Microarray data analysis

Using the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), we downloaded ovarian cancer-related microarray data GSE4122, GSE18520 and GSE38666 as well as the gene annotation files (Table ​(Table1).1). The limma package of R language was employed for differential analysis of the microarray data gene with the screening threshold of |log2FoldChange (FC)|>2.0 and adj.P.Val<0.05, with plotting of the heat map for differentially expressed genes (DEGs). The jvenn (http://jvenn.toulouse.inra.fr/app/example.html) was applied to compare the DEGs from three microarray data, the String database (https://string-db.org/) was employed to analyze the interaction of DEGs, and the Cytoscape 3.6.0 software [19] was used to construct the protein–protein interaction (PPI) of DEGs to screen the DEGs. To further explore the molecular mechanisms incorporating DEGs, a combination of the TarBase (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8%2Findex), miRDB (http://www.mirdb.org/), mirDIP (http://ophid.utoronto.ca/mirDIP/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/search.php), TargetScan (http://www.targetscan.org/vert_71/) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) were employed to predict the regulatory miRNAs of DEGs, and the predicted results were compared using jvenn.

Sample collection

The ovarian tumor samples were harvested from 85 patients following initial diagnosis as ovarian cancer at the Linyi People’s Hospital from July 2014 to April 2016. All enrolled patients were aged between 36 and 83 years with an average age of 59.68±13.72 years, without prior history of radiotherapy or chemotherapy. Moreover, in our study, 35 cases were classified in an early stage while 50 cases were in the advanced stage according to the International Federation of Gynecology and Obstetrics (FIGO) staging classification (2013). All samples were harvested by surgical intervention and the patients were followed up for 3 years. Additional 43 cases of adjacent ovarian epithelium tissues were harvested for this study.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

The total RNA content was extracted using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and integrity of the total RNA content was evaluated by gel electrophoresis. Successful isolation of total RNA content was indicated by clear appearance of the 28S band and 18S band in electrophoretogram, wherein the 28S band was twice the length of 18S. Next, Nanodrop2000 (Thermo Fisher Scientific, Waltham, USA) was used to detect the absorbance at excitation wavelengths of 260 and 280 nm for determining the RNA concentration, and 1 µg total RNA was reversely transcribed into complementary DNA (cDNA) using PrimeScript™ RT reagent kit with the gDNA Eraser kit (RRO37A, Takara Bio Inc., Otsu, Shiga, Japan). Then, RT-qPCR was conducted using the SYBR®Premix Ex Taq™ (Tli RNaseH Plus) kit (RR820A, Takara) in the ABI7500 quantitative PCR instrument (Thermo Fisher Scientific). Additionally, the synthetic C. Elegans (cel)-miR-39 was used as a spike-in control for the total RNA content extracted from EVs. The relative mRNA and miRNA expression, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 respectively, was determined based on the 2^−ΔΔCt method. All primers were provided by GenePharma Co., Ltd., (Shanghai, China) and the primer sequences are shown in Table ​Table22.

Western blot analysis

Phenylmethanesulfonyl fluoride and protease inhibitor (P0100, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) were added into the Radio Immunoprecipitation Assay lysis to extract total protein content from the tissues and cells. Total proteins were lysed at 4 °C for 15 min and centrifuged at 15,000 rpm for 15 min to isolate the supernatant followed by determination of protein concentration using the bicinchonininc acid kit (23227, Thermo Fisher Scientific). After protein quantitation, the proteins were separated by polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Thereafter, the membrane was blocked with 5% bovine serum albumin for 1 h at room temperature followed by overnight incubation at 4 °C with the corresponding primary antibodies MYB (dilution ratio of 1: 2500, ab12296, Abcam Inc., Cambridge, UK), vascular endothelial growth factor (VEGF; dilution ratio of 1: 100, AF-293-NA, R&D Systems, Minneapolis, MN, USA), vascular endothelial growth factor receptor (VEGFR; dilution ratio of 1: 100, ab11939, Abcam) and GAPDH (dilution ratio of 1: 5000, ab8245, Abcam). The following day, the membranes were washed with Tris-buffered saline with 0.1% Tween-20 (TBST) 3 times (5 min for each), incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG; dilution ratio of 1: 20,000, ab205718, Abcam) for 1.5 h at room temperature, and rinsed 3 times (5 min for each) with TBST again. Subsequently, the membranes were developed with the addition of the developer (NCI4106, Pierce, Rockford, IL, USA), followed by quantitative protein analysis using the ImageJ 1.48u software (Bio-Rad, Hercules, CA, USA). The relative expression of proteins was expressed as the ratio of the gray value of proteins to that of GAPDH.

