Human gastric cancer cell MGC-803 and the normal gastric epithelial cell line GES-1 were purchased from Nanjing KeyGen Biotech Co., Ltd. The cells were cultured in RPMI-1640 medium with 10% FBS (both purchased from Thermo Fisher Scientific, Inc.), 100 µg/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator at 5% CO₂. The PI3K inhibitor LY294002 (10 mmol/l; Cell Signaling Technology, Inc.) was dissolved in DMSO (cat. no. D2650; Sigma-Aldrich; Merck KGaA) and diluted with RPMI-1640 medium to 12.5, 25 and 50 µmol/l. LY294002 was cultured with MGC-803 cells at 37°C for 24 h in a humidified incubator at 5% CO₂.

The CYP2E1 construct was synthesized and integrated into a recombinant lentiviral vector GV358 (Shanghai GeneChemCo., Ltd.). CYP2E1 overexpression was accomplished with the recombinant lentiviral vector (Shanghai GeneChem Co., Ltd.), and the empty vector was used as a control. MGC-803 cells were seeded into 96-well plates (5×10³ cells/well in 100 µl complete medium), and three infection gradients (MOI=10, MOI=20, MOI=30) were used to infect the cells using FuGENE^® 6 transfection reagent (Nanjing KeyGen Biotech Co., Ltd). The MGC-803 cells were incubated for 24 and 48 h after transfection for subsequent experiments.

The expression levels of CYP2E1 and the normalization gene β-actin were assessed via RT-qPCR. Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and RNA purity was detected with a NanoDrop™ 2000 spectrometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). In accordance with the manufacturer's protocol, RNA (1 µg) was reverse transcribed into cDNA via a two-step reverse transcription reaction performed (37°C for 15 min and 85°C for 5 sec) using a Takara cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.). RT-qPCR was performed with a StepOnePlus real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and SYBR Green One-Step Real-Time RT-PCR Master Mixes (Thermo Fisher Scientific, Inc.) to measure the expression level of the selected long non-coding RNA. Amplifications were performed with 2 min enzyme activation at 95°C, followed by 40 cycles of denaturation at 95°C for 15 sec, and then annealing/extension at 60°C for 15 sec. At the end of each run, melting curve analysis was performed at 72°C for 30 sec. The comparative Cq method was used to measure the relative fold change in CYP2E1 levels in GC cells. Relative quantitation values of mRNA expressions were calculated using the 2^−ΔΔCq method (14). The primers were obtained from Kingsley Biotechnology Co., Ltd. The mRNA primer sequences for CYP2E1 and the housekeeping gene β-actin were as follows: CYP2E1 forward, 5′-GCCATCAAGGATAGGCAAGA-3′ and reverse, 5′-TCCAGAGTTGGCACTACGACT-3′; β-actin forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′ and reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′.

Total protein was extracted from cells using RIPA lysate reagent (Thermo Fisher Scientific, Inc.), and the total protein concentration was detected by a BCA protein detection kit (Thermo Fisher Scientific, Inc.) Total protein (20 µg/lane) were separated using SDS-PAGE (10%) and then transferred to a nitrocellulose membrane. The membrane was blocked in TBS-Tween-20 (TBST) with 5% milk at room temperature for 2 h and then incubated with primary antibody in 5% BSA-TBST (1:1,000) overnight at 4°C. Then, the membrane was washed with TBST and incubated with secondary antibody (1:10,000) at room temperature for 1 h. Protein bands were detected with an ECL chromogenic kit (Thermo Fisher Scientific, Inc.). Images were captured using a Tanon-5200 chemiluminescence imaging system (Tanon Science and Technology Co., Ltd.), and ImageJ software (v1.8.0; National Institutes of Health) was used for densitometric analysis. Specific primary antibodies against β-actin (cat. no. 4967S), PI3K (cat. no. 4292S), phosphorylated (p)-PI3K (cat. no. 4228T), Akt (cat. no. 9272S), p-Akt (cat. no. 9271T), mTOR (cat. no. 2972S), p-mTOR (cat. no. 2971S), p70 ribosomal protein S6 kinase (P70S6K; cat. no. 9202S), p-P70S6K (Ser371; cat. no. 9204S) and CYP2E1 (cat. no. ab28146; Abcam) and the secondary antibody (anti-rabbit IgG, horseradish peroxidase-conjugated antibody; cat. no. 7074P2) was purchased from Cell Signaling Technology, Inc. The high molecular weight protein marker was supplied by Bio-Rad Laboratories, Inc., and the small molecular weight protein marker was supplied by Thermo Fisher Scientific, Inc.

