Patients and samples
Sixty-six pairs of frozen HCC tissues and corresponding non-cancerous liver tissues were obtained from the Ajou University Hospital (Suwon, South Korea). Patient demographics and clinical characteristics are presented in Additional file 1: Table S1. All experiments were performed according to the Declaration of Helsinki and the study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMR-KSP-16-365 and AJIRB-BMR-SMP-17-189). Anonymous serum samples and clinical data were provided by the Ajou Human Bio-Resource Bank, and the need for informed consent was waived.
22K human protein microarray
Protein microarray experiments were performed using a HuProt human proteome microarray v3.0 (CDI Laboratories Inc., Mayaguez, PR, USA) with 20 human plasma samples [five patients with liver cirrhosis, five patients with HCC (at diagnosis, 6 months before diagnosis, and 1 year before diagnosis)]. Briefly, each protein chip was equilibrated using microarray buffer (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.8 mM KH2PO4 pH 7.4; 0.05% Triton X-100) for 5 min at 22–25°C and then incubated with 5% skimmed milk (BD Biosciences, San Jose, CA, USA) in microarray buffer for 1 h at 22–25°C. To identify autoantibodies in HCC samples, a blocked protein chip was washed three times with microarray buffer for 10 min, incubated with 20 µg/mL of serum in reaction buffer (50 mM Tris-Cl pH 7.5; 2 mM DTT; 2.5 mM MgCl2) for 8 h at 4°C, and washed with microarray buffer for 10 min. The washed chip was then incubated with Alexa Fluor goat-anti rabbit 647-conjugated secondary antibodies (1:5 000) diluted in microarray buffer containing 1% skimmed milk for 30 min at 22–25°C. The chip was then washed three times with microarray buffer, dried by centrifugation in a 50-mL conical tube (200 × g for 2 min), and scanned using a GenePix 4000B instrument (Molecular Devices, San Jose, CA, USA). All spotted proteins were probed using glutathione S-transferase antibodies with Alexa Fluor goat-anti rabbit 546-conjugated secondary antibodies. The signal intensity of each spot was recorded as the ratio of foreground to background signal and normalized to glutathione S-transferase. The mean signal intensity of all proteins on the chip was calculated.
Acquisition and analysis of the expression and methylation data in public omics databases
To analyze WASF2 mRNA expression in HCC, we obtained expression datasets from The Cancer Genome Atlas liver hepatocellular carcinoma project (TCGA_LIHC), the International Cancer Genome Consortium liver cancer RIKEN Japan (ICGC_LIRI), and gene expression omnibus databases from the National Center for Biotechnology Information [GSE6764, GSE12443, GSE77314, GSE89377 (Catholic University of Korea's liver hepatocellular carcinoma project, Catholic_LIHC), GSE93392, and GSE102418].
To analyze WASF2 methylation status, we detected genome-wide DNA methylation in TCGA_LIHC. Level 3 data sets obtained using an Infinium HumanMethylation450 BeadChip were analyzed using R software (http://www.R-project.org/). The correlation between WASF2 methylation and expression was analyzed and visualized using MEXPRESS (https://mexpress.be/).
Cell culture, treatments, and transfection
Human HCC cell lines (Huh-7, Hep3B, PLC/PRF/5, SNU368, SNU398, SNU423, SNU449, and SNU475) were acquired from the Korean Cell Line Bank (Seoul, South Korea). Immortalized normal hepatocytes (MIHA) were provided by Dr Roy-Chowdhury (Albert Einstein College of Medicine, Bronx, NY, USA). HCC and MIHA cells were cultured in RPMI 1640 or Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum (Invitrogen, Waltham, MA, USA) and 100 units/mL penicillin-streptomycin (GenDEPOT, Barker, TX, USA). THLE-2 immortalized normal hepatocytes were obtained from the AmericanType Culture Collection (ATCC, Bethesda, MD, USA) and cultured in bronchial epithelial cell growth medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (Invitrogen), 5 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 70 ng/mL phosphoethanolamine (Sigma-Aldrich), and antibiotics. Cells were grown in a humidified incubator with 5% CO2 at 37°C.
