moderately differentiated (82 cases), poorly differentiated (75 cases), mucinous adenocarcinoma (17 cases) and signet ring cell carcinoma (2 cases). Tumor stages for all patients were rede ned according to pathological outcomes, which were based on the AJCC/UICC TNM classi cation revised in 2010. Five patients were staged I, 98 patients were stage II and 75 patients were staged III cancer.
Immunohistochemical analyses were performed to detect protein expression levels of Apelin in 53 of 178 normal gastric tissues adjacent to tumor tissues, while 88 of 178 tumor tissues were analyzed by immunohistochemical staining (29.78% vs 49.44%, P < 0.001, Table 1.1). Additionally, the immunoreactivity of Apelin exhibited a diffuse cytoplasmic immunostaining in tumor tissues ( Fig. 1.1). Similarly, the percentage of VEGF-positive tumor cells was signi cantly higher in tumor tissues than in adjacent normal tissues, which was consistent with previous studies (55.62% vs 39.89%, P = 0.003, Table 1.1). Furthermore, immunoexpression of VEGF protein exhibited a cytoplasmic pattern consistent with Apelin ( Fig. 1.2). A chi-square test was used to investigate the correlations between clinicopathological factors and Apelin and VEGF expression. Apelin expression was signi cantly positively correlated with vessel invasion and lymph node metastasis (P < 0.01), (P < 0.01) ( Table 1.2). In addition, signi cant positive correlations were observed between Apelin expression and late-staged tumor (T) status, pathological type, and nerve invasion (P < 0.05). Conversely, no signi cant associations were observed between Apelin expression and gender, age, and the site of primary lesion (P > 0.05). VEGF expression was signi cantly high during late T stage (P < 0.01), vessel invasion and positive N stage (P < 0.05); however, no statistically signi cant difference was observed between VEGF expression and gender, age, the site of primary lesion, pathological type and nerve invasion (P > 0.05).  Fig. 1.3).  The OS rates of patients with positive expression of Apelin at 1 year, 3 years and 5 years were 97.73%, 62.50% and 28.41%, respectively ( Fig. 1.4)   Western blotting was performed to assess the expression of Apelin at the protein level in MGC-803, SGC-7901 and HGC-27 cell lines. The lysates of MGC-803 cells had the lowest levels of immunoreactive Apelin based on the results of mRNA expression of Apelin. A signi cant difference was observed between the two groups (P < 0.05).
ELISA was performed to detect the concentration of cellular secretion of Apelin that was secreted by the examined cell lines into the culture medium. The results were similar to those observed in the protein and mRNA expressions of Apelin, accounting for the lowest concentration in MGC-803 cells (132.00 ± 31.97 pg/mL). In addition, a statistically signi cant difference was observed between each two cell lines (P < 0.05, Fig. 2.3). The rate of apoptosis induced by SAHA was substantially increased in treated cells compared to the untreated cells (control) ((15.100 ± 3.997) % vs (7.300 ± 1.745) %, P < 0.05) (Fig. 2.11). Strikingly, the week by the same investigator. Furthermore, the tumor volumes (mm 3 ) were calculated using the formula, V = ab 2 /2 and a tumor growth curve plotted. The neoplastic growth velocity in mice subcutaneously injected with pLV-puro-Apelin signi cantly increased from the fourteenth day (Fig. 3.3 and Table 3.1). A maximum body weight and tumor size of 2.96 ± 0.61 g was observed in the mice injected with MGC-803 cells of overexpressed-Apelin, whereas a minimum body weight and tumor size of 1.70 ± 0.43 g was observed in mice of the control group (Fig. 3.4). The results revealed the function of Apelin in promoting proliferation of gastric cancer cells via in vitro and in vivo experiments. MVD assay by CD34 immunolabeling was performed to explore the mechanism underlying the differences in neoplastic growth velocity between over-expressed Apelin and control, and to establish the relationship between Apelin overexpression and angiogenesis. MVD was signi cantly higher in xenograft tumors with Apelin overexpression (168.833 ± 35.078) than in the control group, which had low MVD (112.333 ± 29.859; t=-2.734, P = 0.021) (Fig. 3.5).

