Human gastric cancer AGS and MGC-803 cell lines were used in the present study. The cell lines were obtained from the Cell Bank of Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The two cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with humidified 5% CO_2.

In order to silence AAED1 expression in gastric cancer cell lines, a lentivirus-mediated transfection method was used. A pLKO.1 TRC cloning vector that was obtained from Addgene Inc. (Cambridge, MA, USA) was used to express shRNA targets against AAED1 (14). The 21-bp targets against AAED1 were 5′-CGGCATTTCCTGTGTTACAT-3′ and 5′-CGCGATAGGAATAGGTTGGAT-3′, respectively. The lentivirus was produced by co-transfecting AAED1-ilencing constructs with psPAX2 and pMD2.G vectors in a ratio of 4:3:1 into 293T cells. Stable AAED1-silenced cell lines were obtained by infecting AGS and MGC-803 cells and subsequent puromycin selection.

Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to measure the cell viability. Briefly, 200 µl medium containing cells (3,000/well) was seeded into 96-well plates. After culturing for the indicated times, CCK-8 solution was added into each well at 37°C. After 2 h, the optical density values of each well were measured using a microplate reader at 450 nm.

AGS and MGC-803 cells (5×10²) stably expressing shRNA targets against AAED1 and its relative control cells were seeded. After cultivating for 10 days, 4% paraformaldehyde was used to fix the cells followed by staining with 1% crystal violet. The colonies were counted subsequently. Briefly, the images of the plates were taken on a gel imager using a light filter, and colonies >500 µm or other appropriate diameters were counted.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Takara PrimeScript RT reagent kit was used for reverse transcription to obtain cDNA (Takara Bio, Inc., Otsu, Japan). The expression status of candidate genes and β-actin were determined by quantitative real-time PCR using an Applied Biosystems^® 7900HT Real-Time PCR system (Thermo Fisher Scientific). β-actin was used as the reference genes. Thermocycling conditions were 40 cycles of 95°C 5 sec and 60°C 30 sec. The method of quantifications were calculated according to a previous report (15). Primer sequences are listed in Table I.

Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris/HCl, pH 8.0 and 10% glycerol) containing protease and phosphatase inhibitors purchased from Selleck Chemicals LLC (Houston, TX, USA). Cell debris was removed by centrifugation at 12,000 rpm for 20 min at 4°C. Protein concentrations of the whole cell lysate were determined using a Thermo Pierce^® BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of 20 µg total protein were subjected to SDS-PAGE separation and then transferred to polyvinylidene difluoride (PVDF) membranes. An antibody against AAED1 was purchased from Sigma-Aldrich (Merck KGaA). Glut1 (dilution factor 1:500; cat. no. 21829-1-AP), Hk2 (dilution factor 1:1,000; cat. no. 22029-1-AP), Ldha (dilution factor 1:3,000; cat. no. 19987-1-AP), Pgk1 (dilution factor 1:1,000; cat. no. 17811-1-AP), ERK1/2 (dilution factor 1:1,000; cat. no. 16443-1-AP), Akt1 (dilution factor 1:1,000; 10176-2-AP), p-Akt1 (dlituion factor 1:1,000; cat. no. 66441-1-lg) and β-actin (dilution factor 1:3,000; cat. no. 60008-1-lg) antibodies were manufactured by Proteintech Group (Rosemont, IL, USA). Phospho-p44/p42 MAPK (Erk1/2) (Thr202/Tyr204) (dilution factor 1:1,000; cat. no. 9101) was purchased from Cell signaling Technology (Danvers, MA, USA).

