Exosomes deliver miR-145 to regulate epithelial ovarian cancer cell metastasis and cancer stem cell-like characteristics

Wenjing Zhang,Jing Wang,Yujin Zhang,Minglian Yu,Hui Wang

Abstract Objective:To investigate the effects of epithelial ovarian cancer (EOC) cell-secreted exosomal miR-145 on the proliferation,migration and invasion abilities and stem celllike characteristics of EOC cells.Methods:Ovarian cancer SKOV3 cells were transfected with miR-145 mimic,miR-145 inhibitor,or miR-145 negative control (NC).Exosomes (Exo)derived from SKOV3 cells and miR-145-overexpressing or miR-145-downregulated SKOV3 cells were isolated and identified.The expression of miR-145 was assessed by RT-qPCR.SKOV3 cells were co-cultured with exosomes from different sources.Cell proliferation,migration and invasion were detected by MTT assay and Transwell assay.The impact of exosomal miR-145 on angiogenic tube formation ability of endothelial cells was tested by tube formation assay.The stem cell characteristics of CD133+/CD44+ cells sorted from SKOV3 cell line were evaluated by tumor sphere formation assay.Results:miR-145 expression was significantly upregulated in SKOV3 cells treated with miR-145 mimic-Exo and downregulated in cells treated with miR-145 inhibitor-Exo.Exo derived from ovarian cancer cells favored the proliferation,migration and invasion of SKOV3 cells (P＜0.05).High levels of exosomal miR-145 decreased the proliferation,migration and proliferation of SKOV3 cells,inhibited angiogenesis in human umbilical vein endothelial cells,and suppressed tumor sphere forming capacity of SKVO3 cells(P＜0.05).However,miR-145-downregula-ted exosomes had opposite effects (P＜0.05).Conclusion:The transfer of miR-145 through exosomes can inhibit the migration,invasion and angiogenesis of epithelial ovarian cancer cells,and regulate the stem cell-like characteristics of epithelial ovarian cancer.

Keywords epithelial ovarian cancer;exosome;miR-145;tumor stem cells

Introduction

Ovarian cancer seriously threats the health of women around the world,accounting for about 5% of all female cancer deaths.Epithelial ovarian cancer (EOC),as the most common type of all ovarian cancer,accounts for 90%of all ovarian cancer cases.Due to the lack of obvious early symptoms and accurate biomarkers,most EOC patients have been in late stage when they were diagnosed,and the prognosis is poor.The overall 5-year survival rate of patients is less than 30% [1-2].At present,the main treatment of EOC is surgery and adjuvant platinum chemotherapy,while recurrent EOC can not be cured [3],so there is an urgent need to develop new strategies and explore new methods to improve the clinical outcome of EOC patients.

Exosomes (Exo) are nanoscale extracellular vesicles with a size of 30-150 nm,which can be secreted by almost all types of cells under physiological or pathological conditions.Exosomes mediate communication between donor and recipient cells by enriching and delivering nucleic acids,proteins,metabolites or lipids from derived cells[4].microRNA(miRNA)is a small non-coding RNA that inhibits gene expression by binding to the 3' untranslated region of the target mRNA.Recent studies have shown that exosome-derived miRNA is an important mediator of cancer-host crosstalk.Exosome miRNA from tumor cells can regulate cell function and phenotype by promoting crosstalk between various cells in tumor microenvironment[5-6].Exosomes are not only structurally stable,but also protect their contents from degradation,and have the advantages of non-cytotoxicity,low immunogenicity and high biocompatibility.Therefore,exosomes are considered as a new ideal drug delivery carrier.

The purpose of this study was to explore the effect of exosomal miR-145 on the proliferation,migration and invasion of EOC cells,and the stem cell-like characteristics of EOC,in order to provide novel and potential therapeutic targets and tools for the treatment of EOC.

