Cell culture and transfection
The subclones of A2780 (CVCL_0134) cells obtained from the Basic Research Institute of Peking Union Medical College Hospital with the highest or lowest invasive/migratory capacity (AHC and ALC, respectively) were isolated and established through a limiting dilution methodology, as we described previously8,20. AHC/ALC cells were cultured in RPMI-1640 medium (Corning) containing 10% fetal bovine serum and antibiotics of 1% penicillin‒streptomycin solution at 37℃ in a humidified atmosphere incubator with 5% CO2. The candidate DEM mimic and negative controls were transfected into AHC cells using the RiboFECT CP Transfection Kit (Ruibo Biology Co., Ltd). All experiments were performed with mycoplasma-free cells in this paper.
Cell line authentication process
All cell lines have been authenticated in the past three years. Take an appropriate amount of A2780 cells and use TIAAmp Genomic DNA Kit to extract DNA. 20 STR loci and gender identification loci were amplified using the MicroreaderTM21 ID system, and the PCR products were detected using the GenReader 7010 gene analyzer. The detection results were analyzed using GeneMapper Software6 software (Applied Biosystems) and compared with the ExPASy database. The STR typing results of the cell line DNA showed that no human cell cross contamination was found in the A2780 cell line.
CCK-8 proliferation assay
Cells were seeded into 96-well plates at a density of 2×103 cells and cultured with medium containing 10% FBS (Corning). Cell proliferation was by CCK8 solution reagent (KeyGEN BioTECH) and assessed according to the optical density (OD) detected by a 96-well plate reader (BIO-RAD Microplate Reader). All experiments were repeated three times with six replicate wells.
EdU assay
An EdU kit (RiboBio) was used according to the instructions. The results were observed and imaged with a fluorescence microscope (Leica DM2500). The experiment was performed three times.
Colony formation assay
Cells were plated into six-well plates (Corning )(500 cells per well) and cultured for 14 days until visible cell colonies appeared. The colonies were fixed with 4% PFA and stained with 0.1% crystal violet. The number of colonies was counted and compared.
Wound healing assay
ALC and AHC cells were cultured in 6-well plates until the cell density reached 90%. The cell monolayers were wounded with a 200 µl pipette tip to scratch a gap on the plates. The plates were gently washed with PBS three times to remove the floating cells, and the culture medium was replaced with serum-free medium. The wound closure area at the first scratch (0 h) and after 24 h were observed and photographed by microscopy with Leica software. The healing area was measured using ImageJ software.
Matrigel migration/invasion assay
For the migration assay, ALC and AHC cells were harvested and resuspended in serum-free medium. Then, the cells (3×106 cells/ml) were seeded into the upper chamber. RPMI-1640 medium with 20% FBS was added to the lower chamber. After incubation for 24 h, the invaded cells were fixed with 4% PFA, washed with PBS, and stained with 0.5% crystal violet. For the invasion assay, the chambers were coated with diluted Matrigel. Then, cells were suspended in RPMI-1640-only medium and loaded in the upper chambers. The following steps were consistent with those of the migration assay. The migratory or invaded cells were photographed and counted under a microscope.
Cell immunofluorescence assay
Cells were cultured on poly-L-lysine-coated coverslips (8×104 cells per well) in 24-well plates. After being cultured for 24 h, cells were fixed with 4% PFA at room temperature for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min. BSA (1%) in PBS was used to block nonspecific binding for 1 h. Then, the cells were treated with anti-E cadherin (CST), anti-N cadherin (CST), and anti-Vimentin antibodies (CST) at 4°C overnight. The cells were stained with goat anti-rabbit secondary antibody (Invitrogen)( at room temperature for 2 h in a dark box and then stained with DAPI for 10 min. The fluorescence of the cells was visualized by fluorescence microscopy.
Western Blot (WB) assay
The cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer with a 1% protease inhibitor. The BCA assay kit (Thermo) was used to determine the total protein concentration. Equal amounts of protein were separated using SDS‒PAGE gel and transferred onto a nitrocellulose (NC) membrane. Then, the membrane was blocked with 5% bovine serum albumin (BSA) dissolved in Tris-buffered saline with Tween-20 (TBST) for 1.5 h at room temperature and incubated with primary antibodies specific to protein at 4°C overnight. The following antibodies against proteins were used: E-cadherin (CST, #3195C), N-cadherin(CST, #13116), vimentin(CST, #5741) ,p-PI3K(CST, #17366S), PI3K(CST, #4255), p-AKT(CST, #4060), AKT(CST, #4685S) and GAPDH(Abcam, ab8245). After being washed in TBST buffer three times, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody(Beyotime Biotechnology ;1:5000) or anti-mouse antibody (Beyotime Biotechnology;1:5000) secondary antibodies for 1.5 h and then washed with TBST buffer three times again. Finally, the protein bands were scanned with an enhanced chemiluminescence kit on an Chemiluminescence Imaging System (Minichemi) with Image Lab software.
Exosomes isolation
Cells were cultured at approximately 60–70% density and washed twice with PBS to eliminate the interference of exosomes in serum. Then, the medium from each culture plate was replaced by 10% exosomes-depleted FBS, which was centrifuged at 110 000 × g and 4°C overnight (> 12 h)27,38. Extraction of exosomes by differential centrifugation. The Pierce TM BCA Protein Assay Kit was used to quantify the protein concentration.
