BC tissues and non-tumor adjacent normal tissues from 30 BC patients (mean age: 51.73 ± 9.66 years) admitted to our hospital from June 2016 to December 2017 were collected. The basic information of the patients was also recorded: age, gender, blood pressure, tumor stage, distant metastasis or not, and lymphatic metastasis or not. In all the patients, BC was the primary lesion and confirmed by pathological examination. Each patient has no history of major systemic disease and was not treated with chemotherapy or radiotherapy before surgery. Informed consent was obtained from each patient.

HUCMSCs purchased from American Type Culture Collection (ATCC), and were cultured in FBS-free medium for 48 h, followed by collection of the supernatant. Centrifugation (2,000 g, 4°C for 30 min) was then performed to remove cell debris and apoptotic bodies and a 0.2 mm filter was used for filtration. Subsequently, the samples were first centrifugated at 110,000 g, 4°C for 70 min and then at 110,000 g, 4°C for 60 min. The collected exosomes were resuspended with phosphate buffer, and later were aliquoted and stored at −80°C.

In order to determine the characteristics of exosomes, the diameter and concentration of exosomes were analyzed by nanoparticle tracking analysis (NTA) using NanoSight NS300 (Malvern, UN) equipped with a 405 nm blue laser.

The morphology of exosomes was observed using a transmission electron microscope (TEM, Hillsboro Oregon, United States) (Tadokoro et al., 2013). Briefly, the exosomes were fixed with 4% paraformaldehyde and adsorbed to a carbon-coated grid. Then the copper grid was immersed in a 2% phosphotungstic acid solution for 30 s. After drying, the samples were examined under TEM at 80 keV. The expression of the marker proteins CD9, CD63, and HSP70 of the exosomes was detected by western blotting.

Human BC cell lines MCF-7, MDA-MB-231, T47D, SK-BR-3, MDA-MB-435, and HCC1937, and human normal breast epithelial cells MCF10A were purchased from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All cells were grown in media supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) with culture conditions of 37°C and 5% CO₂. MiR-224-5p mimic, NC mimic, miR-224-5p inhibitor, and NC inhibitor purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China) were transfected into hUCMSCs, respectively, and the exosomes of each group were collected. MCF-7 or MDA-MB-231 cells were assigned into six groups: (1) Control: cells without any treatment; (2) Exo: cells were co-cultured with hUCMSCs-exo; (3) NC-exo: cells were co-cultured with exosomes transfected with NC mimics; (4) miR-224-5p-exo: cells were co-cultured with exosomes transfected with miR-224-5p mimics; (5) in-NC-exo: cells were co-cultured with exosomes transfected with NC inhibitor; (6) in-miR-224-5p-exo: cells were co-cultured with exosomes transfected with miR-224-5p inhibitor.

MCF-7 and MDA-MB-231 cells in logarithmic growth phase in each group were digested and counted. Then they were seeded in 96-well plates (2,000 cells/well) with five replicates for each sample. For adherent cells after treatment for 24, 48, and 72 h the media were aspirated from each group. Then the prepared MTT solution (Aladdin, United States) was added, followed by another 1-h incubation. The absorbance (490 nm) was measured by a microplate reader.

The treated MCF-7 and MDA-MB-231 cells in each group were digested with trypsin to prepare single-cell suspension. The cell concentration was determined and adjusted to 1,000 cells/ml. Single-cell suspension was evenly seeded in sterile 6-well plates for forming colonies. The medium was changed every 3 days, and the colony formation of the cells was observed after 14 days.

The determination of apoptosis by flow cytometry was performed as shown in the previous report (Jurisic et al., 2011). Cells were centrifuged at 1,000 r/min for 5 min and then the supernatant was discarded. Subsequently, the cells were resuspended and counted. A total of 50,000–100,000 cells were taken and resuspended with 195 μl of Annexin V-FITC binding solution, followed by addition of 5 μl of Annexin V-FITC reagent. The cells were gently mixed with the reagent, and placed at room temperature for 10 min in the dark. After another 10 μl of propidium iodide staining solution was added and mixed gently, the samples were placed at room temperature for 10 min in the dark. Finally, the sample was detected by a flow cytometer.