Cells were starved in serum-free medium for 4 h prior to treatment with human recombinant transforming growth factor-β1 (TGF- β1; 20 ng/mL; R&D Systems, Minneapolis, MN, USA) dissolved in sterile 4 mM HCl supplemented with 0.1% bovine serum albumin (BSA; R&D Systems) for 24 h. Cells were also transfected with 100 nM small interfering RNAs (siRNA) purchased from Bioneer (Daejeon, South Korea) and negative-control RNA duplexes synthesized by Genolution (Seoul, South Korea) for 24–48 h using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
RNA isolation and quantitative real-time polymerase chain reaction analysis
Total RNA was isolated from frozen tissues and cell lines using QIAzol reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized from 500 ng of total RNA in a 10 µL final volume using 5X PrimeScriptTM RT Master Mix (Takara Bio, Shiga, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using amfiSure qGreen Q-PCR Master Mix (GenDEPOT) and monitored in real time using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences used are listed in Additional file 1: Table S2. All assays were performed three times.
Western blot analysis
Proteins were extracted from tissues and cell lysates using radio immunoprecipitation buffer containing Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific). Equal amounts of total proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffer saline and 0.1% Tween-20 for 1 h at 22–25°C, incubated overnight with primary antibodies at 4°C, and then incubated with HRP-conjugated secondary antibodies at 22–25°C for 1 h. Chemiluminescence signals were detected using Clarity™ Western ECL Substrate (Bio-Rad Laboratories) and visualized using ChemiDoc™ (Bio-Rad Laboratories). Antibodies are listed in Additional file 1: Table S3 and S4.
Cell viability and growth assays
To analyze cell viability, two HCC cell lines transfected for 48 h in 60 mm dishes were harvested by trypsinization, stained using 0.4% trypan blue solution (Invitrogen), and counted using a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany).
To analyze cell growth, cells were seeded in 12-well plates, transfected with WASF2-targeting siRNA (siWASF2) or negative control siRNA (siNC), and incubated for 90 min at 37°C with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Biosesang, Seongnam, South Korea) solution. Dark blue formazan products were dissolved in dimethyl sulfoxide (Biosesang) and absorbance was detected using a TECAN SUNRISE Microplate Reader (TECAN, Zürich, Switzerland) at a wavelength of 570 nm.
Clonogenic proliferation assay
Cells were transfected with siWASF2 or siNC in 60 mm dishes for 48 h, reseeded in 6-well plates (2, 4, or 6 × 103 cells per well), and incubated at 37°C in a CO2 incubator for 10 days. Cell colonies were washed with phosphate-buffered saline (PBS), fixed with 1% paraformaldehyde for 30 min, stained with 0.5% crystal violet overnight at 22–25°C, and counted using ImageJ software (Laboratory for Optical and Computational Instrumentation, Madison, WI, USA).
Wound healing assay
Cells were transfected with siWASF2 or siNC in 60 mm dishes for 48 h, reseeded in 6-well plates (1.5 × 106 cells per well), and incubated at 37°C in a CO2 incubator for 24 h. The cell monolayer was scratched manually using a sterile micropipette tip. Initial (0 h after scratching) and residual (48 h after scratching) gap widths were imaged using an Olympus CKX53 (Olympus, Tokyo, Japan) photomicrograph. Each area was measured three times and expressed as a percentage.
Apoptosis and cell cycle assays
Apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (Koma Biotech, Seoul, South Korea). Briefly, cells transfected for 48 h were harvested by trypsinization, rinsed in PBS, and resuspended in 1× binding buffer before being incubated with 1.25 µL of Annexin V-FITC solution at 22–25°C for 20 min in the dark, stained with 10 µL propidium iodide (PI; Sigma-Aldrich), and analyzed using a FACSAria III flow cytometer (BD Biosciences).
