Application of exosome miR-211-3p in prevention and / or treatment of ovarian cancer peritoneal metastasis

Abstract

The invention discloses application of a preparation for detecting exosome miR-211-3p in preparation of a reagent for diagnosing ovarian cancer metastasis, and compared with an ovarian cancer non-metastatic group, the ovarian cancer cell exosome miR-211-3p is highly expressed in a metastatic group; the invention also discloses application of the exosome miR-211-3p as a target in developing or screening medicines for preventing and / or treating ovarian cancer peritoneal metastasis diseases. Researches show that miR-211-3p derived from ovarian cancer cell exosomes can promote a mesenchymal-mesenchymal transition (MMT) process of PMCs by targeting ATP1B2 so as to promote migration of the PMCs and attachment of the PMCs to the ovarian cancer cells, and miR-211-3p can also promote migration of the ovarian cancer cells. The invention provides a new target, namely the exosome miR-211-3p, for preventing / treating peritoneal metastasis of ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of exosome miR-211-3p in preventing and / or treating peritoneal metastasis of ovarian cancer. Background Art

[0002] Ovarian cancer is one of the most deadly female malignancies, with a five-year survival rate of less than 50%. Patients with early ovarian cancer can have a 90% cure rate, but there are no obvious characteristics in the early stages of ovarian cancer. Most cases are found in the middle and late stages, accompanied by extensive peritoneal metastasis and a large amount of ascites, and even drug resistance. In recent years, the survival rate of many cancers has been greatly improved due to advances in chemotherapy and surgical techniques and the development of new targeted drugs, but the survival rate of ovarian cancer patients has not been greatly improved due to these technological advances. Therefore, it is necessary to study the molecular mechanism of ovarian cancer and find new therapeutic targets.

[0003] Most ovarian cancer patients are diagnosed with peritoneal metastasis, and a large number of cancer cells can be found in the ascites. Peritoneal mesothelial cells (PMCs) are the first barrier encountered by cancer cells when metastasizing to the peritoneum, and can be transformed into cancer-associated fibroblasts (CAFs) through mesothelial to mesenchymal transition (MMT). An important effect of tumor embedding in the peritoneal membrane is the accumulation of a large number of cancer-associated fibroblasts (CAFs). Many studies have also shown that MMT of PMCs is observed in peritoneal metastasis of ovarian cancer and is conducive to the metastasis of ovarian cancer cells. However, the specific molecular mechanism leading to MMT has not been fully elucidated.

[0004] Exosomes refer to small membrane vesicles (30-150nm) containing complex RNA and proteins. Nowadays, they specifically refer to discoid vesicles with a diameter of 40-100nm. Exosomes were first identified as microvesicles with 5' nucleotide activity in 1983. A variety of cells can secrete exosomes under normal and pathological conditions. They are mainly derived from multivesicular bodies formed by the invagination of lysosomal microparticles in cells, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane with the cell membrane. Exosomes are rich in proteins, DNA, lipids, coding and non-coding RNAs (ncRNAs), and non-coding RNAs (ncRNAs) are composed of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In addition, the exosomes secreted by cancer cells are at least ten times more than those of normal cells. Exosomes carrying miRNAs released by immune cells, mesenchymal cells and cancer cells in the tumor environment can shuttle from donor cells to recipient cells. In recent years, many studies have pointed out that exosomal miRNA plays a key role in the mesothelial-mesenchymal transition of mesothelial cells.

[0005] MicroRNA is a class of endogenous single-stranded RNA molecules with a length of about 22 nucleotides. Although it cannot be further translated into protein, it can specifically bind to target mRNA, thereby indirectly inhibiting the expression of post-transcriptional genes. Studies have shown that miR-211-3p is highly expressed in chemotherapy-insensitive hypopharyngeal squamous cell carcinoma (HSCC) and promotes the insensitivity of hypopharyngeal squamous cell carcinoma (HSCC) to chemotherapy; compared with non-tumor tissue samples, miR-211-3p is significantly upregulated in liver cancer tissues and liver cancer cells, and miR-221-3p overexpression significantly enhances the proliferation, HBV-DNA replication, migration and invasion of HCC cells. In a study on bladder cancer (BCa), it was found that overexpression of miR-211-3p can inhibit the proliferation, migration, invasion and tumor growth of nude mouse BCa cells and promote cell apoptosis. This shows that miR-211-3p may play different roles in the malignant progression of different tumors. Regarding the malignant development of ovarian cancer, no studies have yet shown the role of miR-211-3p. Summary of the invention

[0006] The purpose of the present invention is to address the above problems and provide a use of exosomal miR-211-3p in preventing and / or treating peritoneal metastasis of ovarian cancer.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of a preparation for detecting exosomal miR-211-3p in the preparation of a reagent for diagnosing ovarian cancer metastasis. Compared with the ovarian cancer non-metastatic group, the exosomal miR-211-3p of ovarian cancer cells is highly expressed in the metastatic group.

