Plasma exosomal miRNA-205 can be used for early diagnosis and prognostic evaluation of ovarian cancer

Ovarian cancer (OC) is a serious threat to women's health around the world and new biomarkers are urgently needed for early detection. Our previous published research have conrmed that miR-205 can promote the invasion and metastasis of OC cells by inhibiting the expression of the tumor suppressor gene TCF21. This study is based on the previous work, using exosomal liquid biopsy technology to detect the expression levels of the four genes miR-205, CA125, HE4 and TCF21 in the plasma exosomes of OC patients; at the same time, combined with the clinicopathological parameters of OC patients data analysis aims to provide ecient and non-invasive laboratory testing basis for the early diagnosis of OC.

Ovarian cancer (OC) is one of the most common gynaecological malignancies, with the leading fatality rate in the female reproductive system, posing a serious threat to women's health worldwide [1]. Due to the lack of early clinical manifestations and effective screening methods, 70%-75% of OC patients are at an advanced stage at the time of diagnosis and miss the best time for treatment, so OC is called the "silent killer" [2]. Therefore, there is an urgent need for more e cient, non-invasive, and early screening methods to improve the prognosis of OC patients. Liquid biopsy technology represented by exosomes is likely to solve this problem.
Exosomes are tiny vesicles secreted by most cells, with their unique lipid bilayer membrane structure, can well protect their contents from degradation, such as mRNA and miRNA [3]. Therefore, exosomes are much more stable than the targets contained in plasma. In the early stages of tumors, cancer cells secrete a large amount of exosomes, reaching 10 12 /mL of blood, which makes the quantitative detection of exosomes easier and makes them possible as a new tumor marker [4]. Our previous published studies have found that miR-205 is up-regulated in OC tissues, and in the mechanism study, we have con rmed that miR-205 can promote the invasion and metastasis of OC cells by inhibiting the expression of tumor suppressor gene TCF21 [5], suggesting that miR-205 has the potential to become a new type of OC diagnostic biomarker. This study is based on the previous work, using exosomal liquid biopsy technology to detect the expression levels of the four genes miR-205, CA125, HE4 and TCF21 in the plasma exosomes of OC patients; at the same time, combined with the clinicopathological parameters of OC patients data analysis to explore the diagnostic value and clinical signi cance of the four exosomal genes for OC.

Study populations
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 con rmed by postoperative pathological examination [6]; (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 A liated 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 signi cant 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).

Plasma collection
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.

Exosome isolation
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 lter 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. Speci cally: 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 quanti ed 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 nally 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-Puri cation 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, speci cally using the Mir-XTM miRNA rst-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 quanti cation 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 Scienti c. Inc, USA). Speci cally: two microliters cDNA products were used for qRT-PCR template, and the nal volume was 25µl. U6 was used as the internal reference gene, and the reaction conditions were: 95°C predenaturation 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 [7].
qRT-PCR analysis for quanti cation 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 Scienti c. 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. Table 1 qRT-PCR primer sequences for each gene

Genes
Forward Sequence Reverse Sequence

Statistical analysis
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 signi cant.

Characteristics of plasma exosomes
The exosomes extracted in this experiment were identi ed by three methods: TEM, NTA and WB. As we proved, the extracted clusters were observed under TEM as cup-shaped or dish-shaped vesicles with a diameter of about 100 nm (Fig. 1a); the WB results showed that the lysates of isolated plasma particles of all groups expressed the speci c exosomal proteins CD63 and TSG101 (Fig. 1b); the NTA results showed that the particle size distribution range of exosomes was 20.7 ~ 345.2 nm, with an average particle size of 98.6 nm, and the total concentration of the exosomes was 1.3E + 12 particles/mL after multiplication by 20,000 times (Fig. 1c).
Comparison of expression levels of plasma exosomal miR-205, CA125, HE4 and TCF21 among the three groups of people The expression levels of four genes in the three groups of subjects were shown in Table 2 and Fig. 2. The results showed that the expression level of plasma exosomal miR-205 in the OC group was signi cantly higher than that of the benign group and the control group (P < 0.01) (Fig. 2a); the expression level of plasma exosomes CA125 in the OC group was the highest among the three groups, which was signi cantly different from the benign group (P < 0.01) (Fig. 2b); the expression levels of plasma exosomal HE4 in benign group was signi cantly lower than OC and control group( P < 0.01) ( Fig. 2c);the expression level of plasma exosomal TCF21 in the OC group and the benign group was signi cantly lower than that of the control group (P < 0.05) (Fig. 2d). In addition, there was no signi cant difference in the expression levels of four genes among the other groups (P > 0.05).  Table 3 for details.

