Twenty primary breast cancer biopsy specimens and corresponding paracancerous tissues were collected from patients at the Taizhou Hospital of Zhejiang Province (Taizhou, China) who underwent surgery between December 2016 and July 2017, and were immediately stored in liquid nitrogen until use. All patients provided written informed consent for the use of these clinical materials in research, and the Institutional Ethics Committee of the Taizhou Hospital of Zhejiang Province ethically approved the project. The human breast cancer cell lines MDA-MB-231 and SKBR3, in addition to the normal human mammary cell line MCF-12A, were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences), 100 U/ml penicillin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 100 g/ml streptomycin (Sigma-Aldrich; Merck KGaA). All cell lines were cultured in a humidified incubator in an atmosphere of 5% (v/v) CO₂ at 37°C.

Total RNA was isolated from cell lines or tumor samples using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. miRNA was converted to cDNA using the TaqMan miRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. The expression levels of miR-27a were determined using the TaqMan miRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and calculated using the 2^−ΔΔCq method by normalizing to the signal for U6 expression (25). Complementary DNA synthesis of mRNA was performed using Moloney murine leukemia virus reverse transcriptase kit with oligo dT primers (Promega Corporation, Madison, WI, USA), at 70°C for 5 min, 42°C for 60 min and 70°C for 15 min. The expression levels of FBXW7, Snail, zinc finger E-box binding homeobox 1 (ZEB1), E-cadherin, N-cadherin and Vimentin were evaluated using qPCR with a SYBR green PCR master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and calculated using the 2^−ΔΔCq method by normalizing to β-actin. The thermocycling conditions were as follows: 95°C for 10 min, 45 cycles of 95°C for 15 sec and 60°C for 1 min, for both miRNA and mRNA PCR. All of the reactions were performed in triplicate and the primer sequences are listed in Table I.

MDA-MB-231 and SKBR3 cells were seeded into a 6-well culture plate and grew to 100% confluence in DMEM supplemented with 10% FBS. Subsequently, the cells were starved in serum-free medium overnight. A plastic pipette tip was used to produce a clean wound area across the center of the well. The cell debris were washed away using 1X PBS and the cells were allowed to migrate in DMEM with 10% FBS for 24 h or 48 h. Images of the wound healing process were captured and the gap distance normalized to the control was examined at 0, 24 and 48 h.

For the migration assay, a Transwell system (24 wells; 8-µm pore size with a poly-carbonate membrane; Corning Inc., Corning, NY, USA) was used according to the manufacturer's protocols. The cells were seeded into the upper chambers and cultured in serum-free DMEM. The lower compartment was filled with DMEM with 10% FBS as a chemoattractant. Subsequent to incubation for 24 h, the cells remaining in the upper chamber were removed. The cells at the bottom of the insert were fixed with 70% ethanol for 30 min at room temperature, stained with 0.5% crystal violet for 30 min at room temperature and counted under an inverted fluorescence microscope at ×40 (Olympus Corporation, Tokyo, Japan). A total of three independent experiments were performed to determine the mean number of migrated cells.

The hsa-miR-27a mimics, inhibitor, negative control (NC) mimics and NC inhibitor oligonucleotides were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequences for these oligonucleotides were as follows: 5′-UUCACAGUGGCUAAGUUCCGC-3′ (sense) and 5′-GGAACUUAGCCACUGUGAAUU-3′ (antisense) for miR-27a mimics; 5′-GCGGAACUUAGCCACUGUGAA-3′ for miR-27a inhibitor; 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense) for NC mimics; and 5′-CAGUACUUUUGUGUAGUACAA −3′ (sense) for the NC inhibitor. The cells (3×10⁵/well) were seeded in a 6-well plate and cultured for 24 h prior to transfection. The cells were transfected with hsa-miR-27a mimics/inhibitor or NC mimics/inhibitor (50 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for induction. After 24 h, the cells were collected to perform the in vitro assays.