Cellular glycolytic capacity and mitochondrial function were determined using the XF96 Extracellular Flux Analyzer (Seahorse Bioscience; Agilent Technologies, North Billerica, MA, USA), according to the manufacturer's instructions included in the Seahorse XF Glycolysis Stress Test kit (cat. no. 103020) and the Cell Mito Stress Test kit (cat. no. 103015). ECAR shows the ability of glycolysis. Before adding glucose, the ECAR value is known as the basic value, which reflects the acid production through the non-glycolytic pathway. When glucose is added, ECAR reflects the glycolysis capacity of the cells. When oligo is added, oxidative phosphorylation is inhibited, and the increased value of ECAR reflects the total glycolysis capacity of the cells, which is also known as the potential glycolysis ability of the cells. The total numerical value is the maximum glycolysis ability of the cells. 2-DG, which is finally added, is an inhibitor of glycolysis, and the ECAR change after this step reflects the acid production through the non-glycolytic pathway. In OCR assay, the basal value before adding oligo reflects the basic oxygen consumption of the cells, which includes two components; mitochondrial oxidative phosphorylation and proton leakage oxygen consumption. Protons form electrical potential energy on the mitochondrial membrane via the respiratory chain. Some of protons reflux and ATP is formed through ATP synthetase, and the potential energy is transformed into energy in the form of ATP. Another part of the proton is oxidized by the proton in the mitochondrial membrane, and the potential energy is not used to synthesize ATP, but to heat energy. After the addition of ATP synthase inhibitor oligo, the decrease in oxygen consumption reflects the oxygen consumption of the body in the process of ATP synthesis, which indirectly reflects the ATP production of the cells. FCCP is a kind of uncoupler that can be regarded as a proton carrier, which allows a large number of protons to flow back. Oxygen consumption after the addition of FCCP reflects the largest mitochondrial oxygen consumption capacity, indirectly reflecting the maximum breathing capacity. Finally, two types of agents, amphoterin A and rotenone, were added, and they were respiratory chain inhibitors, which completely prevented the oxygen consumption of mitochondria.

The 80 clinical tissue samples used in this study were histopathologically and clinically diagnosed at the Shanghai Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China) between June 2014 and May 2017. The age distribution of patients (47 males and 33 females) was between 28 and 75 years. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained. Paraffin-embedded tissue slides were deparaffinized in xylene, rehydrated through a graded alcohol series, blocked in methanol containing 3% hydrogen peroxide, and then incubated with an AAED1 antibody (dilution 1:50; cat. no. HPA021294; Sigma-Aldrich, Merck KGaA). Followed by PBS solution rinsing, the slides were incubated with secondary antibodies and peroxidase reagent at room temperature (cat. no. GK6007; Beijing Genetech, Co., Ltd., China). At the end, the slides were incubated with 3,3′-diaminobenzidine solution at room temperature for 10 min and counterstained with hematoxylin. A scoring scale was used to evaluate the percentage of cells stained (0, <10%; 1, 10–25%; 2, >25–50%; 3, >50–75%; and 4, >75%) and intensity of staining (0, negative; 1, low; 2, moderate; and 3, strong). The overall staining scores were determined by combining the two scores (frequency plus intensity). An immunohistochemical score >6 was defined as high expression, whereas a score ≤6 was defined as low expression (16).

AGS and MGC-803 cells were seeded on 96-well culture plates and transfected with indicated vectors using Invitrogen™ Lipofectamine™ 2000 (Thermo Fisher Scientific). HIF1α activity was assessed using HRE-luciferase firefly luciferase construct (Addgene plasmid 26731) (17). pRL-TK (Promega, Madison, WI, USA) was used as an internal control. Firefly and Renilla luciferase activities were measured using a Dual-Luciferase system (Promega), as described in the manufacturer's protocol.

Intracellular ROS were detected using a Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology, Haimen, China). In brief, cells were washed with PBS. Then, cells were stained with 10 µmol/l DCFH-DA at 37°C for 20 min according to the manufacturer's instructions. DCFH-DA was deacetylated intracellularly by non-specific esterase, which was further oxidized by ROS to the fluorescent compound 2,7-dichlorofluorescein (DCF). DCF fluorescence was detected using a FACScan flow cytometer (Becton-Dickinson; BD Biosciences, Franklin Lakes, NJ, USA).

All data are presented as the mean ± SD; experiments were repeated at least three times. Two-tailed unpaired Student's t-tests and one-way analysis of variance (ANOVA) and post hoc tests were used to evaluate the data. SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. Differences were considered significant at *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as indicated in the figures.

To verify the expression status of AAED1 in gastric cancer, IHC staining was performed using an AAED1 antibody. As shown, AAED1 was significantly higher in gastric tumor samples than that in normal paratumor samples (Fig. 1A). The scoring criteria was listed as negative, weak, modest and strong (Fig. 1B). Results indicated that AAED1 expression was significantly higher in gastric tumor samples than that in the paratumor samples (Fig. 1C). Taken together, these results suggested that AAED1 may be a tumor-promoting marker in gastric cancer.

