Introduction
Despite promising advancements in breast cancer therapeutic approaches, metastasis makes it the leading cause of cancer-related mortality [
1], so the establishment of effective and safe therapeutic strategies is needed. Epithelial–mesenchymal transition (EMT) and angiogenesis are two critical processes for cancer cell metastasis.
MicroRNA (miRNA) profiling and deep sequencing indicate that aberrant expression of miRNAs in various cancer types is associated with cancer metastasis [
2]. Restoring normal miRNA expression in cancer cells can change cancer phenotype. [
3]. However, the administration of efficient and safe delivery systems is one of the main challenges in miRNA-based therapy [
4].
Exosomes are membrane-bound nanovesicles produced by almost all cell types [
5]. Unlike synthetic nanocarriers, exosomes do not face toxicity and immunogenicity as major drawbacks [
6]. Low immunogenicity, high biosafety, and natural tumor tropism of mesenchymal stem cells (MSCs)-derived exosomes characterize them as an appropriate candidate for delivery of anti-cancer agents [
7].
miR-218 is one of the tumor-suppressing miRNAs in different types of cancers [
8‐
12]. There are conflicting reports on the role of miR-218 in breast cancer progression. Despite the studies showing the tumor-suppressing role of miR-218 in breast cancer [
13‐
16], some studies suggest the tumor-promoting role of miR-218 [
17,
18]. Therefore, the evaluation effects of miR-218, as a miRNA targeting EMT and angiogenesis, on breast cancer cells needs further studies.
In the present study, ADMSC-exosomes were used to overexpress miR-218 in breast cancer cells. We focused on RUNX family transcription factor 2 (Runx2) and RPTOR-independent companion of MTOR complex 2 (Rictor), two potential targets of miR-218 that play important role in EMT and angiogenesis.
Methods
ADMSCs isolation
To achieve enough number of cells from the best sources, adipose tissues were separately obtained from healthy donors (aged between 22 and 35 years) undergoing surgical procedures. They all signed an informed consent form approved by the ethics committee of Shahid Beheshti University of Medical Sciences (Ethical code: IR. SBMU.REC.1400.010).
Briefly, after washing adipose tissues from lipoaspirate samples with phosphate-buffered saline (PBS), they were digested with 0.1% collagenase I (Sigma, USA) for 40 min (min) at 37 °C with gentle agitation. At the end of the incubation time, collagenase was neutralized by adding FBS-containing medium and digested samples were centrifuged at 1200 RPM for 20 min (Hettich, Germany). The resultant cell pellet was resuspended in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 15% FBS and 1% penicillin and streptomycin, seeded in culture flasks and maintained in a humidified atmosphere at 37 °C and 5% CO2. After 24 h, cells were washed to discard non-adherent cells and the fresh medium was replaced. During the expansion, half medium refreshment was done twice per week for optimal growth.
ADMSCs characterization
Immunophenotyping of ADMSCs was performed using flow cytometry. At the third passage, 1 × 106 ADMSCs were suspended in PBS and then incubated with primary antibodies including CD45-FITC, CD14-FITC, CD34-PE, CD90-FITC, CD73-PerCP, and CD105-PerCP (eBioscience, USA) for 30 min. Identification of ADMSCs’ surface markers was performed by FACSCalibur flow cytometer (BD Biosciences, USA).
The osteogenic and adipogenic differentiation potential were assessed using respective induction media and protocols [
19]. ADMSCs (1 × 10
4 cells/well) at passage 3 were seeded into 24-well plates and cultured in DMEM/F12 with 10% FBS. After 24 h, the differentiation media were replaced and refreshed every 3 days. 21 days after osteogenic induction, cells were fixed with 10% neutral formaldehyde and stained with 0.1% Alizarin red S dye (Sigma-Aldrich, USA). For adipogenic differentiation, after 14 days, cells were fixed and stained with 0.5% Oil Red O dye (Sigma-Aldrich, USA). The differentiated cells were observed by the light inverted microscope (Olympus, USA).
