Cell lines culture
The human esophageal cancer cell lines (Eca109, Eca9706, TE-1, TE‐10, TE‐11, KYSE140, and KYSE150) were obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (HyClone, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco-BRL), 100 µg/ml streptomycin and 100 units/ml penicillin G (Beyotime Biotechnology, Shanghai, China) in 5% CO2 and humidified atmosphere at 37℃. Cells at the third generation of the exponentially growing state were used for all experiments.
IHC analysis in ESCC and adjacent tissues
Samples were collected from 41 patients with ESCC, who underwent complete surgical resection at the Department of Cardiothoracic Surgery of the Affiliated Hospital of Southwest Medical University from January 2017 to March 2018. The clinicopathological features of the ESCC patients are summarized in Table 1. Tissue sections were incubated with rabbit anti-human KDM6B antibody (1:100; ab38113; Abcam, Cambridge, UK), IHC scoring was blindly assessed by two Pathology doctor. The immunohistochemistry procedure and scoring
of KDM6B expression were performed as previously described. Samples with IHC score < 3 were defined as low expression, while samples with IHC score ≥ 3 were defined as high expression.
Cells were lysed in Lysis buffer (Beyotime Biotechnology, Shanghai, China) and centrifuged at 4 ℃ for 15 min. Protein concentrations were measured through BCA Protein Quantitation Assay (Beyotime Biotechnology, Shanghai, China). Total cell lysates were equally loaded on 8–15% SDS-polyacrylamide gel for running and then transferred to polyvinylidene difluoride (PVDF) membranes (Amersham, Buckinghamshire, UK). After blocking for 1 h at room temperature with 5 % non-fat milk in Phosphate buffered saline with Tween 20 (PBST), the membranes were incubated for overnight with the primary antibodies anti-KDM6B (abs103127, Absin, 1:1000), anti-Tri-Methyl-Histone H3(Lys27) (C36B11) Rabbit mAb (#9733, Cell Signaling, 1:1000), anti- GAPDH (AG019, Beyotime Biotechnology, 1:5000), anti- Histone H3 Mouse Monoclonal Antibody (AF0009, Beyotime Biotechnology, 1:1000). After staining with corresponding horseradish peroxidase (HRP)-linked secondary antibodies, signal detection was performed using a chemiluminescence phototype-HRP Kit(Beyotime Biotechnology, Shanghai, China) and exposed to film in a dark room.
Total RNA from esophageal tissues was extracted with TRIzol (Invitrogen, Carlsbad, CA) and cDNA was synthesized from 1 µg of total RNA using the ReverTra Ace qPCR RT Master Mix ( TOYOBO, Japan) following the manufacturers' instruction. The mRNA levels of target genes and the internal standard glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by RT-qPCR in triplicate using the QuantiNova SYBR Green PCR kit (Qiagen, Inc.) and the StepOne™ real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.).The specific primers for the genes are listed in Table 2.
Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen). Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel. We used 1 µg of total and polysome-associated RNA fractions for RNA-seq from two independent biological sample replicates. The RNA-seq was performed in an Illumina HiSeq platform using RNA-seq kit according to the manufacturer’s recommendation (Illumina, Inc.).The sequences were processed and analyzed by GENEWIZ.
Establishment of KDM6B knockdown cell line
Gene-specific shRNA sequences were obtained from the Sigma website (http://www.sigmaaldrich.com/), and the hairpin sequences were cloned into the lentiviral vector pLKO-Tet-On with Puromycinas as a selection marker,shRNAs targeting KDM6B were screened.
Lentiviral particles were packaged in HEK 293 T cells by transient transfection. Briefly, 10 µg of shRNA encoding lentiviral plasmid, 5 µg of helper pMD2.G plasmid and 10 µg of helper psPAX2 plasmid were mixed with 30 µl PEI transfection reagent (Sigma-Aldrich). And co-transfected into HEK 293 T cells at 70–80% confluency in a 10 cm Petri dish according to the manufacturer’s protocol. At 72 h post-transfection, the supernatant containing virus particles was collected and filtered through a 0.45 µm PVDF Millex syringe filter.