To confirm the role of AAED1 in gastric cancer cell proliferation, we firstly silenced AAED1 expression in gastric cancer cell lines using a lentivirus-mediated method. Two shRNA targets that efficiently suppressed AAED1 mRNA expression in AGS and MGC-803 cells were generated (Fig. 2A). Immunoblotting with AAED1 antibodies further verified the silencing effect (Fig. 2B). Next, a CCK-8 cell viability assay was performed, and the results indicated that silencing of AAED1 expression inhibited cell viability in the AGS and MGC-803 cells (Fig. 2C). Finally, the impact of AAED1 on colony formation capacity was assessed. As shown, silencing of AAED1 expression significantly inhibited colony formation capacity (Fig. 2D and E). Collectively, these results suggested that AAED1 may have positively regulated proliferation in gastric cancer cells.

Aberrant cancer cell metabolism is pronounced in cancer cells to facilitate uncontrolled proliferation. Aerobic glycolysis is the best characterized metabolic reprogramming that regulates cancer cell proliferation. Thus, we analyzed the impact of AAED1 on gastric cancer cell metabolism. Through glycolysis, cancer cells utilize glucose to produce lactate and increased levels of lactate could produce an acidic environment that could be measured by extracellular acidification rate (ECAR). Our ECAR measurement results demonstrated that silencing of AAED1 expression decreased ECAR levels, indicating that AAED1 is a positive regulator of aerobic glycolysis (Fig. 3A and B). During glycolysis, mitochondrial respiration was impaired and was measured using the oxygen consumption rate (OCR). In AAED1-silenced AGS and MGC-803 cells, we observed an increase in OCR levels, which further reinforced the impact of AAED1 on aerobic glycolysis (Fig. 3C and D). In conclusion, our results suggested that AAED1 is a positive regulator of glycolysis, and AAED1 supports the proliferation of gastric cancer cells.

To search for the molecular pathway that participates in aerobic glycolysis by AAED1 in gastric cancer cells, the changes in HIF1α expression in AAED1-knockdown AGS and MGC-803 cells were examined. As observed, HIF1α protein levels were observably decreased in the AAED1-silenced cells. Activation of ERK1/2 and Akt1 has been reported to positively regulate aerobic glycolysis and to regulate HIF1α and HIF1α-transcriptional activity. Thus, we performed immunoblot analysis to observe changes in the ERK1/1 and Akt1 activation status in the AGS and MGC-803 cells. As shown, silencing of AAED1 inhibited the activation of ERK1/2 and Akt1, which may account for the regulation of HIF1α by AAED1 in gastric cancer cells (Fig. 4).

AAED1 may play an antioxidant role in cancer cells. As observed above, AAED1 regulated ERK1/2 and Akt1 activation, two signaling pathways that may regulate ROS generation and antioxidant response. Thus, we measured the influence of AAED1 on ROS generation in AGS and MGC-803 cells. As shown, silencing of AAED1 expression in these two gastric cancer cell lines decreased intracellular ROS levels (Fig. 5A and B).

Aerobic glycolysis is a multiple step enzymatic process to metabolize glucose. Among the many glycolysis genes, GLUT1, HK2, PGK1 and LDHA are reported to catalyze the rate-limiting steps and are direct HIF1α target genes. Thus, quantitative real-time PCR (qPCR) analysis was performed to assess the impact of AAED1 on the expression status of GLUT1, HK2, PGK1 and LDHA. As shown, AAED1 knockdown significantly decreased the expression of these HIF1α-targeted, glycolysis genes (Fig. 6A-D). Furthermore, western blot analysis results were consistent with the qPCR results, and the protein levels of GLUT1, HK2, PGK1 and LDHA were decreased in the presence of AAED1 knockdown (Fig. 6E and F). GLUT1, HK2, PGK1 and LDHA are HIF1α target genes and conserve hypoxia response elements on their promoter region. HIF1α transcription activity could be assessed by using HRE-luciferase activity. Then, we performed a Dual-Luciferase assay, and our results demonstrated that AAED1 significantly regulated HRE-luciferase activity in a dose-dependent manner (Fig. 6G and H).

In conclusion, the results of the present study demonstrated that AAED1 levels were increased in patients with gastric cancer. In vitro results suggested that AAED1 positively regulated ERK1/2 and AKT1 activation and the relevant HIF1α upregulation. Increased HIF1α resulted in increased expression of glycolysis genes and enhanced glycolysis, which favors uncontrolled proliferation of gastric cancer cells (Fig. 7).