[0009] In the application technology scheme, the reagent for diagnosing ovarian cancer metastasis includes detecting the transcription of miR-211-3p in ovarian cancer exosome samples based on high-throughput sequencing methods and / or based on quantitative PCR methods and / or based on probe hybridization methods, or detecting the expression of target genes regulated by miR-211-3p in ovarian cancer exosome samples.

[0010] The PCR primers used in the quantitative PCR method are: forward primer: 5′-ACCAAGGCAGGGACAGCAA-3′; reverse primer: 5′-ACTGCAGGGTCCGAGGTATT-3′.

[0011] The target gene regulated by miR-211-3p in the ovarian cancer exosome cancer sample is ATP1B2, and miR-211-3p negatively regulates the expression of ATP1B2.

[0012] The PCR primers used in the quantitative PCR detection of ATP1B2 are:

[0013] Forward primer: 5′-CCTGGGGCTGTGAAGTTCC-3′;

[0014] Reverse primer: 5′-CGATGTTGGCGGCGTTGATG-3′.

[0015] The second aspect of the present invention provides the use of exosomal miRNA as a target in the development or screening of drugs for preventing and / or treating peritoneal metastasis of ovarian cancer, wherein the miRNA is miR-211-3p.

[0016] The drug inhibits the expression of miR-211-3p.

[0017] The third aspect of the present invention provides use of a miR-211-3p inhibitor in the preparation of a medicament for preventing and / or treating peritoneal metastasis of ovarian cancer.

[0018] The inhibitor downregulates miR-211-3p and / or the transcription of miR-211-3p and / or blocks the activity of miR-211-3p.

[0019] Antisense oligonucleotides, antagomiRs, miRNA sponges, miRNA Erasers, Target Masking and / or multi-target antisense oligonucleotides are used to downregulate miR-211-3p and / or the transcription of miR-211-3p and / or block the activity of miR-211-3p.

[0020] The beneficial effects of the present invention are:

[0021] The present invention shows that miR-211-3p derived from ovarian cancer cell exosomes can promote the mesenchymal-mesenchymal transition (MMT) process of PMCs by targeting ATP1B2, thereby promoting the migration of PMCs and the attachment to ovarian cancer cells. MiR-211-3p can also promote the metastasis of ovarian cancer cells. The present invention provides a new target for the prevention / treatment of peritoneal metastasis of ovarian cancer - ovarian cancer cell exosome miR-211-3p. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The isolation and identification of exosomes from ovarian cancer cells: a. Exosomes were isolated from the supernatant of three ovarian cancer cell cultures, and the concentration of nanoparticle analysis represents the number of nanoscale particles per 1 ml of culture medium; b. Western blot was used to detect exosome positive markers CD9 and TSG101, and negative marker Calnexin, with HEPG2 cells as a control; c. Transmission electron microscopy was used to detect the exosome structure of Sk-ov-3 cells (scale bar, 100 nm); d. Laser confocal microscopy was used to detect the exosomes of Sk-ov-3 cells (DiI labeled) after co-culture with mesothelial cells Met-5A (DiO labeled); (scale bar, 20 μm).

[0023] Figure 2The results show that exosomes derived from ovarian cancer cells induce mesothelial-mesenchymal transition of MeT-5A in vitro: a. Cell migration experiment after Met-5A cells were pretreated with 50 μg of exosomes extracted from Sk-ov-3 cells for 48 hours (scale bar, 100 μm); b. Cell migration experiment after Met-5A cells were pretreated with 50 μg of exosomes extracted from HEY and A2780 cells for 48 hours (scale bar, 100 μm); c. Cell adhesion experiment after Met-5A cells were pretreated with 50 μg of exosomes extracted from Sk-ov-3 cells for 48 hours (scale bar, 250 μm); d. Cell adhesion experiment after Met-5A cells were pretreated with 50 μg of exosomes extracted from HEY and A2780 cells for 48 hours. A. Cell adhesion experiment after 48 hours, (scale bar, 250μm); e. WB experiment to detect the expression of Met-5AMMT markers (N-cadherin, E-cadherin, Vimentin) after co-culture with A2780 cell exosomes; f. WB experiment to detect the expression of Met-5AMMT markers (N-cadherin, E-cadherin, Vimentin) after co-culture with HEY cell exosomes; g. WB experiment to detect the expression of Met-5AMMT markers (N-cadherin, E-cadherin, Vimentin) after co-culture with HEY cell exosomes, with β-actin as the internal reference.

