Primary cells
The co-culture cell lines of normal human esophageal epithelial cells (HEEC) and esophageal fibroblast (HEsF), ESCC cells (EC109) and esophageal fibroblast (HEsF) were constructed. In the co-culture experiment, CAFs or NFs were mixed with cancer cells in a ratio of 3:1, and the cancer cells were inoculated on a 6-well plate, then CAFs or NFs were inoculated in the upper chamber of Boyden chamber. All procedures were supported by the Ethics Committee of the First Affiliated Hospital of Soochow University.
Quantitative real-time PCR
Total RNA was extracted from co-cultured HEsF cells using TRIzol reagent (Invitrogen, MA, USA). RNA and HiScript Q RT SuperMix for qPCR reverse transcription (Vazyme, Jiangsu, China). Quantitative RT-PCR was performed using ABI Prism7900 sequence detection system (Applied Biosystems, Canada) and SYBR Green PCR Master Mix (Vazyme). GAPDH was used as internal control, and the relative mRNA expression was calculated using the 2-ΔΔCt method. 2μg total RNA was incubated in a 1×reaction buffer at 37°C for 20 min. The RNA was purified using the RNeasy MinElute cleaning kit (Qiagen,Valencia,CA) and then reversely transcribed into cDNA.
MS-PCR
The DNA of the cells was extracted using BGenomic DNA Extraction Kit. The concentration and purity of the DNA were determined by ultraviolet spectrophotometry and stored in a refrigerator at −80°C for use. Then MS-PCR was performed to detect promoter methylation using the DNA Methylation-GoldTM kit.
siRNA interference construction
RNAi is a powerful new technology that can quickly identify components of cellular signaling pathways. According to the DNMT3A (DNMT1, DNMT2 and DNMT3B) gene sequence in GenBank, DNMT3A siRNA and negative control primers were designed and cloned into PGLV3//H1//GFP+Puro vector. The lentiviral vector was successfully constructed by cell transformation and positive cell cloning plasmid sequencing.
RNA pull-down
This assay confirmed binding of miR-29b-3p with DMNT3A. RNA was transcribed in vitro using the MEGAscript™T7 transcription kit and biotinylation using the Pierce RNA 3'-end desulfurization biotinylation kit. Proteins are extracted from the cells using the Pierce IP Lysis Buffer. Then, the PEARCE magnetic RNA protein pull-down kit was used for RNA pull-down determination. Biotinylated RNA was captured with streptavidin magnetic beads and incubated with cell lysates at 4°C for 6 h. Then, we washed and eluded the mixture. Finally, the presence of DMNT3A in the complexes was detected by qRT-PCR.
Western blot
Total protein concentrations isolated from cells were measured using a BCA assay kit (Thermo Fisher Scientific, USA). The proteins were then isolated with 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). 5% skim milk was used to close membrane 2 hours and incubate overnight. Primary antibodies included CD63, CD81 and HSP70 (Sigma, USA, 1:1000) and GAPDH (Santa Cruz Biotechnology, USA, 1:1000). GAPDH was served as an internal control.
Immunofluorescence
Cell lines were cultured on fibronectin coated cover slides for two days. After washing with PBS 2 times, the cells were immobilized in 4% paraformaldehyde for 15min and then permeated with 0.25% Triton X-100 at room temperature. Cells were sealed with 1% bovine serum for 20min and incubated overnight with primary antibody at 4℃. After washing with PBS for 3 times, they were incubated with goat anti-rabbit IgG secondary antibody at room temperature for 1h. Nucleic acids were stained with DAPI and images were taken with fluorescence microscope.
Exosome extraction and identification
Exosome precipitate was placed in 2.5% glutaraldehyde droplets and fixed overnight at 4℃. The samples were rinsed in PBS buffer (3 times, 10min each) and fixed in 1% osmium tetroxide at room temperature for 1h. The sample was then embedded in 10% gelatin, fixed in glutaraldehyde at 4℃, and cut into several pieces (less than 1mm3). The samples were dehydrated for 10 min in progressively increasing concentrations of alcohol (30%, 50%, 70%, 90%, 95% and 100%). Next, pure ethanol was exchanged with propylene oxide and the concentration of Quetol-812 epoxy resin mixed with propylene oxide was increased at increasing concentrations (25%, 50%, 75% and 100%), soaking at each step for at least 3h. The samples were embedded in pure fresh Quetol-812 epoxy resin and polymerized at 35°C for 12h, 45°C for 12h and 60°C for 24h. The ultra-thin slices (100nm) were cut with Leica UC6 ultra-thin slicer and stained with uranoyl acetate for 10min and lead citrate for 5min at room temperature, then observed with FEI Tecnai T20 transmission electron microscope at 120kV.
CCK‑8 assay and transwell assay
After transfection for 72h, cells were inoculated with 2000 cells/well density in 96-well plates and cultured for 0, 24, 48 and 72h, respectively. At the end of impregnation, 20µL CCK-8 was added to each well (Dojindo Molecular Technologies, Inc., Japan). Incubate for 1h under the condition of 5% CO2 at 37℃ in an incubator, and measure the absorbance at 450nm wavelength.
After transfection for 72h, cells were inoculated into the 6-well transwell upper chamber with a density of 25000 cells/well. The transwell assay was conducted based on the manufacturer's instructions. The transwell chamber was then placed in a humidified incubator with 5% CO2 at 37℃ for 48h. The lower chamber was stained with hematoxylin and photographed.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA) and SPSS 17.0 (IBM, Chicago, IL, USA). Student’s T-test or one-way analysis of variance (ANOVA) were used to analyze the differences and statistical significance between the two groups of data. P<0.05 was considered statistically significant.