Human breast cancer MDA-MB-231 and MCF-7 cells (both purchased from Peking Union Medical College) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Human normal mammary epithelial cells (MCF-10A, purchased from Peking Union Medical College) cells were cultured in growth medium consisting of DMEM/F-12 (1:1; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. Cells were incubated at 37°C in a 5% CO₂ atmosphere. Cells were cultured at saturated humidity and 37°C, changing culture medium every 2 days. When cells grew to 90%, they were trypsinized (25%) and then centrifuged for 10 min at 4°C and 18,750 × g before resuspension. The suspension was added to a new petri dish.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) or the Ultrapure RNA kit (CoWin Biosciences) according to the manufacturer's protocols. RNA (0.5 µg) was subjected to reverse transcription using ReverTra Ace qPCR RT Master mix with gDNA Remover (Toyobo Life Science). After adding oligo(dT) into RNA, it was heated at 70°C for 10 min and put in an ice bath for 1 min; m-mlv, dNTP and rnasin were then added, mixed well and centrifuged, then stored at 42°C for 40–90 min, 95°C for 10 min and finally at 4°C. qPCR was performed using a Bio-Rad IQ5 instrument (Bio-Rad Laboratories, Inc.) with TransStart^® Top Green qPCR SuperMix (Beijing Transgen Biotech Co., Ltd.). The reactions were performed under the following conditions as suggested by the manufacturer: 94°C for 30 sec, followed by 40 cycles of 94°C for 5 sec and 60°C for 30 sec, followed by a dissociation protocol. The following primer sequences were used: miR-223-3p forward, 5′-TAAAGCAACCGAGCACTGAGA-3′ and reverse, 5′-ACGGTAGAGGTCCTTTCCTTTG-3′; and 18S rRNA forward, 5′-AGGCGCGCAAATTACCCAATCC-3′ and reverse, 5′-GCCCTCCAATTGTTCCTCGTTAAG-3′. The relative expression was normalized to 18S rRNA expression (24), used as a loading control. Relative gene expression was analyzed using the 2^−∆∆Cq method (25).

MCF-7 cells were cultured in a 6-well plate at 37°C for 24 h. Transfection was performed when the cell density grew to ~70%. Subsequently, cells were transfected with miRVana miRNA inhibitors of miR-223-3p (miR-223-3p inhibitor; 50 nM; cat. no. 4464084; Thermo Fisher Scientific, Inc.) and negative control (NC; 50 nM; cat. no. R10034; Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, for 6 h at 37°C. Subsequently, the medium was changed to DMEM containing 10% serum and incubated at 37°C for 12 h before subsequent experiments (26).

A Cell Counting Kit 8 (CCK8; Beyotime Institute of Biotechnology) assay was used to detect the proliferation of mammary cancer cells. Briefly, at a cell growth density of ~70%, control (untransfected cells), NC- and miR-223-3p inhibitor-transfected MCF-7 cells were cultured in 96-well cell culture plates. After incubation at 37°C for 12, 24, 48 and 72 h, 10 µl CCK8 reagent was added to each well. The cells were placed in a cell incubator at 37°C for 0.5–1.5 h. Subsequently, the optical density value of each well was determined on a microplate reader at 450 nm.

The cells were cultured in 6-well plates and divided into three groups: Control group (untransfected MCF-7 cells), NC group and miR-223-3p inhibitor MCF-7 group. After transfection, 1×10⁶ cells were collected, washed in 1 ml 1X binding buffer and centrifuged at 4°C and 18,750 × g for 10 min. Subsequently, 1 ml 1X binding buffer was added to the collected cells, and then 10 µl Annexin V-FITC (Sungene Biotech Co., Ltd.) reagent was added to each group of collected cells. After incubation in the dark at 37°C for 15 min, 5 µl PI (Sungene Biotech Co., Ltd.) solution was added for 10 min at room temperature and apoptosis was analyzed by flow cytometry (CytoNova; CapitalBio Technology, Inc.). Results were analyzed using the Kaluza software (v2.1.1; Beckman Coulter, Inc.). The apoptotic rate was calculated as the percentage of early and late apoptotic cells.

Cell migration was measured using a wound healing assay. A total of 5×10⁴ cells were cultured in each well of a 24-well plate and cultured overnight in the incubator at 37°C to enable the cells to adhere to the bottom of the plate. Once the cells grew to ~80% confluence, a straight-line scratch was made with a 200-µl pipette tip, followed by washing with PBS and the medium was replaced with FBS-free medium. Subsequently, the scratch area was imaged using an optical light microscope (Olympus CKX53; Olympus Corporation; magnification, ×100) to record the results at 0 h. The cells were then placed in a cell incubator. After 24 h at 37°C, cell migration area size was analyzed with ImageJ software (v1.8.0; National Institutes of Health), comparing the findings with the previous time point. The experiments were repeated three times.

The invasive ability of cells was examined using a Transwell (Corning, Inc.) assay. The upper surface of the bottom membrane of the Transwell chamber was coated with 50 mg/l Matrigel (1:8) for 30 min at 37°C and air dried at 4°C. The residual liquid in the culture plate was aspirated and 50 µl serum-free culture medium containing 10 g/l BSA (Thermo Fisher Scientific, Inc.) was added into each well at 37°C for 30 min. The chamber was placed in the culture plate, and 300 µl pre-warmed serum-free medium was added to the upper chamber, which was left at room temperature for 15–30 min to rehydrate the matrix gel and absorb the remaining culture medium. Cells were starved in serum-free medium for 12–24 h before preparing the cell suspension. Cells were added to 24-well culture plates and Transwell chambers were placed in them. Moreover, 500 µl medium containing FBS was added to the lower chamber of the orifice plate. The cells were cultured at 37°C for 24 h. The cells invading the chamber were stained with 0.1% crystal violet for 1 h at room temperature, and 3–5 fields of view were randomly selected for imaging. Stained cells were counted under an optical light microscope (Olympus CKX53; Olympus Corporation; magnification, ×100).