[0024] Figure 3 The results show that miR-211-3p is enriched in the exosomes of ovarian cancer cells and promotes the migration of ovarian cancer cells: a. Venn diagram shows the miRNAs that are commonly upregulated in the exosomes of three ovarian cancer cells (A2780, HEY, Sk-ov-3); b. Heat map shows the differences in co-expressed miRNAs in the exosomes of three ovarian cancer cells (A2780, HEY, Sk-ov-3); c. qPCR shows the local expression level of miR-211-3p in three ovarian cancer cells (A2780, HEY, Sk-ov-3); d. Migration experiment shows the changes in cell migration ability after overexpression of miR-211-3p in HEY cells; e. Migration experiment shows the changes in cell migration ability after overexpression of miR-211-3p in A2780 cells; f. Migration experiment shows the changes in cell migration ability after inhibiting the expression of miR-211-3p in Sk-ov-3 cells.

[0025] Figure 4The results show that miR-211-3p promotes the MMT transformation of mesothelial cells Met-5A: a. qPCR experiment shows the expression of miR-211-3p after transient transfection of miR-211-3p-mimics in Met-5A; b. WB experiment detects the expression of MMT markers (N-cadherin, E-cadherin, Vimentin) in Met-5A after overexpression of miR-211-3p; c. Migration experiment detects the changes in the migration ability of Met-5A cells after overexpression of miR-211-3p (scale bar, 100 μm); d. Adhesion experiment detects the changes in the adhesion ability of Met-5A cells and tumor cells after overexpression of miR-211-3p (scale bar, 250 μm).

[0026] Figure 5 The results show that exosomes derived from ovarian cancer cells carry miR-211-3p into mesothelial cells: a. Laser confocal scanning microscopy shows that miR-211-3p (red cy3 labeled) is delivered to Met-5A (DiO labeled) through exosomes secreted by Sk-ov-3 cells; b. qPCR experiment shows the expression of miR-211-3p in Met-5A after co-culture with Sk-ov-3-derived-exos; c. qPCR experiment shows the expression of miR-211-3p in Met-5A after co-culture with Sk-ov-3-derived-exos overexpressing miR-211-3p. The group with PBS added was used as the control.

[0027] Figure 6 It was shown that ovarian cancer cell exosomes carrying miR-211-3p-mimics can promote the MMT transformation of mesothelial cells: a. Migration experiment detected the changes in the migration ability of Met-5A cells after co-culture with miR-211-3p-mimics-exos (scale bar, 100μm); b. Adhesion experiment detected the changes in the adhesion ability of Met-5A cells and tumor cells after co-culture with miR-211-3p-mimics-exos (scale bar, 250μm); c. WB experiment detected the expression of Met-5AMMT markers (N-cadherin, E-cadherin, Vimentin) after co-culture with miR-211-3p-mimics-exos.

[0028] Figure 7The results show that miR-211-3p directly targets the 3'UTR region of ATP1B2: a. Venn diagram showing the intersection of miR-211-3p target genes predicted on two websites; b. qPCR experiment showing the RNA expression level of ATP1B2 in three ovarian cancer cells (A2780, HEY, Sk-ov-3); c. Dual luciferase labeling experiment showing that miR-211-3p directly targets the 3'UTR region of ATP1B2; d. qPCR experiment showing the changes in ATP1B2 RNA expression level in cells overexpressing miR-211-3p; e. WB experiment showing the changes in ATP1B2 RNA expression level in cells overexpressing miR-211-3p f. qPCR experiment shows the change of ATP1B2 protein expression level in cells after inhibiting miR-211-3p expression; g. WB experiment shows the change of ATP1B2 protein expression level in cells after inhibiting miR-211-3p expression; h. qPCR experiment detects the change of ATP1B2 RNA expression level in Met-5A after co-culture with miR-211-3p-mimics-exos; i. WB experiment detects the change of ATP1B2 protein expression level in Met-5A after co-culture with miR-211-3p-mimics-exos. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0031] Example 1

[0032] 1 Materials and methods

[0033] 1.1 Cell lines and cell culture

[0034] Human ovarian cancer cells A2780 and HEY were originally stored in liquid nitrogen in our laboratory. Human ovarian cancer cells Sk-ov-3 were purchased from Boster, Wuhan, China. Human mesothelial cells Met-5A were purchased from Pronocell, Wuhan, China. All purchased cells were STR-identified and mycoplasma tested negative. A2780 was cultured in DMEM medium (Gibico, Brazil) containing 10% fetal bovine serum (FBS, Gibico, Brazil), 100 U / mL penicillin, and 100 μg / mL streptomycin (Biyuntian, China), HEY was cultured in RPMI 1640 medium (Gibico, Brazil) containing the same ingredients, and Sk-ov-3 was cultured in McCoy's 5A medium (Boster, Wuhan, China) containing the same ingredients. Met-5A cells were cultured in a special medium containing M199+10% FBS+0.5% ITS-G (100×)+3.3nM EGF+400nM hydrocortisone+1% P / S (Pricella, Wuhan, China). All cells were cultured at 37°C and 5% CO2.