Cell lines and culture conditions
HUVEC (human umbilical vein endothelial cells), MDA-MB-231 cells (triple-negative breast cancer cell line), and MCF-10A (non-tumorigenic breast cell line) were obtained from the Pasteur Institute of Iran (Tehran, Iran). MCF-10A and MDA-MB-231 cells were grown in DMEM containing 10% horse serum and FBS, respectively. HUVECs were cultured in DMEM/F-12 supplemented with 10% FBS. All cells were maintained in a humidified atmosphere at 37 °C and 5% CO2.
Preparation of ADMSC-conditioned media and exosome isolation
The ADMSCs at the 3rd passage were used for the collection of conditioned medium (CM). When cells reached 70–75% confluence, they were adapted to FBS-free medium containing 1% insulin–transferrin–selenium (ITS; Sigma, USA). Serum-free ADMSC-CM was collected after 72 h and used for exosomes isolation and characterization. AnnexinV/PI staining was performed to evaluate cell viability after 72 h serum starvation.
ADMSC-exosomes were isolated from ADMSC-CM using EXOCIB exosome purification kit (Cibbiotech, Iran) according to the manufacturer’s instructions. Briefly, the serum-free-CM was centrifuged at 3000 RPM for 10 min at room temperature to remove debris. Exosome precipitation solution was added to ADMSC-CM at a 1:5 (v/v) ratio and mixed by vortexing the tubes for 5 min and incubated overnight at 4 °C. Then, the samples were centrifuged at 3000 RPM for 40 min at 4 °C. After removing the supernatant, exosomes were resuspended with PBS for the following experiments.
Exosome characterization
The size distribution of extracted exosomes was determined using dynamic light scattering (DLS) Zetasizer (Malvern, UK). The morphology and size of exosomes were observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For TEM imaging (Zeiss EM900), after processing of ADMSC-exosomes (fixation, dehydration, and sectioning), the ultrathin sections were prepared and stained using uranyl acetate and lead citrate and visualized under electron microscopy. For SEM (KYKY-EM3200, China), after fixation and dehydration of exosomes, they were left on the glass substrate to dry at room temperature and then were analyzed by scanning electron microscope.
Loading ADMSC-exosomes with miRNA mimic
To load miRNA-218-5p mimics (Bioneer, Korea) into ADMSC-exosomes, electroporation method was used. ADMSC-exosomes at a final concentration of 100 µg/µl protein (measured by BCA) were mixed with electroporation buffer (in a 1:1 ratio) and 100 pmol of synthetic miR-218 mimics or negative control (Scramble), and electroporated at 0.400 kV using an electroporation instrument (Eppendorf, Germany). To evaluate the efficiency of the loading protocol, MDA-MB-231 cells were incubated with 100 µg/ml of manipulated exosomes for 48 h and miR-218 encapsulation in exosomes was quantified using qRT-PCR.
The experimental groups included MDA-MB-231 cells treated with miR-218 or scramble containing exosomes, cells treated with unmodified exosomes, and untreated cells.
Total RNA from MDA-MB-231 cells treated with modified exosomes and their controls was extracted after 48 h using Hybrid-R
™ (GeneAll, Korea) according to the manufacturer’s instructions. Using RT-Stem loop [
20] and random hexamer primers, the RNA of miR-218 and related genes [
Runx2,
Rictor, vascular endothelial growth factor A (
VEGF), cadherin 1 or E-cadherin (
CDH1), cadherin 2 or N-cadherin (
CDH2)] were, respectively, transcribed to complementary DNA (cDNA).
Relative expression of miR-218 and target mRNAs was evaluated using TaqMan® probe and SYBR Green I Master Mix (Amplicon, Germany), respectively, in a StepOne instrument (Applied Biosystems, USA). SNORD47 and β-actin were considered as the internal references for miR-218 and mRNAs, respectively. The 2 –ΔΔCt method was employed to compute the relative expression of miRNA and mRNAs.