Materials and methods

Cells and reagents

EOC cell line SKOV3 and human umbilical vein endothelial cell(HUVECs)were purchased from Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.miR-145 mimic,miR-145 inhibitor and miR-145 NC sequences were synthesized and constructed by Shanghai GenePharma Co.,Ltd.Fetal bovine serum,penicillin,streptomycin and RPMI-1640 medium were from Gbico Company.Insulin,B27,human basic fibroblast growth factor(bFGF)and epidermal growth factor (EGF) were purchased from Sigma Company.HiPerFect trasfection reagent kit was purchased from Qiagen.Exosome extraction kit was purchased from Shanghai Bestbio Co.,Ltd.Mi Pure Cell/Tissue miRNA kit was purchased from Nanjing Vazyme Biotech Co.,Ltd.Trizol,reverse transcription kit and real-time PCR kit were from Takara.MTT kit was from Sigma-Aldrich.Transwell chamber was from Pierce.Matrigel glue was purchased from BD,and antibodies CD9,Alix and TSG101 were purchased from Wuhan BOSTER Biological Technology Co.,Ltd.

Cell culture and transfection

SKOV3 cells were resuscitated in water bath and cultured in RPMI-1640 medium containing 10%fetal bovine serum and 1% penicillin/streptomycin at 37 ℃with 5% CO2.The cells in logarithmic phase were selected and inoculated in 24-well plate with 1×105cells per well.According to HiPerFect transfection reagent instructions,miR-145 mimic,miR-145 inhibitor,or miR-145 NC were transfected into the SKOV3 cells,and the normal cultured and untransfected SKOV3 cells were used as a control group.After transfection,the cells were cultured for 48 h for follow-up study.

Exosomes extraction

After the transfected SKOV3 cells were cultured at 37 ℃and 5% CO2for 48 h,the supernatant was kept after centrifuging for 15 min,and the supernatant was centrifuged at 2,000 r/min for 20 min at 4 ℃.The supernatant was transferred to a new centrifugation tube,and the exosome extraction reagent was added to mix evenly.On the next day,the mixture was centrifuged at 3,000 r/min at 4 ℃for 30 min,the supernatant was discarded and the precipitation was redissolved;then centrifuged at 10,000 r/min for 10 min at 4 ℃;PBS was added to the precipitate,centrifuged again for 10 min,and the exosome was obtained by re-suspension precipitation with exosome preservation solution.

Transmission electron microscope

The exosome samples extracted from 50 μL were added to the copper mesh with 200 meshes,incubated at room temperature for 5 min.The excess liquid at the edge of the copper mesh was absorbed with filter paper;the sample was negatively stained with 1% phospho-tungstic acid for 1 min;the copper mesh was washed with distilled water for 2 times,3 min each time;and then the excess liquid was absorbed by filter paper and irradiated with incandescent lamp for 8 min.After it was fully dried,the morphology of particles in the copper mesh was observed by transmission electron microscope.

Exosome size analysis

The exosome sample was diluted with PBS,and the concentration was adjusted to 3×107particles/mL.The size of exosomes was detected by nanoparticle tracking analysis (NTA),using ZetaView PMX 110(Particle Metrix,Meerbusch,Germany).

Western blotting

The total protein of exosome was extracted by RIPA lysate.The supernatant obtained after centrifuging at 12,000 r/min for 30 min.The protein concentration was determined by BCA.A 10% SDS-PAGE gel was prepared,and the protein was electrophoresed.The separated protein was transferred to PVDF membrane.After blocking for 1 h at room temperature,primary antibodies as CD9,Alix,and TSG101 were incubated overnight at 4 ℃.Corresponding secondary antibody was incubated at room temperature for 2 h,and TBST was used to wash the membrane for three times.After developing color with ECL reagent,image J analyzed the expressions of exosome markers CD9,Alix and TSG101.

RT-qPCR

The total RNA of transfected cells was extracted by Trizol,and the total RNA was extracted from exosomes by exosome RNA purification kit.The total RNA extracted was reverse transcribed by cDNA first strand synthesis kit.Using cDNA as template,the expression level of miR-145 was detected by RT-qPCR,and U6 was used as the internal reference gene.The conditions of quantitative amplification were as follows:95 ℃3 min;95 ℃12 s,62 ℃40 s,95 ℃15 s(45 cycles).The sequence of primers was as follows:miR-145 upstream primer:5'-GTCCAGTTTTCCCAGG-3',downstream primers:5'-GAGCAGGCTGGAGAA-3';U6 upstream primer:5'-CTCGCTTCGGCAGCACA-3',downstream primers:5'-AACGCTTCACGAATTTGCGT-3'.The experiment was repeated for 3 times,and the expression level of miR-145 was calculated by 2-△△Ct.