The treated MCF-7 and MDA-MB-231 cells in each group were seeded on coverslips. With cell confluency of about 70%, the coverslips were rinsed with PBS three times (10 min each time). Then the coverslips were fixed with 4% formaldehyde for 15 min and rinsed again with PBS. After that, 0.5% Triton X-100 was added for 10-min incubation, followed by rinsing step by PBS and 1-h blocking step using 100 mL/L calf serum albumin. The coverslips were then cultured with LC3 antibody (Abcam, Cambridge, United Kingdom; Dilution, 1:50) overnight at 4°C. After rinsing with PBS again, human FITC-labeled immunofluorescence antibody was added for 1-h incubation at 37°C. Finally, the coverslips were rinsed with PBS and distilled water, and, were mounted with glycerol. LC3 spots in each group were observed using a confocal microscope (Leica, Germany) with 400 × magnification.

Activated cells were collected and washed twice with PBS. Cells were cross-linked for 15 min by adding 10 mL PBS and formaldehyde with the final concentration of 0.01%. Then 1.4 mL of 2 mol/L glycine was added and mixed for 5 min, followed by centrifugation at 1,500 r/min for 5 min. The supernatant was discarded and then cells were washed twice with PBS again and lysed by RIPA. The cell lysates were equally assigned into two parts. Subsequently, one part with addition of 4 μg of AGO2 antibody (Abcam) was as the experimental group, while the other part with addition of the amount of normal rabbit IgG as the control group. Both groups were cultured overnight at 4°C, and then the cells were collected. RNA was extracted using TRIzol reagent and was reversely transcribed into cDNA. Finally, the expression of miR-224-5p and HOXA5 was detected with RT-qPCR

The binding site of miR-224-5p to HOXA5 was predicted by software TargetScan. The 3’ UTR of the wild-type HOXA5 gene was cloned into a luciferase vector (HOXA5-WT), and the binding region of HOXA5 to miR-224-5p was mutated to obtain the mutant plasmid (HOXA5-MUT) (GenePharma, China). NC mimics or miR-224-5p mimics were co-transfected with HOXA5-WT/MUT into MCF-7 and MDA-MB-231 cells. Transfected pRL-TK was used as a standard. After 36 h of transfection, cells were harvested. The luciferase activity of MCF-7 and MDA-MB-231 cells was detected according to the instructions of luciferase activity assay kit. The experiment was repeated three times.

Total RNA was isolated from cells and tissues using Trizol reagent (Invitrogen). A total of 500 ng of RNA was reversely transcribed into cDNA using the cDNA Transcription Kit (ABI). Transcription was subsequently carried out at 16°C for 30 min, followed by incubation at 42°C for 30 min and at 85°C for 5 min for enzyme inactivation. Rapid quantitative PCR was performed using SYBRH Select Master Mix (Invitrogen). The RT-qPCR reaction was performed using the following parameters: 95°C for 2 min, 40 cycles of 95°C for 15 s and 60°C for 30 s. With All results were standardized to the expression of GAPDH or U6 as an internal reference, the obtained experimental data was quantified using the 2^{–Δ Δ Ct} method. The primer sequences used were as follows: miR-224-5p, Forward, 5′-AGCATCCACGAGCAAGAGAC-3′ and reversely, 5′-GATGCTACTAGTGTGGCGGG-3′; U6, Forward, 5′-CTCGC TTCGGCAGCACA-3′ and reversely, 5′-AACGCTTCACGAAT TTGCGT-3′; HOXA5, Forward, 5′-GAAGCTGAGCAG TGAAGCCTAT-3′ and reversely, 5′-GACAACTGTGAGAGCC AGGTT-3′; GAPDH, Forward, 5′-AACGGATTTGGTCGTA TTG-3′ and reversely, 5′-GGAAGATGGTGATGGGATT-3′.