Immunohistochemistry

The tissue samples were made into paraffin sections, hydrated with gradient alcohol, washed under running water for 2 min, with 3% methanol H₂O₂ for 20 min, with distilled water for 2 min, and rinsed with 0.1 M phosphate-buffered saline (PBS) for 3 min. Thereafter, the sections were incubated with normal goat serum sealing solution (C-0005, Shanghai Haoran Bio Technologies Co., Ltd. Shanghai, China) at room temperature for 20 min, and with the primary antibody rabbit anti-human MYB (dilution ratio of 1: 100, ab76009, Abcam) at 4 °C overnight and rinsed with 0.1 M PBS thrice (5 min for each). Next, the sections were added with the secondary antibody goat anti-rabbit IgG (ab6785, dilution ratio of 1: 1000, Abcam) at 37 °C for 2 min. Subsequently, the sections were incubated with HRP-labeled streptavidin ovalbumin working solution (0343-10000U, Imunbio, Beijing, China) at 37 °C for 20 min. The sections were then developed with diaminobenzidine (ST033, Whiga, Guangzhou, China), counterstained by hematoxylin (PT001, Shanghai Bogoo Biotechnology, Co., Ltd., Shanghai, China) for 1 min, added with 1% ammonia water to return to blue, dehydrated with gradient alcohol, cleaned by xylene, and mounted by neutral balsam. Bright field images were acquired using a Nikon Eclipse TE2000 microscope (Nikon, Tokyo, Japan) and NIS-Elements software with four pictures taken from each experiment. Analysis and quantification on images were performed using the ImageJ software.

Cell culture and transfection

The normal human ovarian epithelial cell line HOSEpiC and human ovarian cancer cell lines (SKOV-3, HO8910, A2780) and human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Institute of Cellular Biology of Chinese Academy of Sciences (Shanghai, China). The procured cells were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco by Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and penicillin–streptomycin (Gibco by Life technologies) and incubated at 37 °C with 5% CO₂. Following incubation, the cells were detached with 0.25% trypsin, sub-cultured at the ratio of 1: 3, and seeded into 6-well plates at a density of 3×10⁵ cells/well. After achieving 70–80% cell confluence, cells at the logarithmic phase of growth were selected for subsequent experiments.

The ovarian cancer cells at logarithmic phase of growth were seeded in 6-well plates at a density of 4×10⁵ cells/well. When cell confluence reached 80–90%, the cells were transfected based on the instructions of lipofectamine 2000 (11668-019, Invitrogen). Following transfection, the cells were cultured with 5% CO₂ at 37 °C under the condition of saturated humidity. After 48 h, the original medium in wells was discarded, and the cells were added with RPMI 1640 medium containing 10% FBS for another 24–48 h. Next, the cells were grouped by transfecting with mimic-negative control (mimic-NC), miR-424 mimic, inhibitor-NC, miR-424 inhibitor, NC for short hairpin RNA (sh-NC), and sh-MYB, alone or in combination. All the aforementioned transfection sequences and plasmids were purchased from GenePharma.

Isolation and culture of MSCs and osteogenic/adipogenic identification

Healthy and well-grown C57BL/6 mice were collected, euthanized and soaked into alcohol for 10 min. Under aseptic conditions, the femur and tibia of mice were collected with the leg flesh removed, and then washed and placed in a flat dish containing Dulbecco’s modified Eagle medium (DMEM) (Gibco by Life Technologies) for further use. After that, both ends of the femur and tibia were cut open to isolate the bone marrow cells which were then mixed with DMEM into a 15 mL centrifuge tube using injector followed by centrifugation at 1500 rpm for 3 min, with the supernatant discarded. Subsequently, freshly isolated MSCs were resuspended by DMEM containing 10% FBS (Biowest, Nuaillé, France) supplemented with 100 U/mL penicillin–streptomycin (Gibco by Life Technologies) and cultured with 5% CO₂ at 37 °C and the medium was refreshed after 3 days. Thereafter non-adherent cells were removed and cell morphology of MSCs was observed, photographed, and recorded. After attaining 80–90% confluence, the cells were subsequently sub-cultured in different bottles while cells at passage 3 (P3) were collected for further experimentation.

The MSCs at P3 were collected, detached with trypsin (Gibco by Life Technologies), resuspended, and counted while cell concentration was adjusted to 1×10⁶ cells/mL. Following after, 200 μL of cell suspension was collected and placed into an Eppendorf (EP) tube, mixed with 5 μL different fluorescent-labeled monoclonal antibodies (CD19, CD34, CD45, CD73, CD90, CD105, and HLA-DR), and then incubated at 4 °C for 15 min avoiding exposure to light. After that, cells in each tube were added with 2 mL PBS, triturated, mixed, and centrifuged at 1000 rpm for 5 min, with the supernatant discarded to remove uncombined antibodies. After that, the cells were resuspended with 0.01 M PBS (400 μL) containing 0.5% paraformaldehyde and mixed uniformly. The IgG labeled with the same fluorescence was set as the isotype control while a flow cytometer (BD Biosciences, San Jose, CA, USA) was used to characterize the cells.

