Clinical samples
Thirty pairs of OC and the corresponding adjacent normal tissues of patients undergoing resection operation in XXX Hospital from May 2019 to May 2020 were collected and stored in the liquid nitrogen. This research was ratified by the Ethics Committee of XXX Hospital and obtained the informed consent of patients.
Cell lines
Human normal ovarian epithelial cell line IOSE80 and the OC cell lines OVCAR3, SKOV3, A2780 and HO-8910 were obtained from ATCC. After resuscitated, the cells were re-suspended with pre-heated RPMI-1640 or DMEM medium containing 1% Double-antibodies solution and 10% fetal bovine serum. Then the cells were cultured in the cell incubator (Boxun, China). All reagents used were Gibco brand (Invitrogen, USA).
Quantitative Real-time PCR (qRT-PCR)
The total RNA was separated from tissues, cells or exosomes using Trizol RNA Extraction Kit (Absin, China). The concentration and quality of extracted RNA were assessed using the microplate reader (Thermo Fisher, Multiscan MK3, USA). Then, total RNA was used as the template and the cDNA synthetic reaction system was constructed using the Reverse Transcription Kit (Absin, China). The reaction was conducted at 37℃ for 30 min and 85℃ for 2 min. Next, using cDNA as template, the qPCR reaction system was constructed using the SYBR High-sensitivity qPCR SuperMix (Absin, China). CFX 96 Touch (Bio-RAD, USA) was used to complete the qPCR reaction. The sequence of miR-494-3p primers: Forward: 5’-ACTTAGCCATTAGCCGACTACG-3’, Reverse: 5’-GAACAGTAACTTAGTTACGACAC-3’, the sequence of the internal reference U6 snRNA primers: Forward: 5’-GCAGTATTCAGACTAGACCTA-3’, Reverse: 5’-AAGCATCCATTACAAGTACGTC-3’.
Exosome extraction
Exosomes were extracted by differential centrifugation. Briefly, the supernatant of OC cells was collected and centrifuged at 4℃ with the high-speed refrigerated centrifuge (Thermo Fisher, Micro 17R, USA) at 400×g for 10 min, 2500×g for 10 min, and 10000×g for 30 min, successively. Then the supernatant was filtered with a 0.22 μm filter. Finally, the liquid was centrifuged using the ultracentrifuge (Beckman Coulter, Optima L-100 XP, USA) at 4℃ and 100000g for 70 min. The precipitate was the obtained exosomes.
miR-494-3p loading
MiR-494-3p was loaded into exosomes by electroporation. Specifically, 1 μmol of miR-494-3p mimic and 10 μg of the extracted exosome were mixed with the electroporation buffer (BTX, USA) and transferred to a 0.4 cm electrode cup for electroporation using MicroPulser (Bio-RAD, USA). After that, the mixture was centrifuged at 4℃ and 100000g for 70 min to remove the unloaded miR-494-3p, and the obtained miR-494-3p Exo was re-suspended by PBS and cryopreserved.
Characterization of the morphology and the particle diameter of exosomes
For the Transmission Electron Microscopy (TEM) analysis, 10 μL of exosomes was dropped on copper wire and dried, then 10 μL of 2% uranyl acetate was dropped for 5 min staining, the excess liquid was sucked up. The Unload Exo and miR-494-3p Exo were observed by TEM (FEI, TECNAI G2, USA).
For Nanoparticle Tracking Analysis (NTA) of the exosomes, 10 μL of exosomes were mixed with PBS and injected into the measuring cell of Zeta View PMX 110 (Particle Metrix, Germany) and then analyzed with the built-in analysis software.
Protein detection of cells and exosomes
RIPA lysis buffer (Lab-bio, China) added with the PMSF (Yuanye, China) were injected into the OC cells and exosomes. After ice bath for 30 min, the lysates were centrifuged at 12000×g and 4℃ for 8 min. The concentration and quality of the extracted protein was determined by spectrophotometry method. Then 15 μg of the protein was added to the loading well of 12% prefabricated gel and the electrophoresis was conducted. After that, the separated proteins in the gel were transferred to the nitrocellulose (NC) membrane (Millipore, USA). The NC membrane was soaked in the blocking buffer (1% BSA diluted by TBST) for 1 h. Then the NC membrane was immersed in different primary antibodies at 4℃ for overnight. After rinsing with TBST, the NC membrane was immersed in the secondary antibody solution for another 1 h. In the end, ECL solution (Invitrogen, USA) was dropped onto the membrane, and then development was performed with Bio-Rad gel imaging system (USA). Antibodies used in this research were all purchased from Abcam (UK), including anti-TSG101 (ab228013, 1:2000), anti-CD63 (ab321975, 1:1000), anti-GRP94 (ab3674, 1:3000), anti-Bax (ab263897, 1:2500), anti-Bcl2 (ab194583, 1:1500), anti-Cleaved Caspase-3 (ab231289, 1:800), anti-Cleaved Caspase-9 (ab2324, 1:800), anti-MMP2 (ab181286, 1:1000), anti-MMP9 (ab283575, 1:1500), goat anti-rabbit IgG (ab97051, 1:40000).
