Tissue samples
Tumor samples were collected during surgery, and the excised tumors were quick-frozen in liquid nitrogen and stored at -80°C until use. All patients were diagnosed by two senior pathologists and none had received chemotherapy or radiotherapy prior to surgery. A total of 16 cervical cancer samples were selected for this study (Table 3). The samples were obtained from the First Affiliated Hospital of Xi’an Jiaotong University between January 2015 and December 2018.
Bioinformatics analysis
GEPIA database (http://gepia.cancer-pku.cn/) was used to detect the CCL22 and CCR4 mRNA expression levels in CC and normal cervix tissues, the correlation between CCL22 and CCR4 expression were also studied by the GEPIA database.
Cell culture
SiHa, HeLa and C33A cell lines were purchased from the Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai. The tumor cells were cultured in Dulbecco’s minimum essential medium (DMEM) (HyClone, USA) and supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel), in a humidified 37℃ incubator containing 5% CO2. The medium was replenished every 2 days and the cells were passaged every 3 days.
Plasmid transfection
Transient and stable transfections with plasmids were according to the Lipofectamine 2000 transfection reagent (Invitrogen, USA) manufacturer’s instructions. The short-hairpin (sh)RNA against EZH2 gene (PGPU6/GFP/Neo-EZH2-homo-488) and corresponding control shRNA (GenePharma, Shanghai) were used for RNA interference. The gene silencing effects were confirmed by western blotting at 48 h after transfection. Stably transduced cells were maintained in culture in the presence of geneticin (G418).
Transfection of siRNA into cells
The siRNA oligonucleotides (GenePharma, Shanghai), targeted to EZH2 and DNMT3A, were used to knock-down EZH2 and DNMT3A. In a 6-well plate, SiHa and HeLa cells were seeded to 50%–60%. Then, cells in were transfected with siRNA specific for EZH2 and DNMT3A with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. In parallel, scrambled siRNA was used as a control for off-target changes in SiHa and HeLa cells. Twenty-four hours after transfection, the medium was changed and cells were incubated for an additional 24 h before being harvested for analysis. The primers used in the following siRNA oligos are listed in Table 1.
CCL22 and DZNep treatment
Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer. Treatment in this study was done using a concentration of 100 ng/ml, 200 ng/ml, the data were collected at 24 h time point. The EZH2 inhibitor DZNep was provided by (Selleck, USA). Stock solution was dissolved in sterilized ddH2O and tested at 5 μM and 10 μM for 48 h.
Neutralization of CCL22 in CC cells
SiHa and HeLa cells were suspended at a density of 1 × 106 cells/ml in a dilution medium containing 1.5 μg and 3 μg of Neutralization of CCL22 antibody (MAB336-100, R&D, USA) per ml and pre-incubated at 37 °C for 24 h.
Quantitative real-time PCR (RT-qPCR) assays
Total RNA was collected using TRIzol reagent (Sigma, USA) as previously [(Zhang et al. 2019)], in briefly,cDNAs were synthesized using the PrimeScript RT Reagent Kit (Taraka, China) according to the manufacturer's instructions. RT-qPCR was conducted in triplicate with LightCycler2.0 (Bio-Rad, USA), and the expression was normalized to GAPDH as the total RNA and cytoplasmic RNA endogenous control. PCR primers primer sequences are listed in Table 1. The relative quantification value for each target gene was expressed as 2-△△Cq.
5‑Aza‑2'‑deoxycytidine (5-Aza-CdR) treatment, DNA extraction, bisulfite modification
5-Aza-CdR (Sigma, USA) was reconstituted in complete DMEM to final concentrations of 5 μM, 10 μM indicated in results. Fresh treatment of 5-Aza-CdR was given to cells daily over 72 h prior to assaying. Genomic DNA of CC cells and tissues were extracted by TaKaRa Mini BEST Universal Genomic DNA Extraction kit (TaKaRa, China) according to the manufacturer’s instructions. 500 ng of genomic DNA was bisulfite-modified using EZ DNA Methylation-Gold™ kit (Zymo Research, USA).