MTT assay
MDA-MB-231 cells were plated in a 96-well plate (3 × 103 cells/well) and incubated in serum-containing DMEM. After overnight incubation, cells were treated with modified exosomes (100 µg/ml) and their controls in a serum-free medium. At defined time points, the medium was removed and cells were incubated with 100 µL of MTT solution in PBS (0.5 mg/mL) for 3 h in the cell culture incubator. After removing the supernatant, 100 µL of dimethyl sulfoxide (DMSO) was added to each well for 2 h to dissolve formazan crystals. The optical density was read at 570 nm using a microplate reader (BioTek, USA).
Annexin V/PI assay
MDA-MB-231 cells were plated in a 24-well plate (5 × 104 cells/well). 48 and 72 h after their incubation with exosomes (100 µg/ml) in respective groups, cells were trypsinized and stained with annexin V/ fluorescein isothiocyanate (FITC) / propidium iodide (PI) kit according to the manufacturer’s instructions (Abcam, US). FACSCalibur flow cytometer was employed for cell analysis (BD Biosciences, USA). The annexin V+/PI− and annexin V+/PI + cells were considered as early and late apoptotic cells, respectively.
Scratch assay
MDA-MB-231 cells (12 × 104 cells/well) were plated in a 24-well plate. On reaching 90–95% confluence, the scratch was made across each well using a sterile 100 µl tip. After washing with DMEM to remove cell debris, cells were treated with 100 µg/ml modified exosomes in serum-free medium. Plates were photographed by an inverted microscope for 48 h and the images were processed and quantified using the ImageJ software (NIH, USA).
Cell migration assay
5 × 104 MDA-MB-231 cells suspended in serum-free medium with modified exosomes (100 µg/ml) were added into the upper chamber of transwell inserts (24-well insert; pore size 8 µm; SPL) and exposed to FBS-containing medium (as a chemoattractant) in the bottom chamber for 48 h. After incubation time, non-migrated cells were removed by scraping the upper surface of the chamber, and inserts were fixed and then stained with crystal violet. Five random fields were selected for counting the number of migrated cells.
Cell invasion assay
5 × 104 MDA-MB-231 cells were seeded to Matrigel (Corning, USA) coated inserts. Matrigel, being prepared by mixing with serum-free DMEM at a ratio of 1:2, was added to inserts and maintained at 37 °C for 2 h to solidify. The next steps were similar to those described for the migration assay.
In vitro angiogenesis assays
HUVECs were plated in a 96-well plate (3 × 103 cells /well) and incubated in DMEM-F12 overnight. Then, the culture medium was replaced with conditioned media of breast cancer cells treated with modified ADMSC-exosomes and their controls. After 24, 48, and 72 h, viable cells were evaluated by MTT assay.
In vitro migration assay was performed as described above. 4 × 104 HUVECs were added in the upper chamber of transwell inserts and cultured in serum-free DMEM-F12. The conditioned media of breast cancer cells treated with exosomes-encapsulated miR-218 and their controls were added to the lower chamber. After 24 h, migrated cells were stained and counted in five randomly selected microscopic fields.
In vitro capillary network formation was evaluated by tube formation assay. 3 × 104 cells were seeded into Matrigel-coated 48-well plate. Next, the cells were incubated with conditioned media collected from breast cancer cells treated with miR-218 containing exosomes and their controls for 24 h. The number of meshes and total branching length were quantified by randomly selecting five fields per well by using angiogenesis analyzer ImageJ plugin (NIH, USA).
Statistical analysis
Statistical analysis was conducted using GraphPad Prism (GraphPad, San Diego, CA). Student’s t-test was used for comparison between two groups while data among multiple groups were compared by one-way ANOVA. All experiments were performed in triplicate. The data were finally presented as mean ± SD and the asterisks show significant p-value: * p < 0.05; ** p < 0.001; *** p < 0.0001; and **** p. < 0.00001).
