The Cancer Genome Atlas (TCGA) is a large-scale cancer genomics program, and it has molecularly characterized 33 primary cancer types comprising esophageal squamous cell carcinoma. GEO is a public genomics platform composited of array- and sequence-based data. Using UALCAN and Oncomine, the expression of ETV5 in ESCC was investigated from TCGA and GEO database.
We purchased human ESCC cell lines, ECA109, KYSE150 and TE1 from the Institute of Biochemistry and Cell Biology of the CAS (Shanghai, China). All cell lines were cultured in DMEM medium (GIBCO) supplemented with 10% FBS (GIBCO) in an incubator containing 5% CO2 at 37℃.
For transient transfection, cells were seed in 6-well plates, 100pmol siRNA-ETV5 or siRNA-SKA1 or siRNA-TRPV2 (GenePharma, Shanghai, China) was transduced into each well. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were applied to detect transfection efficiency at 48h after transfection. For stable transfection, lentivirus vectors that encode a shRNA targeting ETV5 or shRNA non-targeting control were used to transfect ECA109 cells following the manufacturer’s instruction (Genechem, Shanghai, China). In brief, cells were seed in 6-well plates, medium containing viral fluid but without serum was added when the cell density reached 30% and replaced with complete medium 24h later. The transfection efficiency of each vector was detected via western blotting.
For evaluating the proliferation capacity of ESCC cells, the Cell Counting Kit 8 (CCK8) assay was used. We planted 1x103 cells/well in the 96-well plates, and 10µl CCK8 reagent was used to mix cells in every well respectively after cells being incubated for 0, 24, 48, 72 and 96h. After 4h of incubation, we measured the absorbance of cells at 450nm by using a microplate reader.
Migration and invasion assay
To explore the migration ability, we resuspended ESCC cells in 200µl DMEM without serum and added them in upper chamber of the transwell device, with 5x104 cells/well. We then added 600µl complete medium into the lower chamber as the chemical attractants. After incubation for 48h at 37℃, cells on the lower surface of the non-coated membrane were fixed by 4% paraformaldehyde and then stained by Giemsa. Images from five representative fields of each membrane were taken by using a light microscope (100×). The number of migratory cells was counted and the relative migration rate can be calculated. Invasion assay was similar to migration assay, but with the difference that 100µl of 200µg/ml diluted Matrigel matrix (Corning, 356234) was carefully added to the center of each transwell insert and incubated at 37℃ for 1 hour to form a gel before cells were plated.
Wound healing assay
The ECA109 and KYSE150 cells underwent a culturing process in six-well plates and when the cells reached 80% confluence, the monolayers were scraped by the tip of a 200µl pipette, and cells continued to be cultured in DMEM free of serum. At 0, 24 and 48h after scratch, cell migration was photographed by a light microscope (100×). Image J was used to calculate the closure. The formulas are: average scratch width = scratch gap area/length; the relative cell migration rate = (the scratch width at 0h-scratch width after culture)/the scratch width at 0h × 100%.
Real-time PCR assay and RNA sequencing
Total RNAs were extracted from cells by using Trizol solution, according to the instruction of the manufacturer. We used Nanodrop2000 spectrophotometer to measure the RNA concentrations and synthesized complementary DNA (cDNA) from RNA by using a PrimeScript RT reagent kit (Takara, Japan). TaqMan real-time PCR assays for ETV5, SKA1 and TRPV2 were applied following the instruction of Takara Bio. The relevant primers were summarized in supplementary table 1. All reactions, including the no-template controls were run in triplicate. After the reactions, the CT values were determined using fixed threshold settings. Library preparation for RNA sequencing was conducted. Generally, 1 µg high-quality RNA was used, and sequencing was carried out by HiSeq2500 (Illumina Inc., San Diego, CA) at Genechem Biotechnology (Shanghai) Co., Ltd.
Western blotting analysis
After being washed by PBS, all kinds of cell lines were harvested in RIPA buffer. The protein concentrations were measured using Bradford assay. Samples were separated on 8–12% SDS-PAGE gels and then transferred to PVDF membrane (Millipore, USA) and probed with primary antibodies specific for ETV5 (ab102010, Abcam), MMP2 (ab92536, Abcam), MMP9 (ab76003, Abcam), SKA1 (bs-7846R, Bioss) and TRPV2 (bs-10297R, Bioss). β-Actin and GAPDH were used as loading controls for western blots.
Chromatin immunoprecipitation (CHIP) assay
A CHIP assay was performed using an Upstate Biotechnology kit. Briefly, we successively subjected cells to the procedures containing DNA-protein cross-linking, disruption of membrane and cytosol. Samples were digested by MNase and sonicated and then precipitated with antibody against transcriptional factor ETV5. Quantitative real-time PCR was used to measure the amount of bound DNA. According to the relative amount of input and the IgG ratio, the enrichment value was calculated. The primers covering ETV5 binding site of SKA1 and TRPV2 gene promoter region were summarized in supplementary table 1.
Dual luciferase reporter assay
ESCC cells were seeded into 12-well plates at a density of 2.5×104 cells/well. The reporter plasmid containing wild SKA1 or TRPV2 promoter and mutant SKA1 or TRPV2 promoter at the binding site, was transfected into cells, respectively. The cells were also transfected with Renilla luciferase reporter plasmids for signal normalization. After 24h, the luciferase activity was measured by the Dual Luciferase Assay System Kit (Promega, Madison, WI, USA).
This study was approved by the institutional review board of East Hospital, Tongji University. ESCC and corresponding healthy esophageal mucosa (CHEM) tissues of 98 patients were collected. After being fixed by Formalin and embedded by paraffin, tissue sections were cut to 4µm thickness and then placed in xylene and graded alcohols for deparaffinization and hydration. We performed heat-induced antigen retrieval in EDTA (PH 8.0) buffer for 15 minutes by using a microwave oven. To reduce nonspecific staining, we performed blocking with 10% goat serum. After the specific primary anti-ETV5 (ab102010, Abcam) was dropped onto the sections and incubated overnight at 4℃, the slides were counterstained with light hematoxylin, dehydrated, and cover-slipped.
This in vivo study was approved by the animal care and use committee of Tongji University. 20 female BALB/c nude mice (6 weeks old) were used for animal studies. The animals were randomly divided into 2 groups (control and treated groups, 10 mice per group). ECA109 cells were treated with stable transfection. After cell harvest, cells were resuspended and injected into the tail vein of each mouse (2×106 viable ECA109 cells/mouse). At 6 weeks, the lung metastasis was monitored. According to the AVMA Guidelines for the Euthanasia of Animals, we performed intraperitoneal injection of a three-fold dose of barbiturates to euthanize all the mice. After that, lungs were removed, and the lung colonization number was counted. Serial sections of lung tissue were stained with hematoxylin and eosin.
The statistical analysis was performed by SPSS 19.0 (IBM, Armonk, NY, USA). All the experiments were carried out repeatedly three times. Differences between groups were calculated using Student’s t-test, Chi-square test, or Fisher’s exact test. The p value < 0.05 was considered with statistical significance.