Experiment grouping

The density of SKOV3 cells was adjusted and inoculated into 96-well plates with 1×105cells per well.The subsequent experiments were divided into following groups:(1) control group:normal cultured SKOV3 cells;(2) Exo group:adding 10 μg/mL SKOV3 cell-derived Exo medium to culture SKOV3 cells;(3) miR-145 NC-Exo group:adding 10 μg/mL transfected miR-145 NC of SKOV3 cell-derived Exo medium to culture SKOV3 cells;(4) miR-145 mimic-Exo group:SKOV3 cells were cultured with exo-derived from SKOV3 cells transfected with 10 μg/mL miR-145 mimic;(5) miR-145 inhibitor-Exo group:Exo-derived from SKOV3 cells transfected with 10 μg/mL miR-145 inhibitor were added to culture SKOV3 cells.The cells of each treatment group were cultured at 37 ℃and 5% CO2for 48 h,and the cells were collected for follow-up experiment.

Detection of cell proliferation by MTT method

After the SKOV3 cells were treated with exosomes for 24 h,48 h and 72 h,500 μL MTT reagent was added into each well and mixed evenly.After being cultured at 37 ℃and 5% CO2for 4 h,the culture medium was discarded and 600 μL DMSO reagent was added.The shaking table was shaken until the crystal was completely dissolved.The absorbance at 490 nm was detected by enzyme labeling instrument.

Detection of cell invasion and migration by Tran⁃swell assay

A proper amount of diluted Matrigel glue was added to the bottom of the upper chamber of 24-hole Transwell and placed at 37 ℃until it was solidified to completely cover the bottom.The density of SKOV3 cells treated by exosomes was adjusted to 2×104cells/mL,200 μL was added to the bottom of upper chamber coated with Matrigel glue,and 600 μL fresh culture medium containing 10% fetal bovine serum was added in the lower chamber.After being cultured at 37 ℃and 5% CO2for 24 h,the culture medium was discarded,the upper chamber cells were removed.4%paraformaldehyde was added and fixed for 10 min,0.1% crystal violet was added for 30 min,the dye solution was recovered,cleaned by PBS,and placed on glass slides.The images were observed and photographed under light microscope,and the stained transmembrane cells were counted.The cell migration was also detected by Transwell chamber test,and all the steps were consistent with the above operations except Matrigel glue.

Tube formation assay

The HUVECs (2×105cells/mL) were inoculated into the 24-well plate with Matrigel,and cultivated with exosomes from different sources.24 h after incubation,tube formation was assessed under inverted microscope.

Magnetic separation of CD133+/CD44+cells

SKOV3 cells in logarithmic growth phase were washed by PBS and added magnetic bead reagent buffer.The cell density was adjusted to 1×108cells/mL.300 μL cell suspension was labeled with CD133 and CD44 magnetic beads and incubated on ice for 30 min in the dark.After buffer washing and centrifugation,the cells were resuscitated with buffer,sorted by magnetic beads sorting,and CD133+/CD44+cells were sorted by flow cytometry.

Tumor sphere formation assay

Cells were placed onto a 96-well plate at a density of 1×103cells/well in serum-free medium supplemented with 4 mg/mL insulin,2% B27,10 ng/mL EGF,and 10 ng/mL bFGF.Exosomes from different sources were added.After culture for 7 days,the numbers of tumor spheres formation were counted using an inverted microscope.

Statistical analysis

The measurement data of this study were expressed by mean±standard deviation(SD).The data were analyzed by SPSS 23.0 and the statistical chart was drawn by Graphpad prism 8.3 software.ANOVA analysis of variance was used to compare the data between various groups,and LSD-ttest was used to compare the pairwise data between groups,P＜0.05.

Results

Detection of miR-145 expression in SKOV3 cells after transfection

As shown in Figure 1,miR-145 level was greatly increased by miR-145 mimic but decreased by miR-145 inhibitor,which indicated that transfection was effective(P＜0.05).

