Cell culture and human specimens
HeLa and SiHa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco`s modified Eagle`s medium with 10% fetal bovine serum (HyClone, Logan, UT). Human samples were obtained from patients with cervical cancer by biopsy or surgery at Daping Hospital and Research Institute of Surgery. This study was approved by the Ethics Committee of Daping Hospital, Army Medical University. We obtained consent to publish from the participants (or legal parents or guardians for children) to report individual patient data.
Plasmid constructs
The ZEB1 ORF was amplified by PCR from human cDNAs and inserted into the BamHI and HindIII sites of the pcDNA3.1-flag vector for expression in mammalian cells. The E-cadherin promoter region from − 670 to + 92 was amplified using primers with restriction enzyme sites BglII or HindIII at each end and inserted into the upstream region of the firefly luciferase gene of the pGL3-Basic vector (Promega, Madison, WI, USA) 24. APE1 (NM_001641.4) and ZEB1 (NM_001323642.2) expression vectors were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China). All primer sequences used in this study are given in Table S1.
Immunohistochemistry (IHC), immunofluorescence (IF), and western blot
For western blotting, cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Saint Louis, MO, USA), and the protein concentration of the lysate was measured using a Bradford kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins (30 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was blocked for 1 hour in Tris-buffered saline with Tween 20 (TBST) containing 5% skim milk at room temperature (RT), and immunoblotting was performed by incubating the membranes overnight with their corresponding primary antibodies in 5% skim milk at 4°C. The membrane was washed with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at RT. After washing, the proteins were visualized with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific) in accordance with the manufacturer’s recommendations.
For IHC, the tumor tissues were fixed in 10% buffered formalin (Sigma-Aldrich), embedded in paraffin, and sectioned at 4 µM. The tissue sections were deparaffinized in xylene, rehydrated through an alcohol gradient, washed and incubated in 0.3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 15 min. After washing, the tissue sections were blocked with 5% bovine serum albumin in PBS for 1 hour. Then, primary antibodies were applied to the tissue sections overnight at 4°C. The following day, the tissue sections were washed and incubated with secondary HRP-conjugated antibodies for 1 hour at RT and counterstained with Mayer's hematoxylin (Dako, Carpinteria, CA, USA) for 10 seconds. Coverslips were mounted using Permount (Thermo Fisher Scientific). A panel of pathologists reviewed the IHC staining and scored it as follows: score 0, no staining positive tumor cells; score 1, staining positive tumor cells less than 10% of the total tumor cells; score 2, staining positive tumor cells more than 10% of the total tumor cells, but less than 50%; and score 3, staining positive tumor cells more than 50% of the total tumor cells. Scores of 0 and 1 were defined as low expression, and scores of 2 and 3 were defined as high expression.
For IF, cells were grown on coverslips and transfected with the indicated oligonucleotides. After 72 hours of transfection, the cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.3% Triton X-100 for 10 min. The cells were washed with PBS and incubated with 3.5% bovine serum albumin (BSA) for 1 hour followed by incubation with the primary antibody in 3.5% BSA for 1 hour at RT. The cells were washed with PBS and incubated with FITC-conjugated secondary antibody for 1 hour at RT in the dark. Then, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 30 min. Antibodies against APE1, β-actin, E-cadherin, N-cadherin, vimentin, Flag, HA, and GAPDH and secondary antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-ZEB1 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA).
Invasion assay
Cells were transfected with APE1 construct or siRNA (GeneChem Co., Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Sequences of the double-stranded siRNAs are antisense (5’-GUCUGGUACGACUGGAGUACC-3’, 5’-UACUCCAGUCGUACCAGACCU-3’) and nonsense (5’-CCAUGAGGUCAGCAUGGUCUG3’, 5’-GACCAUGCUGACCUCAUGGAA-3’) 21. Nontargeting control siRNA was purchased from Qiagen (Hilden, Germany). After 48 hours of transfection, the cells were subjected to invasion assays. Briefly, 10,000 cells in medium without serum were seeded in the upper wells of invasion chambers (BD Biosciences, San Jose, CA, USA). The lower wells contained the same medium supplemented with 10% fetal bovine serum. After 24 hours, the cells that invaded to the other side of the chamber were fixed with 2.5% glutaraldehyde, stained with 0.1% crystal violet, and counted.