The MSCs at P3 were trypsinized and made into a single-cell suspension, with the cell concentration adjusted to 1×10⁵ cells/mL. Cells in the experimental group were added with 2 mL osteogenic differentiation induction complete medium (MUBMX-90021, Cyagen Biosciences Inc., Guangzhou, China), while the cells in control group were added with equal amounts of DMEM. After 3 days of cell culture, medium in both the experimental group and control group was replaced with a fresh medium. After 2–3 weeks of induction, the medium was removed and cells were rinsed twice with PBS. Cells in each well were added with 2 mL 4% neutral formaldehyde to fix for 30 min, with formaldehyde then removed. After another two rinses with PBS, cells in each well were stained with 1.5 mL alizarin red for 5 min. Lastly, the cells were rinsed with PBS twice, observed, and photographed under a microscope (TE2000, Nikon, Tokyo, Japan).

The MSCs at P3 were trypsinized and made into a single-cell suspension, with cell concentration adjusted to 3×10⁵ cells/mL. When cell confluence reached 80–90%, the supernatant was removed, and the cells were added with 2 mL of adipogenic differentiation medium A (MUBMX-90031, Cyagen Biosciences Inc., Guangzhou, China). After 3 days of induction, medium A in 6-well plate was removed, and the cells were added with 2 mL of adipogenic differentiation medium B. After 24 h of induction, medium B was replaced with medium A to induce cells, while alternate induction was conducted 5 times and then cells were induced with medium B for 7 days, with medium renewed every 3 days. After that, medium B in 6-well plate was removed and cells in each well were fixed with 2 mL of 4% neutral formaldehyde for 30 min. Next, the cells in each well were stained with 1.5 mL oil red O for 30 min. Finally, the cells were observed and photographed under a microscope (TE2000, Nikon, Tokyo, Japan).

Extraction and identification of MSC-derived EVs

The MSCs at P3 were collected and cultured overnight in serum-free DMEM. Upon reaching 80–90% confluence, the cells were sub-cultured in serum-free medium for 24 h with supernatant collected. Thereafter, the cells were centrifuged at 2000×g for 20 min at 4 °C to remove cell debris, and the collected supernatant was further exposed to high-speed centrifugation (100,000×g) for 1 h at 4 °C. Following after, the cells were suspended in serum-free DMDM containing 25 mM HEPES (pH=7.4), precipitated, and followed by high-speed centrifugation once again. After the removal of the supernatant, the cell precipitates were stored at − 80 °C for subsequent use.

Transmission electron microscopy (TEM) was applied to identify the EVs as follows: 30 μL EVs were placed on a copper mesh and allowed to stand for 1 min, with liquid dried using a filter paper. Next, the EVs were counterstained with 30 μL phosphotungstic acid solution (pH 6.8) for 5 min at room temperature, dried using a filament lamp and photographed by means of TEM.

Flow cytometry was further employed to detect the content of EVs surface marker CD63 as follows: the MSCs with good growth conditions were collected and rinsed with PBS. After detachment with trypsin, the cells were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Next, the cells were added with PBS and triturated into a single cell suspension. The cells were then evenly divided into 10 EP tubes (1.5 mL), centrifuged at 1000 rpm for 5 min, with the supernatant discarded. Following after, the cells were added with 1 mL PBS (containing 1% bovine serum albumin [BSA]) to disperse the precipitates, incubated at room temperature for 30 min to block nonspecific antigen, and centrifuged at 1000 rpm for 5 min with the supernatant discarded followed by resuspension with PBS (200 µL/EP tube). Subsequently, the cells were added with CD63-PE antibody and incubated at room temperature for 30 min, with the cells without antibody used as blank control and the PE-labeled anti-human IgG as an isotype control. After that, the cells were centrifuged at 1000 rpm for 5 min with supernatant discarded, resuspended with PBS containing 1% BSA, precipitated, and sampled followed by detection using a Guava easyCyte™ system flow cytometer. Total RNA content of miR-424 from isolated EVs was extracted using a Trizol reagent (Invitrogen), and the isolated miR-424 was identified by means of agarose gel electrophoresis (Additional file 1: Fig. S1). The expression patterns of miR-424 in EVs were detected using miRcute miRNA qPCR Detection kits (SYBR Green; TIANGEN, Beijing, China).

Dual-luciferase reporter gene assay

The plasmids PmirGLO-MYB-wild type (WT) and PmirGLO-MYB-mutant (MUT) were constructed and co-transfected with miR-424 mimic and mimic NC respectively into 293 T cells. After 24 h of transfection, the cells were lysed and centrifuged at 12,000 rpm for 1 min with supernatant collected. The luciferase activity was detected using a Dual-Luciferase® Reporter Assay System (E1910, Promega Corp., Madison, Wisconsin, USA), and the luciferase activity=firefly luciferase activity/Renilla luciferase activity.