Also, apoptotic cells were evaluated by Hoechst 33342 and PI staining. The transfected cells were fixed in 4% paraformaldehyde (Biosesang) for 10 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 20 min. Then treated with Hoechst 33342 (10 µg/mL; Invitrogen) for 10 min, stained with PI (20 µg/mL; Sigma-Aldrich) for 20 min, and observed on Olympus IX71 (Olympus).
To analyze the cell cycle, transfected cells were collected, fixed using 70% ethanol for 3 h at 4°C, washed in PBS, and stained using 200 µL PBS containing 10 µg/mL RNase A, 1% Triton X-100, and 30 µg/mL PI (Sigma-Aldrich) for 30 min in the dark. The proportion of cells at each stage of the cell cycle was determined using a FACSAria III flow cytometer (BD Biosciences). Experiments were performed in triplicate.
Migration and invasion assays
For the in vitro cell migration and invasion assays, we used 24-well plates and cell culture inserts (BD Biosciences). Prior to the invasion assay, the upper compartment of the cell culture insert was coated with 100 µL of Matrigel (BD Biosciences) diluted to 0.3 mg/mL in serum-free media. After Huh-7 and SNU475 cells had been transfected with siWASF2 or siNC for 24 h, they were seeded onto the cell culture insert with serum-free media and 5–20% fetal bovine serum as a chemoattractant for 24–72 h at 37°C in a 5% CO2 humidified incubator. Migratory or invasive cells on the lower side of the membrane were stained using a Diff-Quik staining kit (Sysmex Corporation, Chuo-ku, Japan) and imaged using a CKX53 inverted microscope (Olympus) at 200× magnification. Cells were counted in three random fields of view.
Immunofluorescence
Cells were seeded in 24-well plates and fixed in 4% paraformaldehyde (Biosesang) for 10 min at 22–25°C and permeabilized in PBS with 0.2% Triton X-100 (PBST; Sigma-Aldrich) for 10 min. After the cells were washed in PBST, the plates were blocked with 2.5% BSA in PBST for 30 min and incubated with the following primary antibodies: F-actin (1:200; Abcam, Waltham, MA, USA). The plates were washed and then incubated with AlexaFluor-488-conjugated secondary antibodies for 2 h and stained with 4′, 6-diamidino-2-phenylindole. The stained plates were analyzed by Olympus IX71 (Olympus).
Animal experiments
Five-week-old BALB/c female nude mice were purchased from ORIENT BIO (Seongnam, South Korea) and six-week-old athymic female nude mice were purchased from Koatech (Pyeongtaek, South Korea), placed in individually ventilated cages under a pathogen-free environment, and allowed to acclimate for two weeks before being used in experiments.
For in vivo subcutaneous xenograft assay, Huh-7 cells transfected with siWASF2 or siNC (5 × 105 cells) were diluted in 0.1 mL Matrigel/serum-free Dulbecco’s Modified Eagle’s Medium and injected subcutaneously into the flanks of BALB/c nude mice. Tumor diameter was measured three times a week for 18 days in the tumor-bearing mice using a digital caliper. Tumor volume was calculated as follows: \(Tumor volume=0.52 \times length \times {width}^{2}\).
At the end of the experimental period, tumors were removed for weighing and histological analysis. Tumor tissue samples were immediately flash-frozen in liquid nitrogen for RNA and protein extraction and placed in 10% neutral buffered formalin for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC).
For in vivo lung metastasis assay, ras-transformed NIH-3T3 cells transfected with siWASF2 or siNC (3 × 105 cells) were mixed with 0.2 mL serum-free Dulbecco’s Modified Eagle’s Medium and injected into the athymic nude mice through tail veins. The mice were sacrificed 14 days after cell inoculation, and the number of the peritoneal nodules were assessed.
All animals were maintained according to the Guide for the Care and Use of Laboratory Animals and experiments were approved by the Ethics Committee for Laboratory Animal Research Center of Ajou University Medical Center (IACUC-2020-0007).
IHC
Tissue samples from patients with HCC were fixed in formalin, embedded in paraffin, and cut into 5-µm sections that were deparaffinized in xylene, hydrated in graded alcohol, and incubated with primary antibodies (listed in Additional file 1: Table S3) overnight at 4°C. After being washed three times, the sections were incubated with secondary antibodies for 1 h and then a peroxidase substrate until the desired stain intensity had developed.