Discussion
Delivery of miRNAs inhibiting EMT and angiogenesis via clinically applicable vehicles can be a promising therapeutic strategy in metastatic cancers. Recently, numerous studies including our previous reports have focused on the therapeutic potential of MSC-derived exosomes for appropriate delivery of oligonucleotides in cancer therapy [
19,
23,
24]. In the present study, we selected miR-218 as a potential therapeutic candidate and used ADMSC-exosomes for its effective delivery to breast cancer cells. We showed that miR-218 overexpression using ADMSC-exosomes impaired breast cancer cells migration, invasion, and viability and downregulated mediators and markers of EMT and angiogenesis.
miR-218 acts as a tumor suppressor miRNA in various types of cancer and its downregulation is associated with tumor progression and metastasis [
9‐
11,
25]. We demonstrated that miR-218 is downregulated in invasive MDA-MB-231 cells compared to normal MCF-10 cells. This result is in accordance with the previous findings showing that miR-218 is downregulated in breast cancer cells [
14] and in contrast to miR-218 overexpression in breast cancer cells shown by Liu et al. [
17]
.
Runx2 and
Rictor are two potential targets of miR-218. It was previously confirmed that miR-218 targets 3'UTR of
Runx2 and
Rictor [
10,
26] but the effects of their interaction have not yet been reported in breast cancer. In this study, the breast cancer cells expressed high levels of
Runx2 and
Rictor compared to non-tumorigenic MCF-10A cells which were in line with previous studies [
27,
28]. The expression of
Runx2 is disrupted in breast cancer and induces the expression of EMT-related transcription factors and metastasis-related genes [
29,
30].
Runx2 directly or indirectly increases the expression of vascular endothelial growth factor (VEGF), the most important angiogenic factor [
31,
32]. The indirect effect of Runx2 on
VEGF expression is mediated by Rictor, the major component of mTORC2 [
26].
Rictor is amplified and upregulated in breast cancer [
33,
34]. Rictor is involved in multiple myeloma and prostate cancer angiogenesis and its inhibition suppresses tumor angiogenesis [
26,
35].
In this study, we found that restoration of miR-218 using miR-218 containing exosomes downregulates
Runx2 in breast cancer cells at the mRNA levels. Our results were consistent with previous reports in thyroid and ovarian cancers in which miR-218 overexpression via lipid-based transfection downregulates
Runx2 and inhibits cell proliferation, migration, and invasion in vitro [
10,
12]. Furthermore, we indicated that miR-218 overexpression using miR-218 containing exosomes downregulates
Rictor and
VEGF in breast cancer cells. A similar result has been shown by Guan et al
. in prostate cancer. They showed that miR-218 overexpression by lentiviral vectors restrains tumor angiogenesis via targeting
Rictor/
VEGF axis [
26].
Overexpression of miR-218 using miR-218 containing exosomes reduces motility and invasiveness of MDA-MB-231 cells in vitro as previously shown by Setijono
et al [
14]l. The anti-angiogenic role of miR-218 and the pro-angiogenic properties of Runx 2 and Rictor [
31,
32] led us to examine the angiogenic effects of MDA-MB-231 cells treated with miR-218 containing exosomes on endothelial cells. Our data showed that conditioned media of breast cancer cells treated with miR-218 containing exosomes significantly decreased viability, migration, and tube formation of endothelial cells compared to other groups. This finding was consistent with our gene expression analysis in which the expression of
VEGF mRNA in MDA-MB-231 cells treated with miR-218 containing exosomes decreased significantly.
A significant finding in this study was that unmanipulated ADMSC-exosomes reduced the apoptosis of breast cancer cells. This finding can be attributed to the presence of large amounts of negative regulators of the apoptosis process in unmanipulated ADMSC-exosomes [
36]. The anti-apoptotic activity of ADMSC-secretome has also been shown in liver injury [
37]. Moreover, as shown in previous reports, our results indicated that unmanipulated ADMSC-exosomes increase angiogenesis in vitro [
38,
39].
Generally, exosomes derived from MSCs of different tissue origins have shown promising results in miRNA delivery and inhibiting breast cancer development. Exosomes derived from miRNAs-overexpressing bone marrow-MSC inhibit breast cancer cell invasiveness and angiogenesis [
40‐
44]. Delivery of exogenous miRNA by exosomes derived from umbilical cord- MSC suppresses tumor invasion in breast cancer (
23).
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