Co-culture of MSCs/EVs with ovarian cancer cells

To inhibit the secretion of MSC-derived EVs, MSCs were treated with EV release inhibitor GW4869. MSCs were then spread in a 6-well plate at a density of 1×10⁶ cells/well. After achieving 80–90% confluence, the cells were treated with 10% GW4869 (D1692-5MG, Sigma-Aldrich, St. Louis, MO, USA), whereas the controls were treated with 0.005% dimethyl sulphoxide.

Next, the MSCs were spread in the basolateral chamber of 24-well Transwell chamber at a density of 1×10⁴ cells/well, while the ovarian cancer cells were spread in the apical chamber. After 24 h of co-culture, the ovarian cancer cells were isolated to detect the expressions of miR-424 and MYB in cells. The MSCs used for detection were categorized as MSCs without treatment, EVs-depleted MSCs, and MSCs treated with miR-424 mimic or miR-424 inhibitor as well as their NCs. Thereafter, the secretion of MSC-derived EVs treated with GW4869 was observed under TEM.

In accordance with the instructions of PKH67 (Green) dye kits (PKH67GL-1KT, Sigma-Aldrich), MSC-derived EVs were labeled, and then the fluorescence-labeled EVs were co-cultured with the ovarian cancer cell (with 50–60% confluence) supernatant in a 24-well plate followed by 48 h of co-culture. The MSC-derived EVs used for detection were categorized as MSC-derived EVs without treatment, EVs-depleted MSCs, MSC-derived EVs treated with miR-424 mimic or miR-424 inhibitor as well as their NCs. The ovarian cancer cell supernatant was collected and the expressions of miR-424 and the mRNA and protein expressions of MYB were detected.

5-ethynyl-2′-deoxyuridine (EdU) assay

The ovarian cancer cells were isolated, rinsed twice with PBS, and seeded in a 96-well plate at a density of 5×10³ cells/well. After 6 h, the cells were added with EdU medium (100 μL/well) and incubated for 2 h. Next, the cells were incubated with cell fixation fluid (4% paraformaldehyde) (100 μL/well) at room temperature for 30 min, followed by incubation with 2 mg/mL glycine for 5 min and then a penetrant (PBS containing 0.5% TritonX-100) (100 μL/well) for 10 min. Afterwards, the cells were incubated with 1×Apollo staining reaction solution (100 μL/well) for 30 min in dark conditions. Following after, the cells were incubated with 1×Hoechst 33342 reaction solution (100 μL/well) on a decoloring shaking table at room temperature for 30 min avoiding exposure to light. After staining, the cells were added with an anti-fluorescence quenching sealer (100 μL/well). Under a fluorescence microscope (TE2000, Nikon, Tokyo, Japan), the cells were photographed and the number of EdU-labeled cells was recorded. The cells with red-stained nucleus were regarded as positive cells, whereas the positive and negative cells were counted within 3 random visual fields. The EdU labeling rate (%)=the number of positive cells/(the number of positive cells+the number of negative cells)×100%.

Transwell assay

A Transwell chamber (diameter=8 mm; Corning, Corning Glass Works, Corning, N.Y., USA) was used for the detection of cell migration and invasion in a 24-well plate. In the polycarbonate membrane Transwell chamber with or without Matrigel, 600 ml of RPMI 1640 medium containing 20% FBS was added to the basolateral chamber and then balanced for 1 h at 37 °C. After 48 h of transfection, ovarian cancer cells were resuspended in FBS-free RPMI 1640 medium while 1×10⁶/mL cells were seeded in the apical chamber and cultured with 5% CO₂ for 24 h at 37 °C. Subsequently, the Transwell chamber was fixed with 5% glutaraldehyde at 4 °C, and stained with 0.1% crystal violet for 5 min. Lastly, the cells were observed under an inverted fluorescence microscope (TE2000, Nikon, Tokyo, Japan) and photographed in 5 randomly-selected visual fields and the average value considered as the number of cells passed through the chamber.

HUVEC treatment

HUVECs were cultured in a 24-well plate at a density of 1×10⁵ cells/well, followed by treatment with ovarian cancer cell supernatant co-cultured with MSC-derived EVs. After 24 h of treatment, HUVECs were isolated. Next, the HUVECs were respectively treated with MSC-derived EVs, EVs-depleted MSCs, and MSC-derived EVs carrying miR-424 mimic or miR-424 inhibitor as well as their NCs. Subsequently, the expression patterns of VEGF and VEGFR were determined using RT-qPCR and Western blot analysis.