[0035] 1.2 Cell transfection

[0036] miR-211-3p mimics (analogs), miR-211-3 inhibitor (inhibitor) and their respective negative controls were synthesized by Genepharma China. According to the instructions of siRNA-Mate (Genepharma, Shanghai, China), miR-211-3p mimics and its negative control were transfected into A2780, HEY and Met-5A cells, and miR-211-3p inhibitor and its negative control were transfected into Sk-ov-3 cells.

[0037] (1) miR-211-3p double-stranded mimics sequence:

[0038] Sense strand: 5'-GCAGGGACAGCAAAGGGGUGC-3' (SEQ ID NO. 1),

[0039] Antisense strand: 5'-ACCCCUUUGCUGUCCCUGCUU-3' (SEQ ID NO. 2);

[0040] (2) miR-211-3p double-stranded mimics negative control sequence:

[0041] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO. 3),

[0042] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 4);

[0043] (3) miR-211-3p single-stranded mimics negative control sequence: 5′-UUGUACUACACAAAAGUACUG-3′ (SEQ ID NO. 5);

[0044] (4)miR-211-3p inhibitor sequence: 5'-GCACCCCUUUGCUGUCCCUGC-3' (SEQ ID NO.6);

[0045] (5) miR-211-3p inhibitor negative control sequence: 5′-CAGUACUUUUUGUGUAGUACAA-3′ (SEQ ID NO. 7);

[0046] 1.3 Extraction, characterization and internalization of exosomes

[0047] After culturing Sk-ov-3, A2780, and HEY cells in serum-free medium for 48 h, their cell supernatants were collected. Exosomes were extracted from the cell supernatant using an exosome extraction kit (Wayen, China) according to the manufacturer's instructions, resuspended in sterile PBS and stored at -80°C for later use. Transmission electron microscopy was used to detect the morphology of exosomes. Nanoparticle analysis was used to detect the size of exosomes. Exosome positive markers CD9 and TSG101, as well as negative marker Calenxin were detected by Western blotting. The concentration of extracted exosomes was measured by measuring the protein concentration in exosomes using a BCA kit (beyotime, Shanghai, China).

[0048] According to the manufacturer's instructions, Sk-ov-3 cells were incubated with DiI (cell membrane red fluorescent probe) (beyotime, Shanghai, China) for 20 minutes, then the probe was washed with PBS, and Sk-ov-3 cells were cultured in serum-free medium for 48 hours, and the cell supernatant was collected to extract exosomes. Exosomes (DiI makes the membrane structure red) were incubated with Met-5A cells for 48 hours, and then DiI (cell membrane green fluorescent probe) (beyotime, Shanghai, China) was incubated with Met-5A cells for 20 minutes to make the cell membrane green. The confocal dish was placed under a laser confocal scanning electron microscope for observation.

[0049] 1.4 Real-time quantitative PCR (RT-qPCR)

[0050] According to the manufacturer's instructions, total RNA was extracted from ovarian cancer cells and mesothelial cells using an RNA extraction kit (Promega, USA). RNA was then reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). Premix Ex Taq TM qPCR reaction was performed using the CFX96 Real-Time PCR Detection System (Bio-rad, USA) using the reagent II (Takara, Japan). U6 small nuclear (snRNA) was used as the internal reference gene for miR-211-3p, and β-actin was used as the internal reference gene for ATP1B2. Formula 2 was used. –ΔΔCT The relative expressions of miR-21-5p and ATP1B2 were calculated.

[0051] The primers for hsa-mir-211-3p, U6, ATP1B2 and β-actin are as follows:

[0052]

[0053] 1.5 Western blotting

[0054] The total protein of the cells was extracted by lysing, and the concentration was determined and then mixed with loading buffer and heated in a 100°C water bath for 10 minutes to denature the protein, and then stored at -20°C for later use. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated with antibodies containing anti-CD9, anti-TSG101, anti-Calnexin, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-ATP1B2, and anti-β-actin at 4°C overnight. The membranes were washed three times with TBST for five minutes each time, and then incubated with secondary antibodies (goat anti-rabbit IgG) IgG (ab6721, 1:5000, Abcam) for 2 hours. After the secondary antibody incubation was completed, the membranes were also washed three times with TBST for five minutes each time. Then, chemiluminescent liquid was evenly sprayed on the membrane for development.

[0055] 1.6 Transwell experiment

[0056] We used a migration chamber (pore size 8 μm) to evaluate the migration ability of cells. 200 μL serum-free culture medium containing 1*10 5 Cells (Met-5A cells co-cultured with exosomes for 48 hours, A2780, HEY, Met-5A cells transfected with miR-211-3p-mimics, Sk-ov-3 cells transfected with miR-211-3p-inhibitor and their respective control group cells) were inoculated in the upper chamber of the chamber, and 700 μL of culture medium containing 10% serum was spread in the lower chamber of the chamber. After culturing at 37°C for 48 hours, the non-migrated cells were removed, the cells on the chamber were fixed, and stained with 1% crystal violet. After drying, the cells were photographed under a 10x objective lens, and the cells fixed on the chamber were counted using Image J software.

