Cell culture
The human esophageal cancer cell line EC9706 (National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) was cultured in RPMI-1640 (Gibco, USA) medium. HUVECs (Shanghai Institute of Biochemistry and Cell Biology, CAS) were cultured in F12K medium (Gibco, USA). Both of the basal culture media above were supplemented with 10% fetal bovine serum (FBS, Gibco, USA)and 1% penicillin and streptomycin. FBS was centrifuged 10,000× g for 30 min, followed by ultracentrifugation at 200,000×g for 6 h to eliminate bovine-derived exosomes using a Type 70 Ti rotor in L-80XP ultracentrifuge (Beckman Coulter, Brea, Ca, USA). Cells were incubated in a humidified incubator at 37˚C with 5% CO2.
Isolation of exosomes from medium
Exosomes were isolated from cell culture medium after a 48-h of culture by differential centrifugation according to previous studies(32). Briefly, the medium was first centrifuged at 300 × g for 10 min, 800 × g for 10 min, 1,200 × g for 20 min, and 10,000 × g for 30 min to remove any live or dead cells or cellular debris. Afterwards, the supernatant was ultracentrifuged at 100,000 × g for 3 h to pellet exosomes. Finally, the supernatant was disposed, and the exosome pellet was washed with phosphate-buffered saline (PBS) at 100,000 × g for 2 h. All steps were performed at 4˚C. Exosomes were harvested from the pellet and resuspended in PBS. The exosome levels were determined by measuring the total protein content, which was presented as micrograms of total protein in the exosomes. The exosome fraction was measured for its protein content using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Wilmington, DE, USA).
Exosome labeling and transmission electron microscopy assay
The assay of exosome labeling and live-cell fluorescence microscopy as previously reported, firstly, the exosome pellet suspension was diluted with RPMI-1640 medium by adding DiI. Then, 2×105 EC9706 and 1.5 ml complete medium were placed in a 35-mm glass-bottom culture dish for Live Cell Imaging System analysis. After a 24 h culture, the DiI-labeled exosomes were incubated with cells for 2.5h and washed once to eliminate the free exosomes. Cells were then transferred to the cell culture chamber of the microscope.
Cell transfection
HUVEC cells were seeded into 6-well plates and transfected using Lipofectamine RNAiMAX (Invitrogen) and Opti-MEM (Gibco, USA), followed by the manufacturers’ instructions. For miRNA upregulation and downregulation, a 50-nmol dose of miR-21 mimics, inhibitors, and NC was used. Cells were harvested 36h after transfection to isolate total RNA or total cell lysate. Transfer efficiency was determined as fluorescent cell percentage by flow cytometry. The miR-21 expression level was detected by real-time quantitative polymerase chain reaction(RT-qPCR). EC9706 cells were cultured in 100-mm dishes and transfected with miR-21 mimics for the ready of co-cultivation model.
Construction for co-cultivation model
Co-cultivation of donor EC9706 cells and recipient HUVECs were performed in 12-well transwell inserts (cat. no. 3401; Corning Inc., Corning, NY, USA). HUVECs were pre-seeded in the lower chambers at a 1×105 cells/well density. Then, the EC9706 cells, transfected with miR-21 mimics or the negative controls, were scraped off and seeded onto 0.4 μM transwell inserts in the following day.
RNA extraction (isolation) and RT-qPCR
Total RNA was extracted from the cultured cells and exosomes using Trizol reagent (Invitrogen) and mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's protocols, respectively. RNA concentration was analyzed using NanoDrop spectrophotometer (NanoDrop ND-1000; NanoDrop Technologies, Inc., Wilmington, DE, USA). After the reactions were complete, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT values were determined from triplicate PCRs. We used the formula to calculate the relative quantities of target genes. RNU6 was used as the internal control for cells and supernatant, respectively, in miR-21 expression level analysis. β-actin was used as the invariant control for mRNA analysis. The sequences of PTEN primers were as follows: forward, 5'-AATGGCTAAGTGAAGATGACAAT-3' and reverse, 5'-TGCACATATCATTACACCAGTTCGT-3'. The sequences of matrix metalloproteinases (MMP)-2 primers were as follows: forward, 5'-CTGATGGCACCCATTTACA CCT-3' and reverse, 5'-GATCTGAGCGATGCCATCAAA-3'. The sequences of MMP-9 primers were as follows: forward, 5'-TGGGCTACGTGACCTATGACAT-3' and reverse, 5'-GCC CAGCCCACCTCCACTCCTC-3'. The sequences of β-actin primers were as follows: forward, 5'-ATCCGCAAAGACCTGT-3' and reverse, 5'-GGGTGTAACGCAACTAAG-3'. The primers used for the amplification of miR-21, RNU6 and cel-miR-39 were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The PCR reaction was performed at 95˚C for 5 min, which was followed by 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec, and 72˚C for 30 sec. Dissociation curve was analyzed from 60 to 99˚C. Relative transcript quantities for each miRNA were calculated using the ΔΔCt method.