Matrigel-based tube formation assay

Matrigel (356,234, Shanghai Shanran Biotechnology Co., Ltd., Hangzhou, China) was placed in a refrigerator at 4 °C overnight to thaw into a yellow-colloidal liquid. Next, 70 μL of this yellow-colloidal liquid (0.5 mmol/L in thickness) was collected using pre-cooled pipettes and immediately added to a pre-cooled 96-well plate. Afterwards, the cell culture plates with Matrigel were placed in an incubator for 30 min at 37 °C for solidification. After 48 h of transfection, cells with different treatments were collected, and then treated with serum-free starvation for 1 h and resuspended in DMEM to make a cell suspension. Thereafter, the cell suspension (1×10⁵ cells/mL) was seeded in culture wells and added with the corresponding medium, and 3 replicates were set for each group. Subsequently, the cell culture plate was placed in an incubator for 18 h and photographed under a Leica inverted phase-contrast microscope (Leica Microsystems GmbH, Wetzlar, Germany). The Image-Pro Plus software (version 6.0) was employed to calculate the number of a complete capillary tube surrounded by cells, with at least 3 visual fields counted.

Tumor xenograft in nude mice

A total of 60 immunodeficient female nude mice (aged 3—5 weeks, weighing 18–21 g) were procured from the Shanghai laboratory animal center, Chinese Academy of Sciences (Shanghai, China). The obtained mice were maintained under specific pathogen-free conditions with regular ultraviolet radiation. The cage, padding, water, and feed for mice were sterilized and the mice were kept at the temperature of 24–26 °C with a relative humidity of 40–60%. Subsequently, the cultured SKOV-3 cells were transfected with lv-NC (lentivirus over-expressing miR-424 NC) and lv-miR-424 mimic (lentivirus over-expressing miR-424) to obtain stably-transfected cell lines. The lentivirus vector and reagents used for aforementioned transfection were purchased from GenePharma. In addition, the cells were also treated with EVs-mimic NC and EVs-miR-424 mimics.

The stably-expressed ovarian cancer cells at the logarithmic phase of growth were collected, and PBS was used to adjust the concentration of cell suspension to 5×10⁶ cells/mL. One week later, the cells treated with lv-NC and lv-miR-424 mimic were injected into the mice (n=15) via the abdomen (every 0.2 mL cell suspension contained 1×10⁶ cells), with 200 μL cell suspension injected every day. Meanwhile, the cells treated with EVs-mimic NC and EVs-miR-424 mimic (60 μg/mL) were injected into mice (n=15) via the tail vein. After injection for 4 consecutive weeks, all mice were euthanized and dissected, and the tumors were removed. By accurately measuring the short diameter (a) and long diameter (b) of tumors, the tumor volume was calculated based on the following formula: π(a²b)/6, and the tumors were also weighed. Additionally, when the mice were euthanized, peripheral blood samples were collected to prepare serum, and then serum EVs were extracted to detect the miR-424 expressions using RT-qPCR. Tumor tissues were fixed by 10% formaldehyde, conventionally dehydrated, paraffin-embedded, and cut into 4-μm sections for subsequent use. The protein expression patterns of MYB and VEGF were detected by Western blot analysis.

Measurement of microvessel density (MVD)

The MVD in tumor tissues of nude mice was measured using the detection method of immunohistochemistry and the primary antibody rabbit anti-mouse MVD was purchased from Santa Cruz Biotechnology (sc-376975). The criteria for MVD results were as follows: using the Weidner microvessel count method, in endometrial tissues, any brown-yellow stained cells or cell clusters with clear boundary with adjacent microvasculature, and gland tissues were considered as a new blood vessel, while the branches were also regarded as a blood vessel as long as the structure was not connected. However, the formation of tube structure was not necessary for analysis, and therefore we did not count the cells with a large tube and a thick muscular layer or with more than 8 red blood cells in the tube. Meanwhile, the brown-yellow stained vessels were selected and counted with 5 visual fields were randomly selected for each pathological section to calculate the average value, which was then used as MVD. Moreover, MVD was considered as positive when the average value was≥MVD threshold and as negative when the average value was<MVD threshold (MVD threshold was set based on the Weidner criteria) [20].

miR-424 is poorly-expressed in ovarian cancer and targets MYB

Immunohistochemistry was employed to detect the positive expressions of MYB in ovarian cancer, and it was found that the positive expression of MYB was brown-yellow in color and located in the nucleus (Fig. 2a). In comparison with the adjacent ovarian epithelium tissues, ovarian cancer tissues presented with higher positive expression of MYB (p<0.05) (Fig. 2b, c). Next, RT-qPCR was conducted to examine the expression patterns of MYB in normal human ovarian epithelial cell line HOSEpiC and ovarian cancer cell lines (SKOV-3, HO8910, and A2780), which revealed higher expression of MYB in SKOV-3, HO8910, and A2780 compared to that in HOSEpiC, and demonstrated the largest FC in SKOV-3 (p<0.05) (Fig. 2d). These results demonstrated that MYB was highly-expressed in ovarian cancer tissues and cells. Meanwhile, Kaplan–Meier survival analysis (http://kmplot.com/analysis/index.php?p=service&cancer=gastric) (Fig. 2e) indicated that the ovarian cancer patients presenting with higher MYB expression had a lower overall survival rate compared to those with lower MYB expression levels.