Discussion
Local recurrence and metastasis of tumors are the principal factors associated with poor prognosis, and are modulated by various genes and cytokines [21,22]. The demonstrated relationship between tumorous proliferation, metastasis, recurrence and angiogenesis has prompted the discovery of various factors associated with angiogenesis to date. VEGF signaling pathway has been the center of attention in relation to tumor angiogenesis, and VEGF-targeted therapy has provided insights into the treatment of certain cancers such as lung and colon cancers [23,24]. In addition, previous studies have revealed that VEGF signaling pathway has a signi cant role in tumorigenesis and development of GC, and that high VEGF expression indicated a poor prognosis [23][24][25]. However, the clinical bene ts of adding anti-VEGF drug in human GC have not been satisfactory [25]. Therefore, it is necessary to nd a novel biomarker and new antivascular targets as therapeutic targets for GC.
Apelin is an emerging endogenous ligand, which has attracted the attention of researchers because of its role in angiogenesis [9,13,14,26]. Previous studies have revealed that Apelin expression is up-regulated in various human solid tumors [13,26]. However, few studies have been conducted on the role of Apelin expression in tumorigenesis and development of human gastric cancer. In the present study, we investigated the correlation between the level of Apelin expression and clinicopathological parameters in patients with GC from our center. Mortality was signi cantly higher in patients with high Apelin-positive expression than in patients with low Apelin expression. In addition to high VEGF-positive expression observed in tumor tissues, this study revealed a signi cant positive correlation between Apelin expression and VEGF expression, which indicated the function of Apelin in angiogenesis in human GC. Nevertheless, further studies are required to investigate whether there is a synergy between Apelin and VEFG in relation to angiogenetic effect. Feng and his colleagues demonstrated that Apelin in tumor tissues rather than serum Apelin was associated with clinicopathological features, and it was an independent prognostic factor [27]. Although Apelin was not an independent prognostic factor in this study, multivariate Cox regression analysis revealed that patients with high Apelin-positive expression exhibited a considerably poor prognosis compared to patients with low Apelin-positive expression based on previous other studies.
In this study, the correlations between high Apelin-positive expression and late-staged T status, N stage, and the high incidence of vessel invasion implies that Apelin can promote local tumor recurrence and lymph node metastasis.
The mRNA levels of Apelin expression, proteins and lysates in cell culture media of three types of GC cell lines, HGC-27, MGC-803 and SGC-7901 were analyzed using RT-PCR, western blotting and ELISA to elucidate the effects and mechanisms of Apelin on biological behavior in vitro. Results of the present study indicated that Apelin expression in MGC-803 cells was not only low at the mRNA level but also at protein level and cellular secretion. With reference to human colon adenocarcinomas, a study by Picault demonstrated that Apelin presents varying expression levels at mRNA and protein levels in several cell lines [28]. Moreover, a similar observation was made in human non-small lung cancer [15]. exogenous Apelin can enhance cell proliferation and migration, such as in human lung adenocarcinomas cell A549 [29] and human prostate cancer cell LNCaP [30]. Moreover, Apelin is resistant to tumor cell apoptosis in hepatocellular carcinoma cell HepG2 [16] and vascular smooth muscle cell VSMCs [31].
However, a few studies have revealed that neither exogenous Apelin nor overexpression of Apelin could promote human colorectal cancer cell proliferation, but xenograft tumors transfected with cells overexpressing Apelin could promote tumor growth in mice [28].
SAHA, a member of histone deacetylase inhibitor, can induce apoptosis in MGC-803 cells according to a previous study [20]. To our knowledge, the relationship between overexpression of Apelin and malignant biological behavior in human GC cells both in vitro and in vivo has not been studied. Remarkably, SAHA did not affect the apoptotic ability of MGC-803 cell lines transfected with pcDNA3.1-Apelin or treated with exogenous Apelin when compared to the control group in this study.
According to studies conducted in the recent past, both mitogen-activated protein kinase kinase/(MAPK)/ERK and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways have a critical role in the proliferation, migration, angiogenesis and inhibition of apoptosis in tumor cells, including the human GC cells [32,33]. Tumor cells promote ERK phosphorylation, thereby resulting in transcription of cyclin D1 that largely stimulates tumorous growth in the ERK signaling pathway. Cyclin D1 promotes cell cycle transition from G1 to S phase and exerts mitogenic effects on cell proliferation [34]. Both the MAPK/ERK and PI3K/Akt signaling pathways participate in tumor invasion primarily by up-regulating MMP-9 transcription, which has been reported to be associated with cancer invasion and metastasis [35,36]. The anti-apoptotic properties were re ected in the MAPK/ERK and PI3K/Akt signaling pathways through stimulation of the expression of anti-apoptotic peptide belonging to Bcl-2 family or inhibition of a caspase-mediated pathway.