Total protein was extracted with RIPA buffer (Sigma Aldrich, United States) from cells and tissues as previously reported (Son et al., 2019). The concentration of the total extracted protein was determined by a BCA protein assay kit (Bio-rad, United States). The proteins were boiled and separated using sodium dodecyl sulfatepolycylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred to PVDF membranes (300 mA, 80 min), followed by blocking step using 10% TBS (Sigma Aldrich, United States). Subsequently the membranes were incubated with primary anti-rabbit monoclonal antibody CD9 (ab92726, Abcam), rabbit polyclonal antibody LC3-II (ab51520, Abcam), rabbit monoclonal antibody p62 (ab56416, Abcam), rabbit monoclonal antibody Beclin-1 (ab207612, Abcam), rabbit monoclonal antibody CD63 (ab8219, Abcam), rabbit monoclonal antibody HSP70 (ab181606, Abcam), and rabbit monoclonal antibody HOXA5 (ab140636, Abcam) at a dilution of 1:1,000 at 4°C overnight. Rabbit monoclonal antibody β-actin (ab8226, Abcam) is the internal reference protein. After incubation, the membranes were washed three times and then incubated with polyclonal goat anti-rabbit IgG (ab6721, Abcam; 1:2,000) secondary antibody conjugated to horseradish peroxidase (HRP) (Sigma Aldrich, United States) at room temperature for 1 h. Then the membranes were washed for several times. The proteins were developed by an enhanced chemiluminescence regent. Images were subsequently quantified with Image J (NIH).

Twenty nude mice were reared at constant temperature (25–27°C), constant humidity (25–50%), and specific pathogen-free (SPF). MCF-7 cells in logarithmic growth phase were prepared into single-cell suspension by trypsin digestion, followed by centrifugation at 1,000 r/min for 10 min. Then the precipitate was collected and washed twice with phosphate buffered saline (PBS) to prepare cell suspension with a density of 1 × 10⁷ cells/mL. A total of 200 μL of the suspension were aspirated with a sterile trocar and inoculated subcutaneously in the disinfected armpit skin of nude mice. After 10 days of inoculation, the mice were randomly divided into control group, Exo group, in-NC-exo group, and in-miR-224-5p-exo group, with five nude mice in each group. Reagents in each group were dissolved in 200 μL PBS and were injected every 2 days for a total of five injections. The long and short axes of the tumors were measured with a vernier caliper every 7 days to calculate tumor volume and draw tumor growth curve. All treatment was completed after 35 days, and all the mice were then sacrificed. Subsequently, the xenografts were removed and weighed. Each tumor tissue was equally assigned into 3 parts, with 2 parts stored in liquid nitrogen for subsequent detection, and the other part fixed in 10% neutral formalin and embedded in paraffin for subsequent immunohistochemical staining.

All tumor tissues were fixed in 10% neutral formalin and embedded in paraffin. IHC staining was performed according to the instructions of IHC kits with rabbit anti- Ki-67 (ab15580, Abcam) monoclonal antibodies. The tissues were developed with diaminobenzidine (DAB) (Tokyo Chemical Industry Co., Ltd., Japan) colorimetric solution, counterstained with hematoxylin, and mounted with neutral resin. Finally, the cell staining was observed under an optical microscope with 200× magnification.

SPSS 22.0 was applied for data analysis. The results were expressed as mean ± standard deviation (SD). Comparison between the two groups was analyzed by t-test. Multi-group comparison of data was carried out using the one-way ANOVA followed by least significant difference (LSD) t-test. Pearson analysis was used to analyze the correlation between the expression of miR-224-5p and HOXA5. Each experiment was repeated at least three times. P < 0.05 was considered statistically significant.

The clinical characteristics of the 30 included BC patients are shown in Table 1. The results of RT-qPCR assay confirmed that the expression of miR-224-5p was significantly elevated while the expression of HOXA5 was significantly decreased in BC tissues (Figures 1A,B) and cells (Figures 1C,D). Pearson correlation analysis revealed a negative correlation between miR-224-5p expression and HOXA5 expression (Figure 1E). These results confirmed that miR-224-5p and HOXA5 may be involved in the occurrence of BC. Since the expression of miR-224-5p expression was higher while HOXA5 expression was lower in MCF-7 and MDA-MB-231 cells, these two cell lines were selected for the subsequent experiments.

