Cell culture

ESCC cell lines KYSE30 (BW-S1092), KYSE150 (HT-X2475), EC109 (BW-5999), and EC9706 (BW-6316) were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and kept in RPMI-1640 medium (#11875-093, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; #16000-044, Grand Island, NY, USA) at 37 °C in a 5% CO2 cell culture incubator. Human lymphatic endothelial cells (HLECs, CP-H026, Procell) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in endothelial cell medium (ECM) with 5% FBS and endothelial cell growth medium supplements (CC-3121, Lonza, Basel, Switzerland). Immortalized esophageal epithelial NE-1 cell line (CP-H031, Procell) was obtained from State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center and cultured in Dulbecco’s modified Eagle medium (DMEM; #11885-076, Gibco, Grand Island, NY, USA) with 10% FBS in a 5% CO2 atmosphere at 37 °C.

Cell transfection

Specific short hairpin RNAs (shRNAs) targeting circ_0026611 (sh-circ_0026611#1/2) and PROX1 (sh-PROX1#1/2) together with their negative control shRNA (sh-NC) were constructed to silence circ_0026611 or PROX1 expression. For the overexpression of circ_0026611 and PROX1, the whole sequences were synthesized and subcloned into pcDNA3.1 vector, with pcDNA3.1 empty vector as the negative control (vector). In line with the supplier’s protocols, transfections were conducted with Lipofectamine 2000 (Invitrogen). Related sequence information is included in Additional file 2: Table S1.

Quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR)

Total RNA of cells was extracted by using TRIzol reagent (#15596018, Invitrogen, Carlsbad, CA, USA). Synthesis of complementary DNA (cDNA) for circRNAs and messenger RNAs (mRNAs) was synthesized by SuperScript III First-Strand Synthesis SuperMix (11752050, Invitrogen, Carlsbad, CA, USA). RT-qPCR reaction was achieved with SYBR Green Kit (#1725085, Bio-Rad Laboratories, Hercules, CA, USA) and real-time PCR system (ABI 7500, Thermo Fisher, Rockford, IL, USA) followed by 2−ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were considered as internal controls in this process.

Exosome isolation

Exosomes were isolated from supernatants of NE-1, KYSE30, and EC109 cells using ultracentrifugation methods. In brief, the cells were cultured in a complementary medium until 80% confluence, and then the medium was replaced with the defined medium without FBS. After 2 days of culture, the supernatants were harvested and centrifuged at 300g for 15 min, 2000g for 15 min, and 10,000g for 30 min. The supernatants were filtrated through a 0.22 μm polyvinylidene fluoride (PVDF) filter (Millipore, USA). Then the supernatants were collected to isolate exosomes by ultracentrifugation at 120,000g for 70 min (Beckman Coulter) twice. Particle Metrix (PMX), transmission electron microscopy (TEM), and western blotting were used to identify the exosomes.

Tube formation assay

Serum-free ECM starved human umbilical endothelial cells (HUVECs) were planted into 24-well plates coated with Matrigel (BD Biosciences) at a density of 2 × 104 cells per well. Conditional medium was collected by incubating exosome-treated or transfected HLECs cells with serum-free Dulbecco’s modified Eagle medium (DMEM) for 24 h and added into each well at 200 μl per well. The images of tube structure were captured under a light microscope, and tube formation was quantified with ImageView 3.7 (Jingtong, China).

Western blot

Total protein extracted from ESCC cell lines (KYSE30-Exos and EC109-Exos) was isolated by radio-immunoprecipitation assay (RIPA) buffer, and after being separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), proteins were transferred to PVDF membranes and cultured in 5% skim milk. The membranes were cultivated with primary antibodies overnight at 4 °C, followed by cultivation with secondary antibody for 1 h. After washing in Tris-buffered saline + Tween 20 (TBST), the secondary antibodies were added and finally assayed by enhanced chemiluminescence (ECL) substrate. The primary antibodies were as follows: anti-CD9 (ab92726, Abcam, Cambridge, MA, USA), anti-CD63 (ab134045), anti-β-tubulin (sc-166729, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VEGF-C (vascular endothelial growth factor C, #2445, Cell Signaling Technology, Boston, MA, USA), anti-VEGF-D (vascular endothelial growth factor D, sc-373866, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VEGFR3 (vascular endothelial growth factor receptor 3, sc-28297, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1, ab219556, Abcam, Cambridge, MA, USA), anti-podoplanin (#9047, Cell Signaling Technology, Boston, MA, USA), anti-PROX1 (prospero homeobox 1, sc-81983, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH (ab9485, Abcam, Cambridge, MA, USA).

Transwell assays

ESCC cells (KYSE30-Exos and EC109-Exos) were planted on the top of 24-well transwell chambers (#353097, BD Falcon, Bedford, MA, USA) coated with Matrigel (FAL354483, Corning, Shanghai, China) for invasion assay or without Matrigel for migration assay. The lower chambers were loaded with complete medium. Twenty-four hours later, cells in the upper layer were removed with caution by a cotton swab and then fixed in methanol solution for 15 min. Crystal violet was adopted to stain the membranes for 10 min, and the invaded or migrated cells were observed and counted under a microscope (10 × 10).

Dual-luciferase reporter assay

VEGFR3 promoter was subcloned into the pGL3 luciferase vector (E1751, Promega, Madison, WI, USA) and then cotransfected with circ_0026611 and Vector into HLECs using Lipofectamine 2000. All luciferase intensities were examined by Dual Luciferase Report Assay System (E1910, Promega, Madison, WI, USA) 36 h after transfection.

RNA pulldown assay

Biotinylated circ_0026611 was taken and treated with structure buffer to form a secondary structure. Streptavidin beads were added to biotin-labeled and denatured circ_0026611 and spun at 4 °C for 2 h. Cells were lysed with lysis buffer, and then the lysates were incubated with specific biotin-labeled probes for 2 h. After that, the mixtures were incubated with streptavidin-coated magnetic beads to pull down the biotin-labeled RNA complex. After washing, the RNA complex was extracted with TRIzol and then detected by western blot analysis. The experiments went through three independent repeats.

RNA-binding protein immunoprecipitation (RIP) assay

With the EZMagna RIP kit (17-701, Sigma-Aldrich, St. Louis, MO, USA), RIP assay in HLECs was carried out with the specific antibodies and normal control anti-IgG antibody (CS200621, Millipore, Billerica, MA, USA). Lysates were obtained from HLECs using RIP lysis buffer. The lysis was incubated with the magnetic beads conjugated with the NAA10 antibody (#9046, Cell Signaling Technology, Boston, MA, USA) or IgG antibody (negative control). The precipitated RNAs were analyzed by RT-qPCR.

Statistical analysis

All data from experiments including three biological replications are presented as mean ± standard deviation (SD). Data analysis was achieved by Student’s t-test and one-way/two-way analysis of variance (ANOVA), applying SPSS 19.0 software. **P < 0.01 indicates that related data contained statistical significance.