[0057] 1.7 Tumor cell adhesion assay

[0058] Previous studies have shown that the ability of mesothelial cells to adhere to tumor cells can be determined by adhesion experiments. We pre-plated Met-5A cells that had been treated differently in 24-well plates, and after they grew into a monolayer of cells, they were incubated with HEY cells treated with DiI (cell membrane green fluorescent probe) at 37°C for 30 minutes. After that, the unattached HEY cells were washed with PBS, and the cells in the 24-well plates were fixed with 4% paraformaldehyde. The cells were photographed under a fluorescence microscope with a 10x objective lens, and the HEY cells attached to the surface of Met-5A cells were counted using ImageJ software.

[0059] 1.8 Dual luciferase reporter gene assay

[0060] Plasmids with wild-type or mutant binding sites of the 3'UTR region of ATP1B2 and miR-211-3p were constructed. The plasmids were co-transfected with miR-211-3p-mimics or mimics-NC into A2780 cells using a transfection reagent according to the manufacturer's instructions. After 48 hours, the luciferase activities of firefly and Renilla were determined using a dual luciferase system (Promega), and the relative luciferase activity was calculated by the ratio of the luciferase activities of firefly and Renilla.

[0061] 1.9 Detection of Cy3-labeled miR-211-3p transfer

[0062] In order to directly observe the transfer of miR-211-3p from ovarian cancer cells to mesothelial cells through exosomes, we constructed cy3-labeled miR-211-3p single-chain mimics, transfected cy3-labeled mimics into Sk-ov-3 cells, cultured at 37°C for 36 hours, switched to serum-free medium and continued cultured for 48 hours, then collected the cell supernatant to extract exosomes. After incubating exosomes with Met-5A cells for 48 hours, Met-5A cells were plated in confocal dishes and adhered to the wall and incubated with DiI (cell membrane green fluorescent probe) for 20 minutes, and the probe was washed off with PBS and imaged under a laser scanning confocal microscope.

[0063] 1.10 Statistical analysis

[0064] All statistical analyses were performed using GraphPadPrism software and expressed as mean ± standard deviation (SD). The differences between the two groups were analyzed using a two-tailed unpaired Student's t test. For multiple group comparisons, one-way ANOVA was used. P<0.05 was considered statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0065] 2 Experimental results

[0066] 2.1 Isolation and identification of exosomes from ovarian cancer cells

[0067] We isolated exosomes from the supernatant of cultured ovarian cancer cells and examined them by scanning transmission electron microscopy and nanoparticle analysis. Figure 1 a) It can be seen that the particle size of the exosomes is about 110nm. Then the extracted exosomes were subjected to protein identification ( Figure 1 b), it can be seen that the extracted exosomes clearly express positive markers, while negative markers cannot be detected. Figure 1 c), it can be seen that the structure of Sk-ov-3 cell exosomes is cup-shaped. After the extracted Sk-ov-3 ovarian cancer cell exosomes (DiO stained cell membrane is red) and mesothelial cells (DiI stained cell membrane is green), laser scanning confocal scanning electron microscopy was used to take pictures, and it can be seen that the exosomes can enter the mesothelial cells ( Figure 1 d).

[0068] 2.2 Exosomes derived from ovarian cancer cells promote the migration of mesothelial cells and their adhesion to ovarian cancer cells, and promote their mesothelial-mesenchymal transition

[0069] In recent years, many studies have shown that tumor-derived exosomes play an important role in tumor metastasis. Therefore, we co-cultured A2780-exos, HEY-exos, and Sk-ov-3-exos with mesothelial cells Met-5A to evaluate the different abilities of ovarian cancer cell exosomes to induce Met-5A in vitro. Figure 2 a, b) It can be seen that ovarian cancer cell exosomes enhance the migration ability of mesothelial cells. WB experiment ( Figure 2 .e,f,g) Detection of MMT markers in mesothelial cell proteins after co-culture revealed that the expression of MMT positive markers N-cadherin and Vimentin increased after co-culture, while the expression of negative marker E-cadherin decreased. Figure 2 c, d) demonstrated that Met-5A treated with ovarian cancer cell exosomes was more conducive to further adhesion to ovarian cancer cells (HEY) compared with the control group. The above in vitro experiments showed that exosomes derived from ovarian cancer cells promoted the MMT transformation of mesothelial cells.