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Fig. 2

miR-424 is downregulated in ovarian cancer and specifically binds to MYB. a The expression of MYB in ovarian cancer tissues and adjacent ovarian epithelium tissues examined by immunohistochemistry (scale bar=25 μm), adjacent ovarian epithelium tissues n=43, ovarian cancer tissues n=85. b The mRNA expression of MYB in ovarian cancer tissues and adjacent ovarian epithelium tissues detected by RT-qPCR; *p<0.05. c The protein expression of MYB in ovarian cancer tissues and adjacent ovarian epithelium tissues examined by Western blot analysis. d The expression of MYB in normal human ovarian epithelial cell line HOSEpiC and ovarian cancer cell lines (SKOV-3, HO8910, A2780) detected by RT-qPCR. e The survival rate of ovarian cancer patients with higher MYB expression analyzed by Kaplan–Meier survival analysis. f The expression of miR-424 in ovarian cancer tissues and adjacent ovarian epithelium tissues detected by RT-qPCR; *p<0.05. g The correlation between MYB and miR-424. h The binding site between miR-424 and MYB predicted using an online data base. i The fluorescence activity determined by dual-luciferase reporter gene assay. j The mRNA expression of MYB in cells after treatments with miR-424 mimic or inhibitor detected by RT-qPCR. k The protein expression of MYB in cells after treatments with miR-424 mimic or inhibitor examined by Western blot analysis; *p<0.05 compared with cells treated with mimic-NC, ^#p<0.05 compared with cells treated with inhibitor-NC. All data above were measurement data and were presented as mean±standard deviation. A comparison between tumor tissues and adjacent ovarian epithelium tissues was conducted using a paired t-test, and data comparison between two groups was performed using the unpaired t-test. Data among multiple groups were compared by one-way ANOVA, and Pearson correlation analysis was used to analyze the correlation between miR-424 and MYB. All experiments were repeated 3 times independently

RT-qPCR was further performed to detect the expression patterns of miR-424 in ovarian cancer tissues, and miR-424 was found to be significantly down-regulated in ovarian cancer tissues relative to adjacent ovarian epithelium tissues (Fig. 2f). By correlation analysis, a negative correlation between MYB and miR-424 was found (Fig. 2g). Further application of the online analysis software indicated the presence of a specific binding region between the MYB gene sequence and miR-424 sequence (Fig. 2h). Subsequently, dual-luciferase reporter gene assay verified that MYB was indeed the target of miR-424. As shown in Fig. 2i, the fluorescence activity was significantly reduced after co-transfection of miR-424 mimic and MYB 3′UTR-WT compared with co-transfection of mimic-NC and MYB 3′UTR-WT (p<0.05), while the fluorescence activity exhibited no pronounced differences following co-transfection of miR-424 mimic and MYB 3′UTR-MUT in comparison with the co-transfection of mimic-NC and MYB 3′UTR-WT (p>0.05), suggesting that miR-424 specifically bound to MYB. Furthermore, SKOV-3 cells were treated with miR-424 mimic or inhibitor followed by RT-qPCR and Western blot analysis to examine the MYB expression in SKOV-3 cells. It was found that in comparison with cells treated with mimic-NC, the cells treated with miR-424 mimic presented with significantly reduced mRNA expressions of MYB (Fig. 2j) and protein expressions of MYB (Fig. 2k), while the cells treated with miR-424 inhibitor exhibited markedly increased mRNA expressions of MYB (Fig. 2j) and protein expressions of MYB (Fig. 2k) relative to the cells treated with inhibitor-NC (all p<0.05). The above-reported results suggested that miR-424 regulated the expression of MYB, and augmented miR-424 suppressed the MYB expression.

miR-424 inhibits the cell proliferation, migration and invasion of ovarian cancer by suppressing MYB

