Patients and tissue samples
A total of 88 HCC and adjacent non-tumor tissues were collected from patients who underwent liver cancer hepatectomy at the Affiliated Cancer Hospital of Zhengzhou University (Zhengzhou, China) from July 2016 to December 2017, of which 81 samples were paired. Detailed information of the sample refers to Additional file 1: Table S1. The ethics committee of Zhengzhou University approved the protocol (18 March 2016; Approved No: 2016-0177) and all the patients gave written informed consent. In addition, all specimens were subjected to RT-qPCR after RNA extraction, and 36 and 20 samples were used for western blot and immunohistochemical verification, respectively.
Cell culture and reagents
Human liver cell lines SMMC-7721, HepG2, MHCC-97H, Huh7, Bel-7402 were donated by Dr. Tingting Liu (Sino-American Hormelian Cancer Research, Zhengzhou, P.R. China) and Prof. Ping Xu (State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing, P.R. China). HCC-LM3 was purchased from China Center for Type Culture Collection (Wuhan, China). All cell lines were cultured at 37 °C with 5% CO2 and grown in Dulbecco’s modified Eagle’s medium (BI, Shanghai) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. 3-Methyladenine (3-MA) was provided by Santa Cruz Biotechnology (sc-205596). N-Acetyl-l-cysteine (NAC) and the CYP2E1 inhibitor clomethiazole (CMZ) was purchased from Yuanye Bio-Technology (Shanghai, China).
RT-qPCR was performed according to our previous study  and the primers used are listed in Additional file 1: Table S2. Briefly, total RNA was extracted by the RNAiso Plus kit (Takara Bio Inc.). The PrimeScript RT reagent kit (Takara Bio Inc) was used to synthesize the cDNA from 1 μg of total RNA. The transcript levels of the target genes were measured by an ABI 7500 Fast Real-Time PCR system. GAPDH was used as a reference gene.
Western blot assays were performed as described recently . Briefly, cells were lysed on ice and proteins were fractionated by SDS-PAGE. Then the protein was transferred onto a PVDF membrane, which was incubated with the indicated antibody and ECL solution. The signal was visualized with an ECL detection system. Antibodies used include anti-CYP2E1 (ab28146, from Abcam, UK); anti-c-Myc (10828-I-AP), Goat anti-rabbit IgG (HL) (SA00001-2), anti-β-Catenin (66379-1-Ig), goat anti-mouse IgG (HL) (SA00001-1), anti-LEF1 (14972-1-AP) (from Proteintech, Wuhan, China); anti-GAPDH (6C5, sc-47724), anti-β-actin (C4, sc-47778), anti-Dvl2 (D-11, sc-390303), anti-TCF-4 (D-4, sc-166699), anti-KLHL12 (D-1, sc-514874), anti-Ub antibody (P4D1, sc-8017) (from Santa Cruz Biotechnology, Inc, USA); anti-E-cadherin (WL01482), anti-Twist (WL00997), anti-N-cadherin (WL01047), anti-Snail (WL01863), anti-Vimentin (WL01960), and anti-Slug (WL01508) from Wanlei Life Sciences, Shenyang, China.
Gene expression manipulations
Vector lentivirus and lentivirus encoding human CYP2E1 (EX-I0304-Lv201 and EX-NEG-Lv201) were provided by GeneCopoeia (Guangzhou, China). Control or CYP2E1 vectors were first mixed with packaging plasmids (Lenti-Pac™ HIV, GeneCopoeia company) and then were co-transfected into HEK293T cells by Lipofectamine 2000 (Thermo Fisher Scientific Inc). Culture supernatants were collected and purified (LPR-LCS-01, GeneCopoeia, Guangzhou, China) at 48 h after transfection. In the presence of 8 μg/ml polybrene, MHCC-97H and SMMC-7721 cells were infected with the virus particles and finally, puromycin was used to select the infected cells. The overexpression of CYP2E1 in transfected cells was verified by RT-qPCR and western blot. To overexpress Dvl2, the full-length Dvl2 plasmid (GV417-Dvl2) was transiently infected into the CYP2E1 stable cells by VigoFect (Vigorous Biotechnology).
Cell migration assay
For cell migration assay, HCC cells were plated in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 10 μl pipette tip and exchanged with serum-free DMEM after washing twice with PBS. The images of the wounds were recorded at the same position using an inverted microscope (Nikon ECLIPSE TS100, Japan) at 0 h, 24 h, 48 h and 72 h, respectively. The width between wounds was detected by Image J software (National Institutes of Health).
Cell invasion assay
For cell invasion capacity, the Transwell chamber was coated with 100 μl 1: 8 diluted Matrigel (BD BioCoat, Corning, USA) and incubated for 5 h. Next, 5 × 104 cells in serum-free medium (200 μl) was seeded at the upper chamber, and 600 μl of DMEM medium containing 10% FBS was added to the lower chamber. After 36 h incubation at 37 °C, the matrigel in the upper chamber was gently removed using a cotton swab. The invaded cells were fixed with paraformaldehyde (4%) and stained with DAPI. The cells were photographed using fluorescence microscopy and counted from 5 random fields of microscope.
200 μl of cell suspension (5 × 103 cells) were added into 96-well plates and incubated at 37 °C. At the appointed time, each well was added with 10 μl CCK-8 reagent (Dojindo Laboratories, Japan) and the absorbance at 450 nm was determined by a microplate spectrophotometer (Bio Tek, USA).