The proliferation ability of CYP2E1-overexpressing MGC-803 cells was determined with a Cell Counting Kit-8 (CCK-8; Yisheng Biotechnology Co., Ltd.). In line with the manufacturer's protocol, cells were seeded into 96-well plates (5×10³ cells/well in 100 µl complete medium) in a CO₂ incubator at 37°C for 6, 12, 24, 36, 48 and 60 h, followed by addition of 10 µl of the CCK-8 mixture to each well. After incubation for 2 h at 37°C in an incubator, the absorbance was measured at a wavelength of 450 nm using a microplate reader (Gene Company Ltd.).

For flow cytometry analysis, cells were seeded on 6-well plates at 5×10⁵ cells/well, with three replicate wells per experimental group. After a 48 h culture period at 37°C, the cells were harvested and washed twice with phosphate buffer, resuspended in 300 µl of binding buffer, and reacted with 5 µl of Annexin V-allophycocyanin (APC) and 5 µl of hypotonic propidium iodide (PI) solution for 10 min at room temperature. Then, 300 µl binding buffer solution was added and the cells were examined via flow cytometry for 10 min. The cells were analyzed using a BD flow cytometer (BD Via; BD Biosciences) (15). FlowJo software (v10.0; BD Biosciences) was used to analyze these data. The apoptosis rate was calculated as the percentage of early + late apoptotic cells. An Annexin V-APC/PI apoptosis kit (MultiSciences Biotech, Co., Ltd.) was used for the cell apoptosis assay.

Cell migration was assayed using Transwell inserts (Corning Inc.). MGC-803 cells transduced for 96 h were collected and washed three times with PBS. MGC-803 CYP2E1 cells were resuspended in RPMI-1640 medium containing 1% FBS (Thermo Fisher Scientific, Inc.) and inoculated into the upper Transwell chamber at a density of 5×10⁴ cells/well (hole, 8 µm). Next, RPMI-1640 medium containing 10% FBS was added to the lower Transwell chamber, and the cells were cultured at 37°C in a humidified incubator containing 5% CO₂. The chamber was removed after 24-h incubation. The cells were fixed for 10 min at room temperature with 95% ethanol, stained with 0.1% crystal violet for 10 min at room temperature, and then washed with PBS. The chamber was air-dried, and 5–10 fields in each chamber were randomly selected for cell counts. Images were acquired for later analyses. The number of cells that passed through the chamber was compared between the MGC-803 CYP2E1 and MGC-803 NC groups. Visualization was performed with an FSX100 biological imaging system (magnification, ×500; Olympus Corporation), and the images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The invasive ability of MGC-803 CYP2E1 and MGC-803 NC cells was examined with Matrigel (Matrigel Gel and serum-free medium preparation, 1:8) at 4°C overnight using a Transwell package. MGC-803 cells transduced for 24 h were collected and washed three times with PBS. MGC-803 CYP2E1 cells were resuspended in RPMI-1640 medium containing 1% FBS and seeded at a density of 1–5×10⁵ cells/well into the top of the chamber (pores, 8-µm; the chamber was removed from 4°C before use and placed at 37°C for 1 h to coagulate the matrix). Medium containing 10% FBS was added to the bottom of the Transwell chamber. After 24-h incubation at 37°C, the remaining cells in the upper chamber were scraped away using cotton swabs. The invasive cells were fixed with 95% ethanol for 10 min at room temperature, stained with 0.1% crystal violet for 10 min at room temperature, and then washed with PBS. The chamber was air dried and imaged with an FSX100 Bio Imaging Navigator (magnification, ×500; Olympus Corporation), and the number of cells that penetrated the chambers was determined as described above for the cell migration assay.

The statistical significance of the RT-qPCR, protein expression, flow cytometry, migration and invasion assay results were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.). Data are expressed as the mean ± SD. Student's t-test and one-way ANOVA followed by Dunnett's test were used to assess the statistical significance of differences. P<0.05 was considered to indicate a statistically significant difference.