The results obtained from our analysis demonstrated that the protein expression levels of ERK phosphorylation (pERK) signaling pathway, cyclin D1 that is associated with cell proliferation and MMP-9 involved in invasion were increased by both overexpression of Apelin and exogenous Apelin in comparison to the control group. Strikingly, there was no signi cant difference in the expression of Akt phosphorylation (pAkt) between the treatment and control groups. According to a study by Picault and his colleagues, Apelin/APJ signaling pathway suppressed apoptosis through phosphorylation of Apelin [28]. MG132 is a proteasome inhibitor, which performs a crucial role in the activation of apoptosis by downregulating pAkt in multiple tumorous cell lines through an intrinsic pathway [37]. Studies have indicated that Apelin/APJ induces Akt phosphorylation to counteract apoptosis activated by MG132 in human colorectal LoVo cells [28]. Remarkably, exogenous Apelin neither improved the expression of pERK nor increased proliferation rate in LoVo cells [28]. Yang et al. reported that Apelin-13 induced cell proliferation and promoted autophagy through phosphorylation of ERK, subsequently activating the downstream transduction cascades in human lung cancer cell line A549 [29]. Moreover, exogenous Apelin induces phosphorylation of ERK and Akt, resulting in cell migration and inhibition of apoptosis rather than cell proliferation in human lymphatic endothelial cells [31].
The results of the present study corresponded to the ndings of most previous studies, implying that Apelin can stimulate proliferation of tumor cells. Conversely, anti-apoptotic effect of Apelin that is activated by SAHA and Akt phosphorylation in vitro was not observed. There might be an anfractuosity relationship between SAHA and the MAPK/ERK and PI3K/Akt signaling pathways. This suggested that Apelin modulated apoptosis signaling by participating in the MAPK/ERK signaling pathway in human GC cells. Therefore, further studies should be conducted to investigate if there is a negative correlation between Apelin and SAHA. Notably, a positive feedback between Apelin expression and ERK activation was observed. Nevertheless, we failed to down-regulate Apelin expression levels in human GC cell line HGC-27 due to lack of an effective SiRNA molecular marker, but we are trying to apply other approaches to delete the Apelin gene.
To the best of our knowledge, this is the rst study to establish human GC cell xenograft models with overexpressed Apelin. The ndings of the present study suggest that Apelin overexpression promotes growth of human GC cells and angiogenesis in vivo when compared with the control group. Apelin has been characterized as a novel angiogenic factor, which can stimulate vascular endothelial cell proliferation and migration through extrinsic and intrinsic pathways. In addition, knocking down Apelin or APJ can e ciently block angiogenesis [38]. Sorli et al. revealed that Apelin overexpression increased the formation of tumorous vessels and accelerated tumor growth, in addition to the paracrine role in tumor neoangiogenesis [39]. Kidoya et al. demonstrated that the Apelin/APJ signaling pathway induced maturation of tumor vascular morphology and function [40]. MVD is the gold standard for estimating tumor angiogenesis, which is closely associated with tumor invasion and metastasis, and is an independent prognosis factor [39]. Berta et al. observed that Apelin overexpression could signi cantly stimulate tumor growth, increase MVD and capillary perimeter [15]. Muto et al. demonstrated that Apelin-13 antagonist could block tumor proliferation or angiogenic activity in hepatocellular carcinoma xenograft model, which con rmed the role of Apelin overexpression in tumourigenesis through angiogenesis [41]. A common observation made among the mentioned studies including the present study was that implantation of tumor cells transfected with overexpressed Apelin increased tumor growth in nude mice and simultaneously enhanced the expression of MVD in comparison with the control group.

Conclusion
Overall, Apelin was highly expressed in human gastric cancer tissues compared to adjacent normal tissues. The expression level of Apelin was associated with vessel invasion, N stage, T stage, VEGF and

Declarations
Ethics approval and consent to participate All procedures performed in study involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication
Informed consent was obtained from all individual.

Availability of data and materials
The datasets used or analyzed during the current study were available from the corresponding author on reasonable request.

Competing interests
None of the authors have any con icts of interest.

Funding
This work was not supported by any research fund. Supervision. All authors agreed to be accountable for all aspects of the work.