To identify the functional effects of hUCMSCs-exo on MCF-7 and MDA-MB-231 cells, we first extracted and identified hUCMSCs-exo. Transmission electron microscopy confirmed the morphological characteristics of the exosomes as oval membranous vesicles (98 nm) (Figure 2A). And nanoparticle tracking analysis demonstrated that the diameter of most particles ranged from 49 to 160 nm (Figure 2B). In addition, the expression of protein markers CD9, CD63 and HSP70 of exosomes was up-regulated in the hUCMSC-exo compared with hUCMSCs (Figure 2C). Hsp70, CD9 and CD63 are the three protein markers of exosomes (Fernando et al., 2017). These results indicated a successful separation of exosomes. Further, after further overexpression or interference with the expression of miR-224-5p in hUCMSCs, exosomes were collected and then co-cultured with MCF-7 or MDA-MB-231 cells. As shown in RT-qPCR results, miR-224-5p expression was elevated in MCF-7 and MDA-MB-231 in the miR-224-5p-exo group, while was decreased in the in-miR-224-5p-exo group (Figure 2D). Collectively, it was proved that hUCMSCs-exo could transport miR-224-5p into BC cells to change miR-224-5p expression.

To determine whether miR-224-5p could be transported into BC cells via hUCMSCs-exo and thus affecting cell function, after transfection of miR-224-5p mimics and inhibitor into hUCMSCs, respectively, exosomes were collected and co-cultured with MCF-7 or MDA-MB-231 cells. As shown in the results of MTT (Figures 3A,B) and cell colony formation assay (Figures 3C,D), the proliferation rate and viability of cells in the miR-224-5p-exo group were significantly increased in MCF-7 or MDA-MB-231 cells compared with the NC-exo group, while the proliferation rate and viability in the in-miR-224-5p-exo group were significantly decreased compared with in-NC-exo. Flow cytometry (Figures 3E,F) showed a significant increase of apoptosis in the miR-224-5p-exo group, while a decrease in the in the in-miR-224-5p-exo group.

The results of immunofluorescence staining indicated that in MCF-7 and MDA-MB-231 cells, the number of LC3 spots was significantly increased in the miR-224-5p-exo group compared with the NC-exo group, while was decreased in the in-miR-224-5p-exo group compared with the in-NC-exo group (Figure 4A). Western blot assay then showed a marked increase of the protein expression of LC3-II and Beclin-1 in MCF-7 and MDA-MB-231 cells and a reduction of p62 expression in the miR-224-5p-exo group compared with the NC-exo group, while opposite changes were found in the in-miR-224-5p-exo group (Figures 4B,C). These results confirmed that miR-224-5p could promote autophagy in BC cells.

Fifteen downstream target genes (Figures 5A,B) regulated by miR-224-5p were predicted by starBase, miRDB and TargetScan. HOXA5, which is lowly expressed in BC, was also one of the target genes of miR-224-5p (Figure 5C). Luciferase reporter gene assay and RNA binding protein immunoprecipitation assay further proved that miR-224-5p targeted HOXA5 (Figures 5D,E). In addition, western blotting also confirmed that miR-224-5p could down-regulate the expression of HOXA5. In MCF-7 and MDA-MB-231 cells, the protein expression of HOXA5 was significantly decreased in the miR-224-5p-exo group compared with the NC-exo group, while was significantly increased in the in-miR-224-5p-exo group compared with the in-NC-exo group (Figure 5F).

Xenograft models in nude mice were constructed by subcutaneous injection of MCF-7 cells in nude mice, followed by injection of saline (control group), Exo, in-NC-exo, and in-miR-224-5p-exo. The results showed that compared with the in-NC-exo group, the tumor tissue of mice in the in-miR-224-5p-exo group was significantly smaller (Figure 6A), and the tumor weight and volume of mice were decreased (Figures 6B,C). Injection of in-miR-224-5p-exo into the models decreased the expression of miR-224-5p and Ki67 (Figures 6D,E), decreased the protein expression of LC3-II and Beclin-1 and increased the protein expression of p62 in the tumor tissues (Figure 6F). These results confirmed that hUCMSCs-exo successfully transported low-expressed miR-224-5p into MCF-7 tumor-bearing mice to inhibit tumor growth and autophagy.