Transmission electron microscopy (TEM)
The morphology and size of exosomes were observed by TEM using negative staining with copper mesh, as described previously 12,27. Exosomes precipitates were resuspended using 50–100 µL 2% PFA, loaded onto a formvar-carbon-coated grid, and incubated for 20 min. After being washed twice with PBS, the grid was refixed with 1% glutaraldehyde for 5 min. The grid was washed 8 times using double-distilled water. The grid was first contrasted in a solution of uranyl oxalate, pH 7.0, then determined and embedded in a mixture of 4% uranyl acetate and 2% methylcellulose in 100 µl/900 µl. The grid was dried in air for 5–10 min. The grid was observed under an electron microscope at 80 kV.
Nanoparticle tracking analysis (NTA)
NTA measured the concentration and size distribution of exosomes released from AHC and ALC cell. The samples were loaded into the NanoSight NS300 instrument (Malvern, UK) to analyze the size and concentration of exosomes.
Exosomes labeling and uptake assay
The PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma) was used to label AHE. The procedure was conducted according to the instructions. Phallotoxins(Beyotime Biotechnology) were used to label F-actin to show cell morphology.
Quantitative miRNA analysis and identification of candidate DEMs
Total RNA was extracted from ALE and AHE and quantitatively determined by BGI company using BGISEQ-500 sequencing. DEMs were defined by default as those with false discovery rates (FDR) ≤ 0.001 and a fold change of more than 2. Multiple databases, including TargetScan, miRanda, and RNAhybrid, were used to predict the target genes of the DEMs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to predict the biological function of the target genes. The candidate DEMs with a potential regulatory effect on the PI3K/AKT pathway were selected for further evaluation.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen). The RNA was reverse-transcribed to cDNA using the Mir-X miRNA First-Strand Synthesis Kit (Takara, 638315). U6, a small nuclear RNA (snRNA), was used as an internal control to normalize the cell miRNA results. Cel-miR-39-3p (miRB0000010, RiboBio), the standard RNA, was spiked as an exogenous control to normalize exosomes and serum sample miRNA results. miRNA expression was detected by qRT-PCR using the Mir-X miRNA qRT‒PCR TB Green® Kit (Takara, 638314) on a 7500 Real-time PCR System (Applied Biosystems). The 2−ΔΔ Ct method was used to analyze the differences in relative expression levels between groups.
Patient samples
Serum samples from patients with HGSOC and benign ovarian tumors were collected in sterile tubes. Patient clinicopathological characteristics, such as age, menopause state, International Federation of Obstetrics and Gynecology (FIGO) stage, tumor node metastasis (TNM) stage, histologic grade, tumor size, differentiation degree, lymph node metastasis, and pretreatment serum CA125 levels, were collected. Patient serum was centrifuged at 12 000 × g for 30 min at 4°C to remove cellular fractions. The separated serum was aliquoted and stored at -80°C until further processing. Repeated defrosting was avoided when possible. The expression of the candidate DEMs in serum exosomes was determined by qRT-PCR.
Exosomes labeling and uptake assay
The PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma) was used to label AHE. The procedure was conducted according to the instructions. Phallotoxins(Beyotime Biotechnology) were used to label F-actin to show cell morphology.
Quantitative miRNA analysis and identification of candidate DEMs
Total RNA was extracted from ALE and AHE and quantitatively determined by BGI company using BGISEQ-500 sequencing. DEMs were defined by default as those with false discovery rates (FDR) ≤ 0.001 and a fold change of more than 2. Multiple databases, including TargetScan, miRanda, and RNAhybrid, were used to predict the target genes of the DEMs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to predict the biological function of the target genes. The candidate DEMs with a potential regulatory effect on the PI3K/AKT pathway were selected for further evaluation.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen). The RNA was reverse-transcribed to cDNA using the Mir-X miRNA First-Strand Synthesis Kit (Takara, 638315). U6, a small nuclear RNA (snRNA), was used as an internal control to normalize the cell miRNA results. Cel-miR-39-3p (miRB0000010, RiboBio), the standard RNA, was spiked as an exogenous control to normalize exosomes and serum sample miRNA results. miRNA expression was detected by qRT-PCR using the Mir-X miRNA qRT‒PCR TB Green® Kit (Takara, 638314) on a 7500 Real-time PCR System (Applied Biosystems). The 2−ΔΔ Ct method was used to analyze the differences in relative expression levels between groups.
Patient samples
Serum samples from patients with HGSOC and benign ovarian tumors were collected in sterile tubes. Patient clinicopathological characteristics, such as age, menopause state, International Federation of Obstetrics and Gynecology (FIGO) stage, tumor node metastasis (TNM) stage, histologic grade, tumor size, differentiation degree, lymph node metastasis, and pretreatment serum CA125 levels, were collected. Patient serum was centrifuged at 12 000 × g for 30 min at 4°C to remove cellular fractions. The separated serum was aliquoted and stored at -80°C until further processing. Repeated defrosting was avoided when possible. The expression of the candidate DEMs in serum exosomes was determined by qRT-PCR.
Statistical analysis
All statistical analyses were performed using the statistical software SPSS version 22.0 and GraphPad Prism software (8.0). The statistical significance of the results was determined using Student's t test and Mann-Whitney U test. The correlations of serum levels of exosomal miRNAs and clinical pathological characteristics of patients were evaluated by the Spearman rho test. ROC curve is an effective tool for the comprehensive and accurate evaluation of diagnostic experiments39. The diagnostic power of the exosomal miRNAs was analyzed by ROC curves with corresponding statistics40. The cutoff values for the relative expression levels of miRNAs and the clinical markers alone and in combination were determined by the Youden index from the ROC curves. Differences were considered significant when p < 0.05.