Discussion
OC is the fth most common tumor that threatens the health of women around the world, and its mortality rate ranks rst among gynecological tumors [8]. According to the latest cancer report in 2021, the incidence of OC accounts for about 3.4% of all tumors, but the mortality rate is nearly 5%, which means that there are about 310,000 new cases and 200,000 deaths in 2020 [1]. Early detection and early diagnosis are the key to improving the prognosis of OC patients. However, studies have shown that the conventional screening method of serum CA125 combined with vaginal ultrasound cannot reduce the mortality of patients [9,10]. At present, there is no effective screening method for OC, and the diagnosis must rely on the pathological results of tissue biopsy, which brings great pain to patients [11]. The liquid biopsy technology represented by exosomes can obtain disease information through non-invasive or minimally invasive sampling methods, especially in recent years, which has received more and more attention in tumor screening, diagnosis and treatment, and prognosis evaluation [12]. This also provides new ideas for OC screening and early diagnosis.
In this study, we extracted the plasma exosomes of all subjects with commercial reagents, and then used three methods (TEM, WB, and NTA) to identify the extracted products. It was visually observed under TEM that the particles were in the form of cup-shaped vesicles with a diameter of about 100 nm; WB results showed that the particles expressed exosomal characteristic proteins CD63 and TSG101; NTA results showed that the average size of the particles was 98.6 nm and contained high concentration (1.3E + 12 particles/mL). These results were consistent with the characteristics of exosomes reported in the previous literature [13], so we can con rm the successful extraction of plasma exosomes. Exosomes are mainly involved in the communication between cells, and they are widely present in biological cells and various body uids [14]. Exosomes derived from cancer cells in the early stages of tumors can be released into the blood in large quantities and are highly stable in peripheral blood, making it possible to be used as new tumor markers. A number of studies have shown that exosomes play an important role in the occurrence, development and drug resistance of ovarian cancer, and they have a certain ability to guide the diagnosis, treatment and prognosis evaluation of OC [15][16][17][18].
After successfully extracting plasma exosomes, we further analyzed the expression levels of the four genes miR-205, CA125, HE4 and TCF21 in plasma exosomes and the concentrations of plasma proteins CA125 and HE4, and found that the expression levels of plasma exosomal miR-205 in OC group were signi cantly higher than in benign groups and control groups, which was consistent with our previous research results in OC tissues and cell lines [5]. At the same time, combined with the analysis of the clinicopathological parameters of OC patients, it was found that the expression level of plasma exosomal miR-205 in OC patients was higher in advanced-stage(stage III-IV) than in early-stage(stage I-II), and the group with lymph node metastasis was higher than the group without lymph node metastasis, and the differences were statistically signi cant. However, the traditional serum markers of plasma proteins CA125 and HE4 have nothing to do with the FIGO staging of OC and lymph node metastasis. Compared with traditional markers, plasma exosomal miR-205 has shown better early diagnosis and prognostic evaluation of ovarian cancer. The big advantage suggests that plasma exosomal miR-205 is expected to become a new tumor biomarker to assist in the screening and early diagnosis of OC. In view of the fact that we found that miR-205 was highly expressed in OC tissues in our previous basic research, and miR-205 mimic in cell lines can promote the invasion and metastasis of OC cells, we highly suspect that the high expression of miR-205 in OC tissues may transmit relevant information through exosomes, thereby accelerating the overall invasion and metastasis process of OC, which also indicates that plasma exosomal miR-205 may become an early diagnostic biomarker for OC and can be used for prognostic evaluation of OC.