Methylation-specific PCR (MS-PCR) and Quantitative Methylation-specific PCR (qMS-PCR) modified DNA templates were used for MS-PCR with Zymo TaqTM PreMix (E2003, Zymo Research, USA) following the instructions. The annealing temperature for the methylated CCL22-CCR4 primer was 55°C and for the unmethylated primer was 55°C. The MS-PCR products were separated on 2% agarose gel, stained with Gelview and visualized under ultraviolet illumination (Bio-Rad, USA). Methylation level was calculated by the ratio of methylated and unmethylated levels. Grey value of each band represented its relative expression and was measured by Image J Software. The qMS-PCR was operated with TB Green® Premix Ex Taq™ II (TaKaRa, China) by a two-step amplification procedure according to the manufacturers’ protocol. For each sample, a relative methylation level was calculated using the difference in Cq values by the standard 2-△△Cq method in which ALU was used as an internal reference gene. The primers used in the MS-PCR and qMS-PCR are listed in Table 1.
Western blotting
Cells were scraped into RIPA lysis buffer. Proteins were running to separate on SDS polyacrylamide gels, electrophoretic ally transferred to PVDF membranes, and incubated with primary antibodies. The primary antibodies are listed in Table 2. Proteins were visualized with second antibody. The secondary antibodies are as follows: HRP-conjugated rabbit anti-mouse IgG (1:5,000 dilution, D110273-0100, BBI Life Sciences, China), HRP-conjugated goat anti-rabbit IgG (1: 5,000 dilution, 31460, PIONEER, China).
Co-immunoprecipitation (Co-IP) assay
The cells were lysed using NP40 buffer and incubated with 50 μl of protein-A/G PLUSA garose beads (Invitrogen, USA), 1 mg of antibodies (details are shown in table 2) at 4℃ overnight. After washing three times with washing buffer, the samples were analyzed by western blotting. The primary antibodies are listed in Table 2.
Chromatin immunoprecipitation (ChIP) assay
The ChIP experiments were executed using the Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, USA) as previously [(Zhang et al. 2020)], in briefly, 1×107 cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. The crosslinking was then quenched with 10×glycine. Chromatin was sonicated in lysis buffer to 200-1,000 bp, and the extraction of ChIP DNA was performed as per the kit's protocol. The antibodies utilized included EZH2, H3K27me3 and DNMT3A (show in Table 2). The primer sequences are given in Table 1.
Tumor Xenograft experiments
24 cohorts of 4-week-old female were divided into 4 groups. EZH2 knocked-down SiHa and HeLa cells (1×107) were injected into the flank of the nude mice. The following treatments were administered in cohorts of three mice when tumors were 15 mm in diameter for each treatment: i) SiHa-shEZH2/SiHa-shNC, ii) HeLa-shEZH2/HeLa-shNC. Tumor growth and mouse weight was monitored until death. Tumor volume was calculated as previously described [(Zhang et al. 2020)], using the following formula: tumor volume (mm3) =0.5×length×width2. Mice were monitored for 15 days, at which time mice were euthanized and tumors and organs were extracted.
Transwell chamber migration assay
5 × 104 cells were resuspended in 200 μl of serum-free medium per sample. Cell suspension was transferred to the upper chamber of the transwell (BD, Franklin Lakes, NJ) for migration through 8.0 μm pore PET membrane in a 24-well plate setting. Migrating cells were fixed with formalin followed by staining with crystal violet dye (0.1%). The stained membrane was then washed and photographed using a light microscope. Five images per chamber were taken from random fields of view and migration was quantified as average area occupied by migrated cells using Image J software.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, USA). A paired t‑test and one‑way ANOVA were carried out on samples within groups, Dunnett's test was used as the post hoc test after one‑way ANOVA. A p values <0.05 was considered to indicate a statistically significant difference. The data are presented as the means ± standard error of the mean (SEM). All experiments were independently repeated at least thrice, with consistent results.