Characterization of exosomes and miR-145 ex⁃pression in exosomes

The exosome preparation was confirmed to contain round vesicles with a diameter rang of 30-110 nm,and the peak particle size was 50 nm,which accorded with the structural characteristics of exosomes (Figure 2A-2B).As shown in Figure 2C,exosome markers CD9,Alix and TSG101 were abundant in our exosome preparations.Compared with exosomes derived from transfected miR-145 NC cells,the miR-145 level was significantly increased in exosomes derived from transfected miR-145 mimic cells whereas decreased in exosomes derived from transfected miR-145 inhibitor cells(Figure 2D).

Figure 1 The miR-145 expression in SKOV3 cells of each group.*P＜0.05 vs. Control group;#P＜0.05 vs. miR-145 NC group.

Exosomal miR-145 inhibits the proliferation,mi⁃gration and invasion of EOC cells

The proliferative activity of SKOV3 cells and the number of migrated and invasive cells in Exo group were significantly higher than those in the control group (P＜0.05).Compared with miR-145 NC-Exo group,the cell proliferation,migration and invasion decreased markedly in miR-145 mimic-Exo group while increased in miR-145 inhibitor-Exo group (P＜0.05)(Figure 3).

Exosomal miR-145 inhibits angiogenesis of HU⁃VECs

Thein vitroendothelial tube formation assay showed that exosomes from SKOV3 cells induced tube formation of HUVECs compared with the control group(P＜0.05).Treatment with miR-145-overexpressing exosomes led to a significant decrease in HUVEC tube-like structure formation,compared to exosomes derived from SKOV3 cells transefected with miR-145 NC(P＜0.05).miR-145-downregulated exosomes had an opposite effect on HUVECs(P＜0.05)(Figure 4).

Figure 2 Characterization of exosomes and expression of miR-145.A:The morphology of exosome-like vesicles was observed under transmission electron microscopy.B:The diameter and concentration of SKOV3 cells-derived exosomes were determined by Nanoparticle tracking analysis.C:The protein expressions of CD9,Alix and TSG101 in exosome-like vesicles were detected by Western blotting.D:The expression of miR-145 in exosomes derived from SKOV3 cells in different groups.#P＜0.05 vs. miR-145 NC-Exo group.

Figure 3 The proliferation,migration and invasion of SKOV3 cells in different groups.A-C:Transwell assays were used to evaluate the migration and invasion of SKOV3 cells (Scale bars,100 μm).D:MTT method was used to assess the proliferation of SKOV3 cells.*P＜0.05 vs.Control group;#P＜0.05 vs.miR-145 NC-Exo group.

Figure 4 Evaluation of HUVECs tube formation after treated with different sources of exosomes(Scale bars,200 μm).*P＜0.05 vs.Control group;#P＜0.05 vs.miR-145 NC-Exo group.

Exosomal miR-145 reduces stem cell-like proper⁃ties of EOC cells

The proportion of CD133+/CD44+cells in the SKOV3 cell line before sorting was only 0.3% and that increased to 83% after sorting.Tumor sphere formation assay indicated that exosomes derived from SKOV3 cells or the cells transfected with mimic NC could significantly increase the tumor sphere forming number compared with control (P＜0.05).Exosomes from overexpressed miR-145 cells formed fewer and smaller spheres compared with exosomes from cells transfected with control mimic(P＜0.05).On the contrary,exosomes with knocked down miR-145 increased tumor sphere formation (P＜0.05).These results suggest that miR-145-overexpressing exosomes can attenuate stem cell-like properties of EOC cells(Figure 5).

Figure 5 Detection of number of tumor spheres formed by EOC cells(Scale bars,50 μm).*P＜0.05 vs.Control group;#P＜0.05 vs.miR-145 NC-Exo group.

Discussion

In recent years,the incidence of ovarian cancer globally continues to increase.EOC,as the fifth leading cause of cancer death in women,is also one of the gynecological malignant tumors with high clinical fatality.Platinum chemotherapeutic drugs and cytoreductive surgery are often used to treat EOC clinically.Although most patients have a high response rate after the first chemotherapy,most patients with advanced EOC will eventually develop drug resistance after chemotherapy and a series of adverse side effects [2,7].In addition,cancer cell metastasis is also an important clinical issue,EOC cells spread outside the ovary,aggravating the pathological process,resulting in death of patients [8].Therefore,elucidating the relevant mechanism of EOC and finding its potential therapeutic targets are of great significance to improve the prognosis of patients with the disease.