Each cell line was subjected to a puromycin tolerance test to determine the optimal concentration for establishing stable cell lines. To generate an shRNA-expressing stable cell line, cells were infected with lentiviral particles with 4 µg/ml polybrene. After 48 h, the cells were selected by 1 µg/ml puromycin (GIBCO) for 5 days. And the knockdown efficiency was tested by WB assay and qRT-PCR.
Plasmid constructs, siRNAs and transfection
To overexpress KDM6B, ESCC cells were transfected with a plasmid containing the full-length cDNA of the human KDM6B gene, which was obtained from Addgene (plasmid ID #24167). The transfections were performed using LipoFiter™ Liposomal Transfection reagent according to the manufacturer’s instructions. The cells were plated in 6 cm dishes and incubated overnight. When the confluence reached 70–80 %, the supernatant of the cell was replaced with fresh RPMI-1640 medium supplemented with 10% FBS, and then transfected with 5 µg of either KDM6B-vector using 16 µl LipoFiter™. The media was replaced 6 h after transfection, and the cells were incubated at 37 ℃ for 48 h. The overexpress efficiencies were confirmed by western blotting and qRT-PCR assay. The indicated siRNAs were synthesized by Guangzhou Riboio Co., Ltd (China). Cell transfection was performed using riboFECT™CP(Ruibo Biotechnology Co., Ltd.) according to the manufacturer’s instructions. Cells were seeded in free-antibiotics media in six-well plates and cultured for 24 h before siRNA transfection. Non-targeting control, siRNA groups were set up when the density of cells reached 50%. Approximately 5 µL of 20 µM siRNA was diluted with 120 µL of 1× riboFECT™ CP buffer and added with 12 µL of riboFECT™ CP reagent. The mixture was incubated for 15 min at room temperature. The prepared riboFECT™ CP mixture was added to 1863 µL of cell culture medium. The culture plate was placed in a CO2 incubator at 37℃ for 48 h, and the cells were then collected. The efficacy of siRNA transfection was evaluated by western blot and real-time quantitative RT–PCR. The siRNA sequences used in this study are as follows:siRNAs against C/EBPβ were siRNA-1:5′-GUAUAUUUUGGG
AAUCUUUTT-3′,siRNA-2:GUCUAUGUGUACAGAUGAAUG and non-targeting control siRNA, 5′-UAGCGACUAAACACAUCAA-3′.
For rescue experiments, plasmids were transfected on day 1 followed by siRNA transfection on day 2 and observation at 48 h after siRNA transfection.
In vitro colony-formation assays
Cells transfected with the indicated vectors were plated at low density (500 cells per 6-cm plate), incubated in a 5% CO2 incubator at 37°C for 10 days. During this period, the culture medium needed replacement 3 d/time, and the culture was stopped when the clone was visible to the naked eye. With the culture medium discarded, the cells were washed two times with PBS, and Cell clones were immobilized by methanol for 15 min and stained with crystal violet solution (Beyotime Biotechnology, Shanghai, China)for 3 min before photographed and counted. Colonies containing more than 50 cells were counted using a microscope.
Cell viability was evaluated using the Cell counting kit-8 (CCK‐8) assay. The cells were detached and inoculated into 96‐well plates at a density of 1000 cells/well (200 µL in each well). Four wells with cells were set for each group, and blank and control wells were also set. The plate was incubated in a 5% CO2 incubator at 37°C for 96 h. After the cells adhered to the wall, With the culture medium discarded, fresh culture medium containing 10 µL CCK‐8 reagent (Beyotime Biotechnology, Shanghai, China) was added for incubation for 2 h. Subsequently, an enzymatic marker (Bio‐Rad, USA) was used to detect the OD value at the wavelength of 450 nm. The cell survival rate was calculated, and the cell growth curve was drawn. The experiment was repeated three times.
Wound scratch based cell migration assay
In the scratch based wound healing assay  cells were seeded in a 12-well plate and cultured until confluence. Then cells were serum-starved for 12 h before scratching. Wound scratching was performed using 200 µl pipette tips, and cells were gently washed twice with PBS. Pictures were taken immediately. Finally, cells were treated with RPMI-1640 medium with 2% FBS. The wound healing rate was measured by a comparison of the images using the closure distance of cells after 0 h and 24 h.