TOPFlash reporter assay
HCC-Ctrl or HCC-CYP2E1 cells were seeded in 24-well plates and incubated for 24 h at 37 °C. Then the cells were transfected with TOPFlash plasmid (800 ng/well) and pRL-TK plasmid (50 ng/well) using VigoFect reagent (Vigorous Biotechnology). The Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) was employed to to measure Luciferase and Renilla activities 48 h after transfection. The luciferase activity of each sample was normalized with the respective Renilla activity.
Detection of reactive oxygen species (ROS)
Cells grown to the appropriate 70–80% confluence in 6-well plates were stained with 20 μmol/l dihydroethidium (DHE, ABP Biosciences, Germany) for 1 h in the dark to assess the ROS level. ROS generation was indicated by red fluorescence and detected at Em: 485 nm and Ex: 610 nm with a fluorescence microscope.
Co-immunoprecipitation (co-IP) experiment
Protein extraction and quantification are as described for western blotting. Briefly, total protein (800 μg) was precleared with Protein A/G agarose (sc2003; Santa Cruz) at 4 ℃ for 30 min to remove non-specific protein. After centrifugation, the precleared supernatant was incubated with 2 μg of primary antibodies at 4 ℃ overnight and then 40 μl Protein A/G agarose was added and incubated at 4 ℃ for 4 h. Next, the agarose beads were collected by centrifugation at 3000 rpm and extensively washed with RIPA lysis buffer. Finally, the bound proteins were mixed with loading buffer and subjected to western blotting analysis. Mouse IgG was used as a negative control.
Liver tissues were embedded with paraffin, sectioned, dewaxed, and dehydrated with gradient ethanol. After antigen repair, the slides were incubated with CYP2E1 antibody (ab28146, Abcam; 1:125 dilution) at 4 °C overnight. Then, the slides were washed with TBST and incubated with HRP-conjugated secondary antibody polymer (SA00001-2, Proteintech, Wuhan, China; 1:250 dilution) followed by the addition of DAB solution and counterstained with hematoxylin.
Immunofluorescence cell staining
The cells seeded on the coverslips in the 24-well plates were cultured for 24 h. After fixing with 4% paraformaldehyde and permeabilizing with 0.5% Triton X-100, the cells were incubated with mouse anti-E-cadherin (2:250 dilution, AF0138, Beyotime) and rabbit anti-vimentin (2:250 dilution, 10366-1-AP, proteintech) at 4 ℃ overnight and then with fluorescence secondary antibodies (CoraLite 594, SA00013-3, goat anti-mouse IgG, Proteintech, 1:500 dilution; CoraLite 594, SA00013-4, goat anti-rabbit IgG, Proteintech, 1:500 dilution) for 30 min at 37 ℃. DAPI was used to stain the nuclei for 5 min and coverslips were mounted. Then, the cells were observed by the fluorescence microscope (OLYMpusBX43).
Tumor xenografts model
Four-week-old male BALB/c nude mice were provided by the Beijing Vital River Laboratory Animal Technology Company. After habituation for 3 days, 100 μl of MHCC-97H-GFP or MHCC-97H-CYP2E1 cell suspension (1 × 107 cells/ml) were injected subcutaneously into the right flanks of mice. Tumor volumes was recorded every 3 days. Tumor volume was calculated with the formula of [0.5 × length × width2]. At 35 days after injection, the nude mice were euthanized and tumors were collected for the subsequent orthotopic liver tumor model. These animal experiments were approved by the Animal Ethics Committee of Zhengzhou University and carried out in accordance with the guidelines of animal ethics.
Mouse liver orthotopic transplantation model
The tumors taken from the above xenograft nude mice were immediately minced into small pieces (about 1.5 mm × 1.5 mm × 1.5 mm). The left lobe of the liver of the 4.5-week-old male mice was extruded under anesthetization and a tunnel was made by a 10 ml syringe needle. Then 2 pieces of the tumors were deposited into the left lobe through the tunnel. Subsequently, the left lobe of the liver was returned to the abdominal cavity, and the muscle and skin were sutured. Ten weeks after the operation, the livers were removed and orthotopic tumors were counted.
Pulmonary metastasis model
200 μl of MHCC-97H-GFP or MHCC-97H-CYP2E1 cell suspension (1 × 106 cells/ml) were injected into the tail vein of 4.5-week-old mice. At the 9th week the nude mice were euthanized and the lungs removed. The lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into slices. The slices were moved onto glass slides, dewaxed, rehydrated in ethanol solution and followed by the addition of hematoxylin and eosin to determine the metastasis of cancer cells.
SPSS version 21.0 and GraphPad Prism 5.0 were used to perform statistical analysis and generate graphs, respectively. The Kolmogorov–Smirnov and Shapiro–Wilk methods were used to assess the normality of the data. For normal distribution data, a two-tailed Student’s t-test was used for pairwise comparisons. The Mann–Whitney U test was used to compare two groups with non-normal distribution data. The overall survival data were analyzed by Kaplan–Meier plots and the cut-off value for defining the subgroups was the median expression level of CYP2E1 mRNA. The sample was then divided into two groups. One group consisted of samples with CYP2E1 expression levels higher than the median, and the other group consisted of the remaining samples. A P value (two-tailed) < 0.05 indicated statistically significant.
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