[0070] 2.3 miR-211-3p is enriched in ovarian cancer cell exosomes and promotes ovarian cancer cell migration

[0071] Through in vitro experiments, we found that exosomes derived from ovarian cancer cells promote the MMT transformation of mesothelial cells Met-5A, but the specific mechanism is still unclear. Exosomes are rich in microRNAs, and many studies in recent years have shown that these microRNAs play an important role in cell communication. Therefore, we focused our research on miRNAs in exosomes. We downloaded A2780, HEY, Sk-ov-3, and IOSE-80 exosome miRNA data from public databases. After analysis, we obtained the differentially expressed miRNAs in A2780, HEY, and Sk-ov-3 exosomes compared with normal ovarian epithelial cell exosomes ( Figure 3 a), the abnormally highly expressed miRNAs in A2780, HEY, and Sk-ov-3 exosomes were intersected to select 12 miRNAs ( Figure 3 b). miR-211-3p has been shown to play an important role in tumor metastasis. We selected miR-211-3p as the research object and performed q-PCR detection on its background expression in three ovarian cancer cells. We found that its expression was highest in the most malignant Sk-ov-3 cells ( Figure 3 c). Then, miR-211-3p-mimics was transiently transfected into A2780 and HEY cells to overexpress miR-211-3p. It can be seen that after overexpression of miR-211-3p, the migration ability of A2780 and HEY cells was enhanced ( Figure 3d, e), After transient transfection of miR-211-3p-inhibitor into Sk-ov-3 cells to inhibit miR-211-3p expression, the migration ability of Sk-ov-3 cells was inhibited ( Figure 3 f). From the above in vitro experiments, we inferred that miR-211-3p plays an important role in the migration of ovarian cancer cells.

[0072] 2.4 miR-211-3p promotes MMT transformation of Met-5A in mesothelial cells

[0073] The above experiments show that miR-211-3p plays an important role in the migration of ovarian cancer cells, but its effect on mesothelial cells is still unclear. We then transiently transfected miR-211-3p-mimics into mesothelial cells Met-5A to overexpress miR-211-3p ( Figure 4 a) To study the effect of miR-211-3p on Met-5A. In vitro experiments showed that overexpression of miR-211-3p promoted the migration of Met-5A ( Figure 4 c), and WB experiments detected that the expression of MMT's positive markers N-cadherin and Vimentin increased, and the expression of the negative marker E-cadherin decreased ( Figure 4 b). Adhesion experiments further showed that the adhesion ability of Met-5A to tumor cells (HEY) was further enhanced after overexpression of miR-211-3p ( Figure 4 d). The above in vitro experiments indicate that miR-211-3p promotes the MMT transformation of mesothelial cells Met-5A.

[0074] 2.5 Exosomes from ovarian cancer cells carry miR-211-3p into mesothelial cells

[0075] We have demonstrated that overexpression of miR-211-3p can promote the migration of ovarian cancer cells and the MMT transformation of mesothelial cells Met-5A, but it is still unknown whether miR-211-3p can be delivered from donor cells to recipient cells through exosomes. Therefore, we labeled miR-211-3p-mimics with cy3, transiently transfected them into ovarian cancer cells Sk-ov-3 cells, and then extracted exosomes from the supernatant of Sk-ov-3 cells. After co-culturing cy3-exos with Met-5A, laser scanning confocal microscopy captured cy3-miR-211-3p (red) entering Met-5A (DiI made its cell membrane green) compared with the control group ( Figure 5a). The total RNA of Met-5A cells co-cultured with Sk-ov-3-exos and cy3-mimics-exos was extracted to detect the expression level of miR-211-3p. As can be seen from the figure, compared with the control group, the expression level of miR-211-3p in Met-5A co-cultured with Sk-ov-3-exos and cy3-mimics-exos was significantly increased ( Figure 5 b,c). The above experiments show that miR-211-3p can be carried by exosomes and delivered from ovarian cancer cells to mesothelial cells.

[0076] 2.6 Ovarian cancer cell exosomes carrying miR-211-3p-mimics can promote mesothelial cell MMT transformation

[0077] Previous experiments have shown that miR-211-3p can be transferred from donor cells to recipient cells through exosomes, but whether it can play a role in mesothelial cells still needs experimental verification. Therefore, we co-cultured miR-211-3p-mimics-exos with mesothelial cells Met-5A. From the in vitro experiments, it can be seen that compared with the control group, the migration ability of Met-5A cells after co-culture is enhanced ( Figure 6 a), and then WB experiments detected that the expression of MMT positive markers N-cadherin and Vimentin increased, and the expression of negative marker E-cadherin decreased in Met-5A cells after co-culture ( Figure 6 c). Compared with the control group, the adhesion ability of Met-5A cells to ovarian cancer cell HEY after co-culture was also enhanced ( Figure 6 b). The above in vitro experiments show that miR-211-3p can promote the MMT transformation of mesothelial cells after being delivered to mesothelial cells via exosomes.