Exosome tracing
Unload Exo and miR-494-3p Exo were suspended in 1 μM PKH26 reagent (Sigma-Aldrich, USA) and stained for 5 min away from light. The excess dye was removed by centrifugation at 4℃, 100000g for 1 h, and the labeled exosomes were re-suspended in 200 μL PBS. Then PKH26-labeled exosomes were added to OVCAR3 and SKOV3 cells at the concentration of 20 μg/mL and co-incubated at 37℃ for 8 h under dark. Afterwards, the cells were rinsed with PBS and fixed with the fixative for 10 min, then the nuclei were stained with DAPI (Absin,China). After rinsing, the ingestion of exosomes by OVCAR3 and SKOV3 cells was observed via the laser confocal microscope (Nikon A1, Japan).
Detection of cell proliferation
The proliferation of OC cells was assessed using the Cell-Light EdU Apollo643 In Vitro Kit (Ribobio, China). OVCAR3 and SKOV3 cells were seeded in 96-well plates with 5×104 cells/ well and treated with PBS, Unload Exo or miR-494-3p Exo for 8 h. Next, the EdU reagent was diluted to 50 μM with serum-free medium. The diluted EdU medium was added with 100 μL/well and treated for 2 h. Then the fixation of OC cells was conducted and the cells were treated with the glycine. After washing, OC cells were permeabilized with 0.5% Triton X-100. Afterwards, 100 μL/well of Apollo staining solution was added and co-incubated for 30 min and keep out light. Then the OC cells were rinsed twice with methanol and once with PBS. In the end, the DAPI stain was applied for the nuclear staining and the cells were observed by confocal laser microscopy immediately.
Detection of cell apoptosis
The apoptosis of OC cells was detected using the commercial Annexin V-FITC/PI kit (Merck, Germany). OVCAR3 and SKOV3 cells of 2×105/well were inoculated into 6-well plates and divided into three groups. When the density reached above 80%, the cells were treated with PBS, Unload Exo or miR-494-3p Exo, respectively. Next, the cells were digested and the concentration was adjusted to 1.5×106 cells/ mL. 5 μL of Annexin V-FITC was co-incubated with the cells for 15 min away from light. After rinsing with the cool PBS, the cells were re-suspended with 200 μL binding buffer. Then 5 μL PI reagent was injected, and the apoptosis was detected via flow cytometry (BD, FACSCalibur, USA) immediately.
Scratch test
OVCAR3 and SKOV3 cells were seeded in six-well plates. When the cells were fully bespread, a scratch was made on the monolayer cells. The scratched cells were rinsed with PBS, and the pictures at 0 h were taken. Then cells in the three groups were treated with PBS, Unload Exo or miR-494-3p Exo, respectively, and the scratch healing was recorded 48 h later. IPP software was used to analyze the images to calculate the percentage of wound healing.
Transwell assay
This experiment was performed to assess the OC cells’ migration and invasion. For evaluating the cell invasion, Matrigel (Corning, USA) was dissolved and mixed with the serum-free medium at 4:1, 50 μL of the mixture was spread on the bottom of the upper room. Then the treated OVCAR3 and SKOV3 cells were inoculated in the upper room, and the complete medium was injected into the lower room. After 24 h, cells and the Matrigel in the upper room were removed and the cells in the lower room were fixed with 4% paraformaldehyde (TCI, China). Next, the crystal violet staining was conducted. After washing and drying, the OC cells were observed and recorded with the microscope. The procedure of migration detection was basically the same as above, except that there was no need to coat the chamber with Matrigel in advance.
Statistical analysis
All experiments were repeated independently for at least 3 times. The data were presented as x ± s. The software SPSS 23.0 was used for statistical analysis. Student’s t test was used to analyze the differences between two groups, and the one-way ANOVA was applied to analyze the multi-groups. P < 0.05 was statistically significant.