In the BALB/c nude mouse model, tumors were harvested, fixed in 10% neutral buffered formalin, and embedded in paraffin blocks before being cut into 5-µm sections and deparaffinized. One section was stained with H&E and another was evaluated by IHC. Both sections were incubated with the antibodies listed in Additional file 1: Table S3.
Bisulfite sequencing polymerase chain reaction (BSP)
Genomic DNA was isolated from human liver tissues and cell lines using a DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. The DNA quality and concentration of each sample was checked using a NanoPhotometer® N60 (Implen, Munich, Germany) and 1 µg of DNA was modified using an EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. Briefly, DNA was heat-denatured and bisulfite-converted using CT-conversion reagent in a thermocycler. The DNA was bound to a Zymo-spin IC column and desulfonated using M-desulfonation buffer. After being eluted from the column using 10 µL of M-elution buffer, 3 µL of bisulfite-converted DNA was used for BSP to amplify the specific WASF2 promoter region (cg24162579) using TaKaRa EpiTaq HS (Takara Bio) with a T100 Thermal Cycler (Bio-Rad Laboratories). The primers used to detect WASF2 CpG island methylation were designed using MethPrimer 2.0 (https://www.urogene.org/cgi-bin/methprimer/methprimer.cgi; Additional file 1: Table S2). PCR was conducted under the following cycling conditions: 40 cycles of 98°C for 10 s, 63°C for 60 s, and 72°C for 30 s. The PCR products were cloned into a pTOP vector and transformed into DH5α-competent E. coli cells using a TOPcloner TA kit (Enzynomics, Daejeon, Korea). Five clones per HCC cell line were expanded overnight and plasmid DNA was extracted using a QIAGEN Plasmid Mini kit (Qiagen). Purified plasmid DNA was sequenced using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) with an M13 primer to analyze the methylation status of specific CpG sites.
Methylation-specific polymerase chain reaction (MSP) and quantitative MSP (qMSP)
MSP and qMSP were performed using primers for methylated or unmethylated DNA designed using MethPrimer. Briefly, 2 µL of bisulfite-treated genomic DNA was amplified using TaKaRa EpiTaq HS (Takara Bio) under the following cycling conditions: 40 cycles of 98°C for 10 s, 60°C for 40 s, and 72°C for 30 s. The PCR products were analyzed using 2% agarose gel electrophoresis. qMSP was performed using an amfiSure qGreen Q-PCR Master Mix (GenDEPOT) and was monitored in real-time using an ABI 7300 Real-Time PCR System (Applied Biosystems) with the following cycling conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 34 s, and 72°C for 30 s, followed by a single cycle of 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s to generate dissociation curves. Relative DNA methylation was calculated using the difference between the Ct values of the methylated and unmethylated PCR products. All measurements were performed in triplicate. The primer sequences used for MSP and qMSP are listed in Additional file 1: Table S2.
Hydroxyurea (HU), 5-aza-2'-deoxycytidine (5-aza) treatment
Cells were treated with 0.5 mM HU (Sigma-Aldrich) or 5 µM 5-aza (Sigma-Aldrich) for 48–72 h at 37°C in a CO2 incubator, with the culture medium replaced daily. The treated cells were harvested and used to detect WASF2 methylation and expression.
Statistical analyses
Receiver operating characteristic (ROC) curves were produced using SPSS version 22 (SPSS, Chicago, IL, USA). OS and disease-free survival (DFS) curves were plotted using GraphpadTM 8.0 software (GraphPad Software, San Diego, CA, USA) using the Kaplan-Meier product limit method. Significant differences between survival curves were determined using the log-rank test. All experiments were performed at least three times. All samples were analyzed in triplicate. Results are presented as the mean ± standard error of the mean (SEM). Significant differences between experimental groups were assessed using paired or unpaired Welch’s t-tests and one-way analysis of variance (ANOVA) in GraphpadTM 8.0 software. P values of < 0.05 were considered statistically significant.