Cell migration assay
A migration assay was performed using Transwell inserts(Corning-Costar, Cambridge, MA) that contained a polycarbonate transwell membrane filter (6.5 mm diameter, 8 µm pore size). Cells were maintained at a concentration of 5×105 cells/ml in serum-free RPMI-1640. A total of 200 µl cell suspension was added into the upper chamber, whilst the lower chamber was treated with 500 µl of RPMI-1640 with 10% FBS at 37°C for 24 h. The medium was discarded, and non-migrating cells on the top surface of the upper chamber were removed gently using cotton swabs. Migrated cells were fixed with pre-chilled methanol for 30 min then stained with 0.5% crystal violet at room temperature for 20 min. Representative images were taken under an inverted microscope (magnification, ×10, and ×20) equipped with a camera(Olympus,Japan).
Cell invasion assay
For the invasion assay, the upper chamber was pre-coated with Matrigel(BD Pharmingen). Ice-cold Matrigel was mixed with ice-cold RPMI-1640 medium at a ratio of 1:8 and spread onto the upper chamber (100 µl/chamber), which was subsequently incubated at 37°C for 2 h. The following steps, including cell plating, incubation, fixing, and de-staining, were conducted as aforementioned for the migration assay.
A total of 24 male BALB/C nude mice(5 weeks of age, 18–20g) were raised in laminar shelves without specific pathogen conditions with constant temperature, constant humidity, and regular disinfection. Bedding, drinking water and feed were replaced regularly under aseptic conditions. When the KYSE150 sh-NC and sh-KDM6B-1 cells were at a logarithmic growth phase, they were subcutaneously injected into the armpit of the forelimb of nude mice (0.2 mL, 2×106 cells). The tumor length (a) and the short diameter (b) were measured using a calliper twice a week, and the tumor growth curve was drawn via V = ab2/2. Three weeks after the injections, the mice were euthanized in a CO2 chamber, followed by cervical dislocation and lack of withdraw reflex to assure animal death. The tumours were removed and photographed.
Tumor metastases were generated by intravenously injecting 2 × 105 KYSE150 sh-NC and sh-KDM6B-1 cells into the tail vein of mice. After 30 days, all mice were euthanized and examined for tumor metastases. Their lungs were removed and fixed in formalin and processed for HE analyses. The methods used in animal experiments were performed following the relevant guidelines.
GSEA (Gene-set enrichment analysis)
GSEA was performed using the Broad Institute web platform by pre-ranking the RNA-seq list based on log2-fold change.
ChIP (chromatin immunoprecipitation) assay
ChIP assays were performed using Simple ChIP Plus Enzymatic Chromatin IP Kits (CST, #9003) according to the manufacturer's instructions. Briefly, 5×106 KYSE150 KDM6B knock-in cells were cross-linked, lysed, and digested to generate DNA fragments to the length of approximately 150–900 bp. ChIP was performed using control IgG or antibodies against H3K27me3. 2% of the chromatin extract was set aside for input. After the immunoprecipitation, cross-link reversal was carried out and the precipitated DNA was purified. All ChIP signals were normalized to the input, and relative fold‐change was compared with IgG controls. The resultant DNA was analyzed with qPCR, the primers of C/EBPβ were: 5′- CATCAAGCAAAACGCTATGGG-3′ and 5′- CTCAGAACCTAGAGCCGGAAA-3′. The IP efficiency was calculated using the equation shown below.
Percent Input = 2%×2(C[T]Input Sample−C[T] IP Sample).
C[T] = CT = Threshold cycle of PCR. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.
ChIP-seq and data analysis
ChIP samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and qualified by Agilent Bioanalyzer 2100(Agilent Technologies, Palo Alto, CA, USA). Next-generation sequencing library preparations were constructed following the manufacturer’s protocol (NEBNext® Ultra™II DNA Library Prep Kit for Illumina®). The sequences were processed and analyzed by GENEWIZ. The ChIP-seq peaks were visualized using IGV(http://www.broadinstitute.org/igv/).
Microsoft Excel and GraphPad Prism software were used for statistical analysis. Comparisons between two groups were conducted by unpaired t-test. The differences among groups were analyzed using a one-way analysis of variance followed by Least Significant Difference posthoc test. P < 0.05 was considered to indicate statistical significance. Data are presented as mean ± SD based on at least three replicates.