MiR-205 is a highly conservative miRNA, which has a dual effect, both as a tumor promote factor and as a tumor suppressor [19]. Studies have found that the expression of miR-205 was reduced in the serum of breast cancer patients [20]; while the expression of miR-205 was up-regulated in OC tissues and cells, and was related to the growth and metastasis of OC [21], which was consistent with our previous research results. In addition, studies have suggested that vascular endothelial growth factor (VEGF) through the increase of miR-205 expression leads to the down-regulation of Ezrin and Lamin A/C, thereby promoting the invasion of OC cells [22]. CHU et al. found that miR-205 regulated the proliferation and invasion of ovarian cancer cells by inhibiting the expression of PTEN/Smad4 [23]. Other studies have shown that miR-205 can directly negatively regulate ZEB1 to promote the clinical progress of EOC patients [24]. A recent study showed that miR-205 derived from OC cell exosomes can promote the transfer of OC by inducing angiogenesis [25]. The above studies have con rmed that miR-205 had abnormal expression in OC and was related to the invasion of OC, which supported the results of our experiment.
In order to further evaluate the diagnostic value of plasma exosomal miR-205 for OC, combined with the current traditional biomarkers plasma proteins CA125 and HE4, through ROC curve analysis, it was found that the AUC of plasma proteins CA125 and HE4 were 0.915 and 0.779, respectively, the sensitivity was 74.3% and 55.6%, and the speci city was 93.7% and 100%, respectively. This experiment con rmed that traditional biomarkers have good diagnostic values for OC, but the sensitivity of both was not high, resulting in some patients missing diagnosis, so neither was an ideal means of screening OC. The study also found that the plasma exosomal CA125 and HE4 expression levels in the OC group were higher than those in the benign group, but there was no statistical difference from the control group, which was inconsistent with the ndings of Fawzy et al. that the expression levels of CA125 and HE4 in OC tissues were higher than those of benign tumor tissues and normal ovarian tissues [26]. In addition, the expression level of plasma exosomal TCF21 in the OC group and the benign group was lower than the control group, but there was no difference between the OC group and the benign group. This was inconsistent with our previous basic research nding that the expression level of TCF21 in OC tissues was signi cantly lower than that of benign tissues [5]. The reason for the analysis may be due to the inherent differences in gene expression patterns in plasma exosomes and tissues. JI J et al. found that the differential expression pro le of serum exosomal mRNA was not completely consistent with the tissue mRNA level, which also illustrates this point well [27]; It may also be due to the selective packaging of the mRNA molecules contained in exosomes, and it has been found that exosomes can regulate the packaging process of their internal RNA through a variety of ways[28]; In addition, the effects of differences in mRNA stability, sample source, sample size, and storage time should also be taken into account, which are for further study.
It should also be noted that although this study initially demonstrated the potential advantages of plasma exosomal miR-205 as a new biomarker for OC, this experiment also had certain limitations. Because the sample size of this study was small and may lead to deviations, in the future need to further increase the sample size, preferably multi-center, prospective research. However, in view of the literature reports that the expression of miRNA had tissue and disease speci city [29,30]; it can be secreted in the form of exosomes and stably exists in the peripheral blood circulation [31; and it can also be delivered to the tumor microenvironment or distant organs through exosomes, and promote tumor angiogenesis and metastasis by targeting gene expression in recipient cells;in addition, the existing research results indicated that plasma exosomal miR-205 may become a new diagnostic marker for OC after con rmation of large samples, which can provide a certain reference value for early screening of OC.

Conclusions
In summary, our research results showed that plasma exosomal miR-205 had a certain diagnostic ability for OC, and its combination with traditional serological tumor markers can improve the diagnostic e ciency of OC; at the same time, plasma exosomal miR-205 was related to OC staging and lymph node metastasis, which can provide a certain reference value for the early diagnosis and prognostic evaluation of OC patients.  The expression levels of plasma exosomal miR-205, CA125, HE4 and TCF21 among three groups of people(Kruskal-Wallis H test) . *p<0.05; **p<0.01; ***p<0.001