Western blot analysis showed that the protein expression level of CYP2E1 in MGC-803 cells was significantly higher than in GES-1 cells (P<0.05; Fig. 1). These results suggested that the expression of CYP2E1 is different between GES-1 and MGC-803.

To further explore the biological function of CYP2E1, a CYP2E1 lentiviral vector was constructed to induce CYP2E1 overexpression. MGC-803 cells were infected with a CYP2E1 lentiviral vector or the empty vector for 24–72 h. MGC-803 cells were efficiently infected with the lentivirus-CYP2E1. RT-qPCR results (Fig. 2A) indicated that the CYP2E1 expression level in MGC-803 cells transduced with the lentivirus-CYP2E1 overexpression vector was significantly increased compared with the MGC-803 NC cells (63-fold; P<0.05). Western blotting showed that protein expression level of CYP2E1 was significantly increased in the MGC-803 cells transduced with the lentivirus-CYP2E1 overexpression vector compared with the MGC-803 NC group (P<0.05; Fig. 2B).

To determine whether CYP2E1 affects GC cell proliferation and apoptosis, a CCK-8 cell proliferation assay and flow cytometry were used to test the proliferative capacity and apoptotic rate of MGC-803 cells overexpressing CYP2E1.

In MGC-803 CYP2E1 cells, cell growth increased from 12 h and was time-dependent compared with negative control cells (P<0.05; Fig. 3A). Annexin V-APC/PI staining combined with flow cytometry showed that the apoptotic rate in the MGC-803 NC group was significantly higher than that in the MGC-803 CYP2E1 group at 24 h (P<0.05; Fig. 3B and C).

In addition, it was investigated whether CYP2E1 plays a critical role in migration and invasion of GC cells. A Transwell assay was used to analyze the invasive and migratory abilities of GC cells after CYP2E1 gene overexpression. The migratory ability of MGC-803 cells was significantly increased after transduction with the lenti-CYP2E1-overexpression plasmid compared with the negative control (Fig. 4A and B). Similarly, the Matrigel invasion assay results indicated that the invasive ability of MGC-803 cells was significantly increased after transduction with the lenti-CYP2E1-vector (P<0.05; Fig. 4C and D).

PI3K/Akt/mTOR is a crucial signaling pathway involved in the carcinogenicity and tumorigenesis of different cancer types, and is closely related to cell biological functions, such as energy metabolism, proliferation, apoptosis and cell invasion (16–19). Previous studies have revealed that certain mRNAs are co-expressed with CYP2E1 and participate in the PI3K/Akt/mTOR signaling pathway (20,21). Therefore, the present study investigated whether CYP2E1 was involved in the regulation of GC cell proliferation, apoptosis and invasion via PI3K/Akt/mTOR signaling proteins. mRNAs and proteins were isolated from lysed MGC-803 cells that were transduced with CYP2E1 overexpression lentivirus or the negative control. RT-qPCR results revealed that the expression level of mTOR mRNA in MGC-803 cells transduced with the lentiviral-CYP2E1 overexpression vector was significantly higher compared with the MGC-803 NC group (P<0.05; Fig. 5A). However, no significant differences in PI3K and Akt mRNA expression levels were observed between MGC-803 CYP2E1 cells and MGC-803 NC cells. Western blotting results revealed that CYP2E1 overexpression increased the relative expression levels of p-Akt, p-mTOR, and p-P70S6KSer371 compared with those in the negative controls (P<0.05; Fig. 5B). Therefore, it is possible that CYP2E1 is involved in the malignant progression of GC cells via p-Akt, p-mTOR and p-P70S6KSer371 proteins in the PI3K/Akt/mTOR signaling pathway.

To further explore the role of CYP2E1 in MGC-803 cells, the PI3K/Akt/mTOR signaling pathway was inhibited using the PI3K inhibitor LY294002. In the LY294002-treated group, PI3K, Akt, mTOR, p70S6K and CYP2E1 protein expression levels decreased with increasing inhibitor concentration compared with the control group (Fig. 6; P<0.05). These results suggested that CYP2E1 could function as an oncogene in GC by participating in the PI3K/Akt/mTOR signaling pathway. CYP2E1 protein expression may be mediated by the PI3K signaling pathway and key downstream mediators.