The miRNA target prediction and luciferase reporter assay
The miRNA target prediction and analysis were performed with the algorithms from DIANA (http://diana.imis.athena-innovation.gr/DianaTools/index.php), TargetMiner (https://www.isical.ac.in/~bioinfo_miu/targetminer20.htm), and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/), RNA22 (https://cm.jefferson.edu/rna22/Interactive/). The reporter plasmid pmiR-PTEN was designed by RiboBio (Guangzhou, China). EC9706 cells in the miR-21 mimics or negative control group were transfected in 96-well plates (1×104 cells/well) with pmiR-Report-WT-PTEN by Lipofectamine RNAiMAX. The renilla luciferase was used as an internal control. At 48 h after transfection, cells were analyzed using a Dual Luciferase Assay Kit (Promega).
Vascular ring formation by HUVECs in vitro angiogenesis assay
An in vitro angiogenesis assay was performed as previously described(33, 34). Briefly, 30 μl Matrigel (BD Biosciences) was added to each well of a 96-well plate and allowed to polymerize at 37˚C for 30 min. Next, the pretreated HUVECs were resuspended in FBS-free RPMI-1640 medium and transferred to each well at a concentration of 4×104 cells per well. After 12 h of culture in a humidified incubator at 37˚C with 5% CO2, the cells were examined under an Olympus FSX100 light microscope (Tokyo, Japan) to assess the formation of capillary-like structures. The total tubule length, number of closed meshes and branch points of the formed tubes, which represent the degree of angiogenesis in vitro, were scanned and quantified in at least ten low-power fields (40×magnification).
Cell proliferation assay
The proliferative ability of HUVECs after different transfections or different exosome cocultures was determined by Cell-Light EdU Apollo in vitro image kit (RiboBio, Guangzhou, China). After pretreatment as described above, HUVECs were incubated in 50 µM EdU for 2 h, and they were then fixed, permeabilized, and stained following the appropriate instructions.
Cell migration assay
The migratory capacity of HUVECs was performed using a transwell insert that contains a polycarbonate filter with 8 μM pore size (cat. no. 3422; Corning). The pretreated HUVECs cells (5×103/well) suspended in 150 μl serum-free RPMI-1640 were added to the 24-well upper chamber, and 600 μl RPMI-1640 that contains 10% FBS was added to the bottom wells of the multiwell insert assembly. Cells were incubated at 37˚C for 24 h to allow cell migration through the membrane. Migrated cells were fixed in 95% ethanol and stained with crystal violet. The number of invaded cells was counted under Olympus FSX100 light microscope (Tokyo, Japan). To minimize the bias, cells in ten randomly selected fields at a 200×magnification were counted to calculate the average cell number.
Western blot analysis
The expression of PTEN was assessed by western blot analysis, and its expression in the samples was normalized to β-actin expression. Cells were lysed in RIPA buffer with freshly added protease inhibitor. Total lysates were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The immunoblots were blocked with 5% fat-free milk and they were incubated at 4˚C overnight with primary antibodies anti-PTEN (1:1000, Cell Signaling Technology), anti-β-actin (1:1000, BM0627; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Akt (Ser473) (1:1000, Cell Signaling Technology) phosphorylation were examined by using phospho-specific antibodies. Total protein was determined using anti-Akt antibodies. After incubation with the secondary antibody, the membranes were visualized with an enhanced chemiluminescence system kit (Millipore, USA), according to the manufacturer’s protocol. To explore whether miR-21 promote angiogenesis in tumor microenvironment by PTEN/Akt signaling pathway, the recipient cells were treated PI3K/Akt inhibitor LY294002 which was obtained from Beyotime Biotechnology (Nantong, China) with a final concentration of 60μM.
Assessment of nitric oxide (NO)
Total nitric oxide concentration in the supernatant of HUVECs medium was detected by measuring the concentration of nitrate and nitrite according to Nitric Oxide Assay Kit by Nanjing Jiancheng Bioengineering Institute (Nangjing, China). The optical densities at 550 nm wavelength were recorded using an ELISA reader (BioTek Epoch, USA) and the concentrations of NO were calculated according to the standard curve.
Statistics
All values are expressed as mean + SEM unless otherwise noted. The results are presented as the average of at least three experiments, each performed in triplicate, with standard errors. Data were described with median values ± SEM and analyzed by using Student’s t test for 2-group comparisons. Differences were considered statistically significant at P < 0.05. In this study, * P < 0.05, **P < 0.01, and *** P < 0.001.