A total of 99 female subjects were included in the study, 36 in the OC group, 31 in the benign group, and 32 in the control group. Cases in the OC group were all from 36 ovarian cancer patients (32 high-grade serous adenocarcinomas, 3 endometrioid adenocarcinomas, and 1 clear cell carcinoma) admitted to Zhejiang Cancer Hospital from September 2020 to July 2021, and the inclusion criteria were: (1) All met the relevant diagnostic criteria for ovarian cancer, and the histological type and the International Union of Obstetrics and Gynecology (FIGO) staging were confirmed by postoperative pathological examination ; (2) None of them received any anti-tumor treatment before drawing blood; (3) patients with complete medical records; (4) patients without other malignant diseases. The benign group came from 31 patients with benign ovarian lesions (including 7 serous cystadenoma, 7 endometriotic cyst, 5 mature teratoma, 3 cases of mucinous cystadenoma, 9 cases of simple ovarian cyst) who were surgically diagnosed in the Affiliated Hospital of Hangzhou Normal University during the same period. The control group consisted of 32 healthy women who participated in the physical examination in this hospital during the same period, and excluded hypertension, diabetes and other diseases, and no history of malignant tumors in all systems of the body, no dysfunction of important organs.
The OC group was 27–90 years old, with an average age of 57 ± 13 years; the benign group was 24–73 years old, with an average of 49 ± 13 years old; the control group was 27–77 years old, with an average age of 51 ± 14 years. There was no significant difference in age between the three groups (P > 0.05). The samples in this study obtained the informed consent of all subjects and were approved by the hospital ethics committee (Ethics No. 2019 (Len 02)-HS-03).
All the subjects were drawn 4mL-6mL of EDTA anticoagulated venous blood on an empty stomach in the early morning, centrifuged at 3 500 r/min for 10 min at 4℃ to obtain plasma, divided into 2 tubes, one tube was stored in the refrigerator at -80℃; the other tube immediately detects the plasma protein CA125 and HE4 concentrations. If the samples cannot be detected in time within 8 hours, they should be stored in a refrigerator at 4°C, and the detection should be completed within 24 hours.
Detection of plasma protein CA125 and HE4
Plasma protein CA125 concentrations were detected using the Chemiluminescence Immunoassay Analyzer (Abbott ARCHITECT i2000SR, USA), and the plasma protein HE4 concentrations were detected using the Electrochemiluminescence Immunoassay Analyzer (Roche Cobas E602,Germany). Use the corresponding kit and strictly follow the manufacturer's protocol for testing.
Plasma exosomes were isolated using the Total Exosome Isolation Reagent (#4484450; Invitrogen, USA) according to the manufacturer’s protocol. General situation: First, the sample was thawed at room temperature, centrifuged at 2000g for 20 min, and centrifuged again at 10000×g for 20 min to completely remove the cells and debris. Next, 400 µL of plasma supernatant was mixed with 120 µL reagents, incubated at 4°C for 30 min, and centrifuged at 10,000 g for 5 min to obtain exosomal pellets. The exosomal samples were stored at -80°C for further analysis.
Transmission electron microscopy(TEM)
Resuspend the exosomal pellets in 1×PBS, and deposit the liquid on the carbon-coated copper mesh for 2 min. The excess liquid was removed, and filter paper was used to drain the grid; a drop was negatively stained with phosphotungstic acid and loaded onto the grid for 5 min. The grid was then dried at room temperature. Finally, the samples were observed by JEOL-1230 TEM at an acceleration voltage of 80 kV .
Nanoparticle tracking analysis (NTA)
The size and concentration of exosomes were used by NTA at VivaCell Biosceinces with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software ZetaView 8.04.02. Specifically: The ZetaView system was calibrated with 110 nm polystyrene particles, then properly dilutes the separated exosome sample with 1× PBS buffer to measure particle size and concentration, and records and analyzes NTA measurements at 11 locations, with temperatures remaining at around 23°C and 30°C during testing.
Western blotting analyses (WB)
The exosomal sample were treated with lysis buffer to obtain total exosomal protein, and the protein was quantified by the BCA method. Load exosomal proteins onto the SDS-PAGE and transfer it to the PVDF membrane. The PVDF membrane was blocked with BSA at room temperature for one hour, and incubated with primary antibodies including CD63 and TSG101 overnight, followed by incubation with the corresponding HRP-conjugated secondary antibodies. The proteins transferred on the PVDF membrane were finally visualized with a gel imaging system(BIO-RAD ChemiDoc XRS, USA).
Extraction of exosomal RNA and reverse transcription synthesis cDNA
Total RNA was extracted from plasma exosomes resuspended using the Multi-type Sample DNA/RNA Extraction-Purification Kit (Sansure Biotech Inc. Hunan, China) following the manufacturer’s instructions. The concentration of RNA was measured using the e-spect Spectrophotometer (Beijing labaid science and technology. Ltd, China), and the OD 260/280 nm ratios of all RNA samples were ≥ 1.8. Divide the total RNA sample into two parts. Part of it was reverse-transcribed into cDNA for the following qRT-PCR of miR-205, specifically using the Mir-XTM miRNA first-strand synthesis kit (#638313; TAKARA Bio Inc, USA); the remaining RNA samples were stored at -80°C and used for qRT-PCR of CA125, HE4 and TCF21.
qRT-PCR analysis for quantification of miR-205
The reagent was TB Green Advantage qPCR Premix (#639676; TAKARA Bio Inc, USA), and the reaction was performed on the 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific. Inc, USA). Specifically: two microliters cDNA products were used for qRT-PCR template, and the final volume was 25µl. U6 was used as the internal reference gene, and the reaction conditions were: 95°C pre-denaturation for 10s, 95°C denaturation for 5s, and 60°C annealing for 20s, a total of 40 cycles to get dissociation curve. Analyze the relative expression level of exosomal miR-205 using the 2−ΔΔCt relative quantitation method.
qRT-PCR analysis for quantification of CA125, HE4 and TCF21
The expression levels of CA125, HE4 and TCF21 were detected by One Step TB Green®PrimeScript™Plus RT-PCR Kit (#RR096A, TAKARA Bio Inc, USA) on the 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific. Inc, USA). Using β-actin as the internal control gene, the reaction conditions were: reverse transcription of RNA into cDNA at 42°C for 5 min, 95°C for 10 sec, denaturation at 95°C for 5 sec, and annealing at 60°C for 34 sec, a total of 40 cycles to abtain the products. The relative expression of genes was calculated by the 2−△△CT method.
The primers used in this study were purchased from Shanghai Biotech Co., Ltd. China, and the primer sequences were shown in Table 1.
qRT-PCR primer sequences for each gene
MRQ 3' Primer
All data were analyzed using the SPSS 26.0 statistical software (IBM Corp, USA) and plotted by GraphPad Prism 7.0 (GraphPadSoftware Inc, USA). All data were expressed as mean ± standard deviation or median (interquartile range) according to data distribution. The Mann-Whitney U test was used to compare the two groups, and the Kruskal-Wallis H test or one-way ANOVA was used for comparison between multiple groups. The area under the curve (AUC) of the receiver operating characteristic curve (ROC) was used to analyze the diagnostic value of plasma exosomal miR-205, CA125, HE4 and TCF21 for OC. P < 0.05 was considered statistically significant.