Subsequently, SKOV-3 cells were treated with interfered MYB to examine changes in MYB expression. As depicted in Fig. 3a, b, the sh-MYB treatment led to significantly reduced expressions of MYB in ovarian cancer cells, while miR-424 inhibitor transfection brought about the opposite results. In addition, EdU assay was applied to detect the proliferation ability of SKOV-3 cells (Fig. 3c), which revealed that cells delivered with miR-424 inhibitor presented with markedly increased red-positive proliferation, while the cells treated with both miR-424 mimic and sh-MYB showed a significant decrease in red-positive proliferation (p<0.05). Meanwhile, the cells delivered with both miR-424 inhibitor and sh-MYB exhibited no pronounced differences in cell proliferation. Transwell assay was then performed to detect the migration and invasion of SKOV-3 cells (Fig. 3d, e), and it was found that cells delivered with miR-424 mimic and with sh-MYB presented with repressed migration and invasion abilities, whereas the cells delivered with miR-424 inhibitor exhibited the opposite results (p<0.05). However, there was no difference in migration and invasion abilities of cells delivered with both miR-424 inhibitor and sh-MYB. Lastly, Western blot analysis was conducted to detect the protein expression patterns of tube formation factors VEGF and VEGFR. The obtained results in Fig. 3f depicted that the protein expression of VEGF and VEGFR was both significantly suppressed after treatment with miR-424 mimic and with sh-MYB, however, an elevation in the protein expression of VEGF and VEGFR was observed following treatment with miR-424 inhibitor (p<0.05). Overall, these results demonstrated that miR-424 suppressed the proliferation, migration, and invasion of ovarian cancer cells by inhibiting MYB, and miR-424 reduced the expressions of VEGF and VEGFR.

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Fig. 3

The proliferation, migration, and invasion of ovarian cancer cells are repressed by miR-424 via inhibition of MYB. The cells used for the following detections were treated with sh-NC, sh-MYB, miR-424 mimic or inhibitor as well as their NCs, and both miR-424 inhibitor and sh-MYB. a The expression of miR-424 and MYB in SKOV-3 cells after different treatments detected by RT-qPCR. b The protein expression of MYB in cells after different treatments examined by Western blot analysis. c The proliferation of SKOV-3 cells after different treatments determined by EdU assay. d The migration of SKOV-3 cells after different treatments detected by Transwell assay. e The invasion of SKOV-3 cells after different treatments examined by Transwell assay. f The protein expression of VEGF and VEGFR in cells after different treatments detected by Western blot analysis, with protein bands assessed; *p<0.05 compared with cells treated with mimic-NC, ^#p<0.05 compared with cells treated with inhibitor-NC; ^&p<0.05 compared with cells treated with inhibitor NC. The data above were measurement data and were presented as sample mean±standard deviation. Data among multiple groups were compared by one-way ANOVA, with Tukey’s post hoc test conducted. Data in each group at different time points were compared by repeated-measures one-way ANOVA. The cell experiment was repeated 3 times independently

The MSC-derived EVs are successfully isolated and identified

The effects of MSCs and MSC-derived EVs in various cancers have been well established, and the current study set out to expand on the role of MSC-derived EVs in ovarian cancer. Firstly, we isolated and seeded MSCs. After 3 days of culture, cells started to be adherent, presented with spindle-like shape and grew in a whirl-shape or cluster, while the nucleus was visible, overall, exhibiting the typical characteristics of MSCs (Additional file 3: Fig. S3A). Thereafter, flow cytometry was applied to analyze the expression patterns of MSC surface antigens, and the expressions of CD73 (91.56%), CD90 (88.93%), CD105 (86.55%), CD34 (21.57%), CD45 (19.63%), CD19 (25.48%), and HLA-DR (27.52%) were found in Additional file 3: Fig. S3B, which conformed to the biological characteristics of MSCs. Moreover, the results of MSCs osteogenic/adipogenic induction were depicted in Additional file 3: Fig. S3C. In addition, after 21 days of osteogenic induction, the cells were noted to be arranged in overlapping layers and presenting with calcified nodules containing a small amount of mineral salt deposition, highlighting the potential of osteogenesis differentiation in cells (Additional file 3: Fig. S3C-a). Moreover, after 25 days of adipogenic induction, significant lipid deposition was observed in the cells, while lipid droplets exhibited larger size and presented as beads-shape, which suggested the potential of adipogenesis differentiation of these cells (Additional file 3: Fig. S3C-b).

Furthermore, MSC-derived EVs were observed using TEM with the diameter of EVs measured by dynamic light scattering (DLS), and it was found that EVs (with size from 30 to 120 nm) presented a round or elliptic membranous vesicle-shapes (Additional file 3: Fig. S3D). Lastly, Western blot analysis was conducted to examine the expression patterns of CD63 and verified the successful extraction of EVs (p<0.05) (Additional file 3: Fig. S3E). These results suggested the successful extraction of EVs from MSCs.

miR-424 is delivered from MSCs to ovarian cancer cells via EVs

RT-qPCR was performed to detect the expression patterns of miR-424 in MSCs and MSC-derived EVs, and the results indicated that compared with MSCs treated with miR-NC, higher expression of miR-424 was noted in MSCs and MSC-derived EVs after treatment with miR-424 mimic, while lower miR-424 expression was found in MSCs and MSC-derived EVs after treatment with miR-424 inhibitor (p<0.05) (Fig. 4a).