To investigate the effects of the APE1 redox inhibitor APX3330 (Selleck, Houston, TX, USA) and the APE1 DNA repair inhibitor APE1 inhibitor III (Merck Millipore, Molsheim, France) on the invasion of cervical cancer cells, cells were pretreated with APE1 inhibitors for 24 hours. After 24 hours, the cells were subjected to invasion assays as described above.
Luciferase reporter assay
Plasmids and/or nucleotides were transfected into HeLa cells that were transfected with a firefly luciferase reporter construct containing the E-cadherin promoter. The Renilla luciferase plasmid was cotransfected as a transfection control (Promega). The cell extracts were processed 72 hours after transfection, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The luciferase activity was normalized to the activity of Renilla luciferase.
Co-immunoprecipitation
HeLa cells were transfected with the indicated plasmids. After 72 hours, the cells were lysed in lysis buffer [1% Nonidet P40, 0.1% SDS, 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM DTT and 0.5 mM EDTA containing protease inhibitors], and the protein concentration of the lysate was measured using a Bradford kit (Bio-Rad). Immunoprecipitation was performed using monoclonal antibodies as indicated. Immunocomplexes were collected on protein A/G-agarose beads (Merck Millipore), washed twice with lysis buffer, and then washed twice with wash buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.2 mM sodium orthovanadate, protease inhibitor cocktail]. The beads were dissolved in SDS loading buffer (Bio-Rad), denatured at 95°C for 10 min, and later subjected to western blot analysis.
Secondary structure prediction
The sequence of ZEB1 was retrieved from UniProt, and the corresponding secondary structure was generated using Jpred (Jnet version: 2.3.1) (http://www.compbio.dundee.ac.uk/jpred/).
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using the LightShift chemiluminescence EMSA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions as described previously 25. Nuclear extracts were isolated from the indicated cells and incubated with 3’-biotin-labeled double-stranded oligonucleotide probes containing consensus sequences for ZEB1 binding sites (Fig. S1), then the samples were separated on a 5% polyacrylamide gel and transferred to a Zeta-Probe GT nylon membrane. The probes were detected by HRP-conjugated streptavidin. The probe sequences are shown in Table S1.
Animal experiments
All xenograft models were generated using GFP-expressing HeLa cells (HeLa-GFP) in 6-week-old female BALB/c nude mice. For the lymph node metastatic model, HeLa-GFP cells (1×106/50 µl PBS per mouse) were directly injected into the footpad of the mice 26. For the abdominal cavity metastatic model, HeLa-GFP cells (1×106/200 µl PBS per mouse) were intraperitoneally (IP) injected into the mice. For the lung metastatic model, HeLa-GFP cells (1×106/100 µl PBS per mouse) were injected intravenously into the tail vein of the mice. One week after the cell injection, the mice were randomly divided into two groups. The control group mice were treated with PBS, and the treatment group mice were treated with APX3330 (12.5 mg/kg body weight) by IP injection once every two days. The mice were treated with APX3330 for 3 weeks for the lung and lymph node metastasis experiments, treated with APX3330 for 2 weeks in the abdominal cavity metastasis experiments, and their body weight was measured every 3 days. Tumor metastasis was monitored by an IVIS Spectrum In Vivo Imaging System (Perkin Elmer, Utah, USA) and MRI (7.0-T MRI, Bruker Biospec 70/20USR, Germany). All animal experiments complied with the Daping Hospital, Army Medical University Policy on the Care and Use of Laboratory Animals.
Statistical analyses
All statistical analyses were performed using Prism 5.0 software (GraphPad). The unpaired two-samples t-test was used to compare the mean of two independent groups, and one-way ANOVA was used to determine differences between the means of two or more independent groups. p values less than 0.05 were considered statistically significant. The data are presented as the mean ± the standard deviation (SD) of at least three independent experiments. The correlation between APE1 expression and lymph node metastasis or E-cadherin expression was tested by the chi-square test.