Proteins were extracted from treated cells using lysis buffer (Beyotime Institute of Biotechnology) and quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 10 µg protein/lane was separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk at room temperature for 60 min and incubated with primary antibodies at 4°C overnight. Rabbit monoclonal primary antibodies against GAPDH (cat. no. ab181602), macrophage stimulating 1 (MST1; cat. no. ab79199), large tumor suppressor kinase 1 (LATS1; cat. no. ab70561), phosphorylated-Yap1 (cat. no. ab76252), Yap1 (cat. no. ab52771) and MMP14 (cat. no. ab51074), and the anti-rabbit immunoglobulin G secondary antibody (cat. no. ab7090) were obtained from Abcam. MMP9 (cat. no. 10375-2-AP), MMP2 (cat. no. 10373-2-AP), CDK2 (cat. no. 10122-1-AP), Cyclin E1 (cat. no. 11554-1-AP), cyclin-dependent kinase inhibitor 1 (p21; cat. no. 10355-1-AP), E-cadherin (cat. no. 20874-1-AP), N-cadherin (cat. no. 22018-1-AP) and vimentin (cat. no. 10366-1-AP) antibodies were obtained from ProteinTech Group, Inc. All antibodies were diluted as recommended in the specifications (dilution ratio, 1:1,000). The blots were then incubated with HRP-conjugated secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h. After extensive washing in TBST, protein bands were revealed with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) and visualized with Image Quant LAS 500 (Cytiva).

All results were obtained from ≥3 independent experiments and data are presented as the mean ± SD. Data were analyzed using SPSS software (version 20.0; IBM Corp.). GraphPad Prism software (version 6.0; GraphPad Software, Inc.) was used to generate figures. One-way ANOVA was used to analyze the significance of differences among different groups. Comparisons between multiple groups were performed using the Scheffe post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

miR-223-3p expression levels were detected in MCF-10A normal breast cells and in two different breast cancer cell lines, MCF-7 and MDA-MB-231 (Fig. 1A). The expression levels of miR-223-3p in breast cancer cells were significantly higher compared with expression in normal cells. As the highest miR-223-3p expression level was observed in MCF-7 cells, these cells were selected for subsequent experiments. After miR-223-3p inhibitor was transfected into MCF-7 cells, miR-223-3p expression was detected (Fig. 1B). The results demonstrated that miR-223-3p inhibitor could inhibit miR-223-3p expression.

To examine the role of miR-223-3p on the proliferation of MCF-7 cells, miR-223-3p inhibitor was transfected into MCF-7 cells and results were compared with the control group. Subsequently, cell viability was detected using a CCK8 assay at different time points, and a growth curve was generated based on the results. As presented in Fig. 2A, there was no significant difference in the proliferation rate between the MCF-7 cell control group and the NC group, whereas the cell proliferation rate of the miR-223-3p inhibitor group was significantly lower compared with that of the control group. By detecting the apoptosis rate, it was found that inhibiting miR-223-3p expression significantly increased apoptosis (Fig. 2B and C).

Subsequently, the expression levels of cell cycle-related proteins CDK-2, cyclin-related proteins Cyclin E1 and p21 were examined to further evaluate the effect of miR-223-3p on cell proliferation. Compared with the control group, the protein expression levels of CDK-2 and Cyclin E1 were downregulated, whereas the expression of p21 was upregulated in the miR-223-3p inhibitor group (Fig. 3). These results suggested that inhibiting miR-223-3p expression could inhibit the proliferation of breast cancer cells.

The effects of miR-223-3p on the migration and invasion of MCF-7 cells were investigated. Results from the wound healing assay demonstrated that loss of miR-223-3p expression significantly inhibited the migratory ability of MCF-7 cells (Fig. 4A and C). Similarly, Transwell analysis indicated that the invasive capacity of cells transfected with miR-223-3p inhibitor was significantly declined compared with that of the control group (Fig. 4B and D). Furthermore, in cells transfected with miR-223-3p inhibitor, the protein expression levels of metastasis-associated proteins, including MMP9, MMP2 and MMP14, were significantly decreased compared with the control group expression levels (Fig. 5). These data suggested that inhibiting miR-223-3p expression may decrease migration and invasion in MCF-7 cells.

The expression levels of EMT-associated proteins were examined to determine the effects of miR-223-3p on EMT in breast cancer cells. The results suggested that inhibiting the expression of miR-223-3p increased the protein expression of E-cadherin, and decreased N-cadherin and vimentin expression levels (Fig. 6). Thus, these data indicated that inhibition of miR-223-3p may reverse EMT in breast cancer cells.

The western blotting results demonstrated that the expression levels of phosphorylated Yap1 were increased in MCF-7 cells transfected with miR-223-3p inhibitor compared with those in control cells (Fig. 7A and D). Moreover, no significant difference was found between the protein expression levels of MST1 in cells transfected with miR-223-3p inhibitor and control cells (Fig. 7A and B). It was identified that LATS1 expression was significantly upregulated in miR-223-3p inhibitor-transfected cells (Fig. 7A and C). These results suggested that miR-223-3p may activate the Hippo/Yap signaling pathway.