Patients and clinical characteristics
In this study, the relationship between CHD4 expression and detailed clinical information was analyzed. First, the values of CHD4 expression between 607 tumor tissues and 130 nonneoplastic tissues were extracted from The Cancer Genome Atlas (TCGA) (https:// cancergenome.nih.gov/). Next, we evaluated the CHD4 expression level in samples of different pathologic types, and the data were extracted from various independent research datasets in the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/). Subsequently, the relationship between CHD4 expression level and clinical characteristics was analyzed. Finally, combining data from TCGA and GEO, the prognostic significance of the expression profile of CHD4 was evaluated.
Cell lines culture
The human ovarian cancer cell lines SKOV3, Caov3, Hey and OVCAR3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SKOV3-ip was kindly gifted by Cui Heng et al. of Pecking University. All cell lines were maintained in RPMI 1640 (Jinuo Co., Ltd, Shanghai, China) medium with 10% fetal bovine serum (Gibco, Life Technologies, Grand Island, NY, USA). Cell numbers were measured with a TC10 Automated Cell Counter (Bio-Rad Laboratories, Inc. Hercules, California, USA). Cells were maintained routinely at 37°C in a 5% CO2 humidified atmosphere.
Plasmids transfections and Lentiviral infection
Knockdown of CHD4 protein expression was achieved by RNA interference with two independent small interfering RNAs (siRNAs, Table S1). For establish a stable CHD4 knockdown cell line, a lentiviral vector-based short hairpin RNA (shRNA) was constructed (Table S1). Furthermore, an expression plasmid containing the full-length EZH2 sequence (Table S1) was generated, and the corresponding lentiviral vectors were constructed by GeneChem (Shanghai, China). Stable clones were obtained by transfecting cells with the corresponding lentiviral vector. The selection medium for transfected cells was supplemented with 1 μg/ml puromycin (Sigma Co., Ltd, USA).
Cell proliferation assays
SKOV3-ip, SKOV3-ip-siControl, SKOV3-ip-siCHD4-1 and SKOV3-ip-siCHD4-2 cell lines were plated in 96-well plates (Corning, NY, USA) at a density of 1 × 103 cells per well and incubated for 0 h, 24 h, 48 h, 72 h or 96 h at 37˚C. At each indicated time, cell viability was evaluated by the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc., Gaithersburg, MD, USA) assay, followed by incubation for 4 h. The absorbance was measured at 450 nm using a BioTeK Reader. Cell proliferation was tested according to the manufacturer’s protocol. Each experiment was repeated at least thrice.
The SKOV3-ip cell line was plated in 96-well plates at a density of 1 × 103 cells per well. After 24 h, the medium was replaced by medium with different concentrations of romidepsin (0 nM, 1 nM, 10 nM, 50 nM and 1 μM, 10 μM, 100 μM, Med Chem Express, New Jersey, USA) for 0 h, 24 h, 48 h, 72 h and 96 h. Romidepsin was dissolved in 0.1% sterile dimethyl sulfoxide (DMSO, Sangon Biotech, Shanghai, China). The control group was treated with 0.1% DMSO solution. At each indicated time point, cell proliferation was evaluated by the CCK-8 assay.
Cell migration and invasion assays
For the migration assay, the cell lines were starved overnight with serum-free medium, and then 2×104 cells in 100 µl of serum-free 1640 medium with/without 10 nM romidepsin were transferred into the upper compartment of uncoated chambers (Corning, New York, USA). The lower chamber was filled with 500 µl of 1640 medium containing 10% FBS with/without 10 nM romidepsin. For the invasion assay, the insert membranes were precoated with diluted (1:8) Matrigel (Corning, NY, USA). After 12 h of incubation, the cells on the upper surface of the membrane were removed. Cells that passed through the membrane were fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China) and stained with 0.1% crystal violet (Beyotime, Shanghai, China). Cells were counted using an Olympus light microscope in 5 random fields at x200.
Quantitative reverse transcriptase (qRT)-PCR
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA), and reverse transcription was performed using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, MA, USA) according to the manufacturer’s instructions. qRT-PCR was performed on the SYBR PrimeScript RT-PCR Kit (TAKARA Bio Inc., Otsu, Shiga, Japan) on an ABI 7900. Data were analyzed through SDS 2.3 software, and the threshold cycle (Ct) values were normalized to GAPDH. The mRNA levels were calculated using the equation 2-ΔΔCt based on experiments performed in triplicate. All experiments were performed in triplicate. The primers used for all genes are listed in Table S2.
Western blot analysis
To collect total (whole-cell) protein, the cells were lysed with RIPA buffer (Beyotime, Shanghai, China) and 1 mM protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China), agitated for 15 minutes at 4°C, and centrifuged at 12000 rpm for 25 minutes. To collect the nuclear protein, the cells were manipulated according to the protocol of nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China). Protein concentrations were measured with the BCA Protein Assay (Beyotime, Shanghai, China). Proteins were electrophoresed on SDS-polyacrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck KGaA, Darmstadt, Germany). The PVDF membrane was blocked for 1 h at room temperature (RT) in 10% milk powder in PBS with 0.2% Tween. The primary antibodies were diluted and incubated overnight at 4°C (the primary antibody information is provided in Table S3). Then, the membranes were washed three times for 10 minutes in PBS with 0.2% Tween. Afterwards, the membranes were incubated in goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilution; MT-bio, Shanghai, China) at RT for 1 h. The membranes were washed again six times and incubated with enhanced chemiluminescence (ECL) reagent (Thermo, MA, USA) for visualization. GAPDH and lamin were used as internal controls to normalize the protein expression levels of the whole-cell extract and nuclear extract, respectively.
Bioluminescent mouse xenograft tumorigenesis studies
All experiments were performed in accordance with the Laboratory Animal Unit (LAU) guidelines and approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of The University of Fudan. Stable luciferase-expressing SKOV3-ip cells were resuspended in 100 µl serum-free culture medium, and mixed with 100 µl Matrigel (Corning, NY, USA). Six-week-old female BALB/c (nu/nu) mice were intraperitoneally injected with the mixture. After allowing tumor formation for 10 days, the animals were randomized into 3 groups as follows: (i) the control group (treated with equal volume of 0.1% DMSO); (ii) the low-dose romidepsin group (intraperitoneally injected with 1 mg/kg romidepsin on days 11, 14, 17, 21, 24 and 27); and (iii) the high-dose romidepsin group (intraperitoneally injected with 2 mg/kg romidepsin on days 11, 14, 17, 21, 24 and 27). Baseline imaging data were recorded for all mice on day 10 (start of drug administration) and reimaged posttreatment on days 20 and 30. For imaging acquisition, the mice were anesthetized with 1.5% pentobarbital (50 µl/10 g, i.p.) and initial imaging of mice was performed with the BRUKER In Vivo Xtreme; after injecting substrate D-luciferin (Sciencelight.biogo. Shanghai, China, 150 mg/kg). Mouse weight was measured every other day to assess the toxicity of the drugs. After 30 days, the nude mice were euthanized by i.p. injection of 200 mg/kg pentobarbital to observe the location and number of metastatic foci. Tumor tissue and liver tissue were excised and processed for histology and immunohistochemistry (IHC). The lesions were stored in 4% paraformaldehyde or RNAStay, respectively.
Statistical analysis
Statistical analyses were performed using SPSS 19.0 software. Means and standard deviations (SDs) were calculated for all independent experiments. All values were analyzed by unpaired Student's t-test. Differences with p-values <0.05 were considered statistically significant.