[0078] 2.7 miR-211-3p directly targets the 3′UTR region of ATP1B2

[0079] To investigate how miR-211-3p plays a role in the progression of mesothelial cell MMT, we used TargetScan (http: / / www.targetscan.org / ) and miRanda (http: / / www.microrna.org / microrna / home.do) online websites to predict potential target genes of miR-211-3p. Figure 7 a), then, ATP1B2 was identified as a potential target. ATP1B2 was expressed at a relatively low level in Sk-ov-3 cells, where miR-211-3p was expressed at a relatively high level ( Figure 7b). A dual luciferase labeling experiment was performed to verify whether ATP1B2 is a direct target of miR-211-3p. The sites of the mutant (MUT) and wild-type (WT) (the former is assumed to contain a site-directed mutation on the miR-211-3p target site) were cloned into a vector containing dual luciferase, and the vector and miR-211-3p-mimics were co-transfected into A2780 cells. As shown in the figure, the luciferase activity of the wild-type vector was significantly reduced in the presence of miR-211-3p, while the luciferase activity of the mutant vector was not reduced in the presence of miR-211-3p ( Figure 7 c). Dual luciferase labeling experiments demonstrated that ATP1B2 is a direct target of miR-211-3p.

[0080] Then, real-time fluorescence quantitative PCR and WB experiments were used to further prove the relationship between ATP1B2 and miR-211-3p. Real-time fluorescence quantitative PCR experiments showed that compared with the negative control, the expression level of ATP1B2 mRNA in the miR-211-3p-mimics group was reduced ( Figure 7 d), ATP1B2 mRNA expression level increased in the miR-211-3p-inhibitor group ( Figure 7 f). WB experiments showed that the expression level of ATP1B2 protein in the miR-211-3p-mimics group was decreased compared with the negative control group ( Figure 7 e), the expression level of ATP1B2 protein increased in the miR-211-3p-inhibitor group ( Figure 7 g), and the RNA and protein expression levels of ATP1B2 were decreased in Met-5A cells co-cultured with miR-211-3p-mimics-exos ( Figure 7 h,i). These results indicate that miR-211-3p inhibits the expression of ATP1B2 protein by degrading the corresponding mRNA.

[0081] 3 Analysis

[0082] Exosomes derived from tumor cells are involved in peritoneal metastasis of ovarian cancer. miRNA is the most studied substance carried by exosomes. Ovarian cancer is the most lethal cancer among female malignant tumors. Most patients are found in the middle and late stages and have extensive peritoneal metastasis, resulting in a poor prognosis. Therefore, it is very necessary to find new therapeutic targets. In these studies, we demonstrated that exosomes derived from ovarian cancer cells promote MMT transformation of mesothelial cells by delivering miR-211-3p. miR-211-3p directly targets ATP1B2 and negatively regulates its expression. The present study is the first to report the effect of miR-211-3p derived from ovarian cancer cell exosomes on the malignant development of ovarian cancer in ovarian cancer research.

[0083] One of the characteristics of advanced ovarian cancer is the presence of extensive peritoneal metastasis and a large amount of ascites, and multiple cell communications are actively involved in the malignant development of ovarian cancer. Exosomes derived from ovarian cancer can be detected in a variety of tissues, such as ascites, serum, and urine. Exosomes have been shown to play an important role in the peritoneal dissemination of ovarian cancer and the formation of pre-metastatic niches. Exosomes derived from ovarian cancer convert fibroblasts, which are essential for normal tissue homeostasis and function, into cancer-associated fibroblasts (CAFs). Exosomes derived from ovarian cancer release CD44 and transmit it to peritoneal mesothelial cells (PMCs). The increase in CD44 in peritoneal mesothelial cells (PMCs) induces them to secrete matrix metalloproteinases (MMPs), thereby destroying the peritoneal barrier and promoting the migration of ovarian cancer. In the present study, after co-culturing exosomes derived from ovarian cancer cells with mesothelial cells, the migration ability of mesothelial cells was enhanced, and the exosomes promoted the mesothelial-mesenchymal transition (MMT) of mesothelial cells, which is a necessary step for peritoneal mesothelial cells (PMCs) to transform into cancer-associated fibroblasts (CAFs). Moreover, exosomes derived from ovarian cancer cells promoted the adhesion ability of mesothelial cells to ovarian cancer, which is conducive to faster intercellular communication between ovarian cancer cells and mesothelial cells.

[0084] Existing studies have shown that miR-211-3p may play different roles in the malignant progression of different tumors. In this study, we downloaded the exosome miRNA data of three ovarian cancer cells (A2780, HEY, Sk-ov-3) from the public database, compared with the exosome miRNA data of normal ovarian epithelial cells, screened out the miRNAs that were upregulated in the exosomes of ovarian cancer cells, and determined miR-211-3p as the research object. The results of qRT-PCR showed that compared with the other two ovarian cancer cells, the expression of miR-211-3p was the highest in Sk-ov-3, which had the highest degree of malignancy. In addition, overexpression of miR-211-3p promoted the migration of A2780 and HEY ovarian cancer cells, and inhibition of miR-211-3p expression inhibited the migration of Sk-ov-3 cells. Overexpression of miR-211-3p in mesothelial cells Met-5A also promoted the migration of Met-5A, the transformation of MMT, and the adhesion to tumor cells. In addition, we also demonstrated that miR-211-3p can be delivered from ovarian cancer cells to mesothelial cells via exosomes, leading to increased miR-211-3p levels in mesothelial cells and promoting the MMT progression of mesothelial cells.