Exosomes are extracellular vesicles with important biological activities,and their role in cancer has been widely reported.Exosomes can mediate communication between tumor cells by transmitting active components such as non-coding RNA,proteins and peptides,and exosomes play their corresponding biological functions after uptake.miRNA,as a non-coding RNA involved in the regulation of tumor process,can be secreted into extracellular space,and play a role in intercellular communication as the main exosome forms.At present,some exocrine-transmitted miRNA have been identified as ideal biomarkers for the diagnosis and treatment of ovarian cancer.Lu et al]9]showed that exosome-mediated miR-34b decreased the proliferation of EOC cell line SKOV3 and inhibited the process of epithelial-mesenchymal transformation.Chen et al]10]proved that hypoxia can induce the high expression of miR-940 in exosomes derived from EOC,while the overexpression of miR-940 delivered by exosomes stimulates the phenotypic polarization of M2 macrophages,which further promotes the proliferation and migration of EOC cells.Li et al]11]found that the expression of miR-429 in multidrug resistant SKOV3 cells and cells-secreted exosomes was higher compared with sensitive A2780 cells and the cells-secreted exosomes.The exosomes derived from SKOV3 were internalized by A2780 cells and transmitted miR-429,which enhanced the proliferation and drug resistance of A2780 cells by targeting CASR/STAT3 pathway.miR-145 participates in a variety of biological processes of cancer by regulating target genes or signal transduction,including tumorigenesis,differentiation,metastasis,angiogenesis and drug resistance [12-13],so it has attracted much attention in tumor research.Garrido et al]14]reported that the overexpression of miR-145 decreased the proliferation,migration and invasion of EOC cells.Li et al]15]also showed that the expression of miR-145 decreased in ovarian cancer tissues,and the increase of miR-145 could inhibit the proliferation and migration of EOC cells.It is speculated that miR-145 may play a role of tumor suppressor in EOC.

Tumor metastasis is a complex multi-step process in which tumor cells spread from primary tumors and form new tumor tissues in different parts and organs of the body,which is regulated by specific genes and signal pathways.EOC cells mainly transfer through the body cavity pathway,spread from the primary EOC tumor,and float freely in the form of globules in the ascites in the abdominal cavity,and then the metastatic cells attach to the mesothelial lining or go deep into the peritoneal organs [16].In addition,metastatic EOC cells can metastasize and extravasate in blood vessels or lymphatic vessels,thus establishing new tumor tissues[17].Deaths from EOC metastases to secondary sites account for about 90%of all ovarian cancer deaths.Thus,it is necessary to explore the potential molecular targets and mechanisms that inhibit the migration and invasion of EOC cells.The results of this study show that after SKOV3 cells were treated with high level of exosomal miR-145,the proliferation,migration and invasion of SKOV3 cells,and the tube formation of HUVECs decreased significantly.The results further suggest that exosomal miR-145 can inhibit the progression of EOC.

Cancer stem cells(CSCs)are cell subgroup that affect the progression of EOC and have strong ability of self-renewal and differentiation.CSCs promote tumor progression,chemotherapy resistance and disease recurrence through their continuous proliferation,invasion of normal tissues,promotion of angiogenesis,escape from immune system and resistance to conventional anticancer therapy [18-19].Therefore,it is important to elucidate the molecular mechanisms that promote CSCs stemness maintenance and treatment resistance.In the present study,we isolated and enriched the CD133+/CD44+sphere-forming cells from SKOV3 cells by magnetic separation.The following results confirmed that the tumor sphere forming capacity of SKVO3 cells was notably reduced after treatment with exosomal miR-145.These founding suggest that exosome-mediated transfer of miR-145 may be involved in the regulation of stem cell-like features of EOC.

In conclusion,we found that exosomes could transfer miR-145 to EOC cells,inhibit the proliferation,migration and invasion of EOC cellsin vitro,and suppress the stem cell-like characteristics of EOC.Exosomal miR-145 may serve as a novel molecular marker for EOC diagnosis.

AcknowledgmentsThis study was funded by National Natural Science Foundation of China (No.81341075).