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Fig. 4

miR-424 is transferred from MSCs to ovarian cancer cells by EVs. a The expression of miR-424 in MSCs and MSC-derived EVs after treatments with miR-424 mimic and inhibitor as well as their NCs. b The uptake of EVs by ovarian cancer cells observed under a confocal fluorescence microscope (scale bar=50 μm). The ovarian cancer cells SKOV-3 used for the following detections were co-cultured with MSCs, EVs-depleted MSCs, and MSCs treated with miR-424 mimic or miR-424 inhibitor as well as their NCs. c The expression of miR-424 and MYB in ovarian cancer cells detected by RT-qPCR. d The protein expression of MYB in ovarian cancer cells examined by Western blot analysis, with protein bands assessed. *p<0.05 compared with MSCs, ^#p<0.05 compared with mimic NC and MSCs mimic NC treatments; & p<0.05 compared with inhibitor NC and MSCs inhibitor NC treatments. The data above were measurement data and were presented as sample mean±standard deviation. Data among multiple groups were compared by one-way ANOVA. The cell experiment was repeated 3 times independently

Subsequently, PKH67-labeled EVs were co-cultured with SKOV-3 cells for 48 h, and the uptake of EVs by SKOV-3 cells was assessed using confocal fluorescence microscopy. The results in Fig. 4b showed that after 48 h of co-culture, there was obvious uptake of PKH67-labeled EVs by SKOV-3 cells, suggesting that EVs could be internalized by ovarian cancer cells SKOV-3.

Furthermore, to determine the effective regulation of MYB expression by MSC-derived EVs carrying miR-424 in SKOV-3, RT-qPCR and Western blot analysis were conducted to examine the expression patterns of miR-424 and MYB in SKOV-3 co-cultured with MSC-derived EVs, respectively. As indicated in Fig. 4c, d, in comparison with MSCs, the MSCs treated with depleted EVs exhibited no significant differences in terms of MYB expression, while the MSCs treated with miR-424 mimic presented with significantly reduced expressions of MYB (p<0.05), suggesting that miR-424 effectively suppressed the MYB expression in SKOV-3 cells in vitro. These results speculated the vital role of EVs transportation as MSCs transferred miR-424 to SKOV-3 cells through EVs.

MSC-derived EVs over-expressing miR-424 suppresses the proliferation, migration and tube formation of HUVECs

HUVECs were treated with the supernatant of ovarian cancer cells co-cultured with MSC-derived EVs and followed by a series of experiments to detect the proliferation, migration, and tube formation of HUVECs. The results of the EdU assay (Fig. 5a) demonstrated that MSC-derived EVs-miR-424 mimic treatment-induced a pronounced decline in the proliferation of HUVECs, while MSC-derived EVs-miR-424 inhibitor treatment significantly enhanced the proliferation of HUVECs (p<0.05). Meanwhile, the results of the Transwell assay (Fig. 5b) showed that migration of HUVECs was suppressed by MSC-derived EVs-miR-424 mimic treatment, while being promoted following treatment with MSC-derived EVs-miR-424 inhibitor (p<0.05). Besides, the results of Matrigel-based tube formation assay (Fig. 5c) demonstrated the enhanced tube formation of HUVECs after treatment with MSC-derived EVs-miR-424 inhibitor (p<0.05). Furthermore, Western blot analysis was conducted to examine the protein expression patterns of tube formation factors VEGF and VEGFR. As revealed in Fig. 5d, the protein expression of VEGF and VEGFR was found to be repressed by treatment with MSC-derived EVs-miR-4242 mimic, while the opposite results were noted after MSC-derived EVs-miR-424 inhibitor treatment (p<0.05). These findings indicated that EVs-packaged miR-424 derived from MSCs repressed the proliferation, migration, and tube formation of HUVECs and inhibited the expression of VEGF and VEGFR.

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Fig. 5

miR-424 from MSC-derived EVs inhibits the proliferation, migration and tube formation of HUVECs. The HUVECs used for the following detections were treated with MSC-derived EVs, EVs-depleted MSCs, MSC-derived EVs after treatments of miR-424 mimic or miR-424 inhibitor as well as their NCs. a The proliferation of HUVECs after different treatments detected by EdU assay. b The migration of HUVECs after different treatments examined by Transwell assay. c The tube formation of HUVECs after different treatments determined by Matrige-based tube formation assay. d The protein expression of VEGF and VEGFR after different treatments detected by Western blot analysis. *p<0.05 compared with HUVECs treated with MSC-derived EVs, ^#p<0.05 compared with HUVECs treated with MSC-derived EVs-mimic-NC; ^&p<0.05 compared with HUVECs treated with MSC-derived EVs-inhibitor-NC. The data above were measurement data and were presented as sample mean±standard deviation. Data among multiple groups were compared by one-way ANOVA. The cell experiment was repeated 3 times independently

We acknowledge and appreciate our colleagues for their valuable efforts and comments on this paper.