[0085] In order to further study the mechanism of action of miR-211-3p in Met-5A mesothelial-mesenchymal transition (MMT), we used the miRNA target gene prediction website to predict the possible target genes of miR-211-3p. After taking the intersection of the target genes predicted by miRNAda and TargetScan, ATP1B2 was selected from the target genes in the intersection as the further research object. ATP1B2 (ATPase Na+ / K+transporting subunit beta 2, ATPase Na+ / K+transporting subunit β2) was first identified as an integral membrane glycoprotein highly expressed in astrocytes, mediating the interaction between neurons and astrocytes. Its expression in glioma cells decreases with the increase of malignancy. Down-regulation of ATP1B2 in Glioma cells withinglioblastoma multiforme (GBM) increases the invasion of GBM (Matthew Z. Sun et al., 2013). In the study of breast cancer, ATP1B2, as a flanking gene of TP53, was also significantly reduced in breast cancer tissues compared with normal tissues (Feng Zeng et al., 2020). In the present study, we first used dual luciferase labeling experiments to prove that ATP1B2 is a direct target of miR-211-3p, and q-PCR and western blot experiments proved that miR-211-3p inhibits the expression of ATP1B2 by degrading the corresponding mRNA. After that, we used bioinformatics analysis to obtain that ATP1B2 was significantly underexpressed in ovarian cancer tissues compared with normal tissues, and the overall survival rate of the ATP1B2 high expression group was significantly higher than that of the low expression group.

[0086] In summary, the present study showed that miR-211-3p in exosomes from ovarian cancer cells can induce MMT transformation of mesothelial cells and directly target ATP1B2, which is lowly expressed in ovarian cancer tissues. miR-211-3p may serve as a new therapeutic target for the malignant development of ovarian cancer.

[0087] In summary, the present study found that miR-211-3p derived from ovarian cancer cell exosomes can promote MMT of mesothelial cells by targeting ATP1B2, and miR-211-3p promotes the metastasis of ovarian cancer cells. It was also found that ATP1B2, which has not been reported in previous ovarian cancer studies, is significantly lowly expressed in ovarian cancer tissues. The present study may provide a potential approach for the study of peritoneal metastasis of ovarian cancer.

Claims

1. Use of a preparation for detecting exosomal miR-211-3p in the preparation of a reagent for diagnosing ovarian cancer metastasis, characterized in that: Compared with the non-metastatic ovarian cancer group, exosomal miR-211-3p was highly expressed in the metastatic group.

2. The use according to claim 1, characterized in that: The reagent for diagnosing ovarian cancer metastasis includes detecting the transcription of miR-211-3p in ovarian cancer exosome samples based on high-throughput sequencing methods and / or based on quantitative PCR methods and / or based on probe hybridization methods, or detecting the expression of target genes regulated by miR-211-3p in ovarian cancer exosome samples.

3. The use according to claim 2, characterized in that: The PCR primers used in the quantitative PCR method are: forward primer: 5′-ACCAAGGCAGGGACAGCAA-3′; Reverse primer: 5′-ACTGCAGGGTCCGAGGTATT-3′.

4. The use according to claim 2, characterized in that: The target gene regulated by miR-211-3p in the ovarian cancer exosome cancer sample is ATP1B2, and miR-211-3p negatively regulates the expression of ATP1B2.

5. The use according to claim 4, characterized in that: The PCR primers used in the quantitative PCR detection of ATP1B2 are: forward primer: 5′-CCTGGGGCTGTGAAGTTCC-3′; Reverse primer: 5′-CGATGTTGGCGGCGTTGATG-3′.

6. Use of exosomal miRNA as a target in the development or screening of drugs for preventing and / or treating peritoneal metastasis of ovarian cancer, wherein the miRNA is miR-211-3p.

7. The use according to claim 6, characterized in that: The drug inhibits the expression of miR-211-3p.

8. Use of miR-211-3p inhibitors in the preparation of drugs for preventing and / or treating peritoneal metastasis of ovarian cancer.

9. The use according to claim 8, characterized in that: The inhibitor downregulates miR-211-3p and / or the transcription of miR-211-3p and / or blocks the activity of miR-211-3p.

10. The use according to claim 9, characterized in that: Antisense oligonucleotides, antagomiRs, miRNA sponges, miRNA Erasers, Target Masking and / or multi-target antisense oligonucleotides are used to downregulate miR-211-3p and / or the transcription of miR-211-3p and / or block the activity of miR-211-3p.

Images