Following transfection, the cells were lysed in lysis buffer (2.1 µg/ml aprotinin, 0.5 µg/ml leupeptin, 4.9 mM MgCl₂, 1 mM orthovanadate, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride). The protein concentration was determined using a bicinchoninic acid assay. Subsequently, protein (20 µg/lane) was subjected to electrophoresis on a 12% or 15% SDS-PAGE gel, proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk at room temperature for 2 h and incubated with Snail (cat. no. ab53519; 1:1,000), ZEB1 (cat. no. ab180905; 1:1,000), E-cadherin (cat. no. ab40772; 1:1,000), N-cadherin (cat. no. ab76057; 1:1,000), Vimentin (cat. no. ab8978; 1:1,000) and FBXW7 (cat. no. ab109617; 1:1,000) primary antibodies at 4°C overnight. The corresponding horseradish peroxidase (HRP)-conjugated secondary antibody was added and incubated at room temperature for 2 h. Signals were visualized using an enhanced chemiluminescence reaction with a HRP substrate (Pierce; Thermo Fisher Scientific, Inc.). All primary antibodies used in the present study, except the β-actin antibody, were purchased from Abcam (Cambridge, UK) and the secondary antibodies [goat anti-mouse IgG-HRP (cat. no. sc-2005; 1:10,000) and goat anti-rabbit IgG-HRP (cat. no. sc-2004; 1:10,000] were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The antibody against β-actin (cat. no. A8481; 1:5,000) was obtained from Sigma-Aldrich (Merck KGaA) and used as a loading control in clinical specimen western blotting only. GAPDH (cat. no. ab8245; 1:1,000) was used as loading control for all other western blotting. Densitometric analysis of the protein bands was performed using ImageJ software 1.49v (National Institutes of Health, Bethesda, MD, USA).

Next-generation sequencing and TargetScan (www.targetscan.org/vert_71/) was used to predict the target genes of miR-27a. The coding sequences of human FBXW7 mRNA were synthesized and subcloned into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The integrity of the respective plasmid constructs was confirmed by DNA sequencing. The transfection of the FBXW7 plasmid using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was performed according to the manufacturer's protocol. pGL3-FBXW7 wild-type and pGL3-FBXW7 mutant plasmids were constructed for the 3′-UTR reporter assays. The cells were transfected with 1.2 µg plasmid using Lipofectamine^® 2000 or with pGL3 empty vector, which was used as a negative control. A total of 24 ng PRL-CMV (Promega Corporation), encoding Renilla luciferase, was included in all transfections to normalize the transfection efficiency. Cells were washed and lysed with the passive lysis buffer from the Dual-Luciferase Reporter Assay system (Promega Corporation), 24 h after transfection. Luciferase activity was measured in each cell lysate using a FLUOstar Galaxy plate reader (BMG Labtech GmbH, Ortenberg, Germany).

All data are expressed as the mean ± standard deviation from at least three separate experiments. Histograms were produced using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). All statistical analyses were performed using GraphPad Prism 5.0 and statistical significance was determined using a two-sided Student's t-test for all data except the basal miR-27a levels in the cell lines, for which one-way analysis of variance followed by the Bonferroni post hoc test was performed to determine the statistical significance. P<0.05 was considered to indicate a statistically significant difference. Survival curves based on the Kaplan-Meier method were compared using a log-rank test (Kaplan Meier Plotter; http://kmplot.com/) (26).

To determine whether miR-27a was increased in human breast cancer or not, a total of 20 paired samples of human breast cancer and corresponding paracancerous tissues were obtained and their miR-27a levels were evaluated using RT-qPCR. The results revealed that miR-27a was significantly increased in breast cancer tissues compared with paracancerous tissues (P<0.01; Fig. 1A). In addition, miR-27a expression levels were additionally measured in breast cancer cell lines. As presented in Fig. 1B, miR-27a expression levels were higher in the breast cancer cell lines MDA-MB-231 and SKBR3 compared with the normal mammary cell line MCF-12A. These results indicated that miR-27a was notably overexpressed in breast cancer tissues and cell lines compared with their respective control counterparts.

The basal migratory activity of breast cancer cells was determined using a wound-healing assay in MDA-MB-231 and SKBR3 cells. As presented in Fig. 1C, it was revealed that SKBR3 cells exhibited a significantly accelerated migration rate of cells into the wounded area compared with MDA-MB-231 cells at 48 h (P<0.01). Thus, these data illustrated that SKBR3 cells were more aggressive compared with MDA-MB-231 cells, while expressing a higher level of miR-27a. These results suggested that the upregulation of miR-27a may be associated with the onset of breast cancer migration.

To identify the physiological function of miR-27a in cell migration, miR-27a mimics or inhibitors were applied in breast cancer cell lines. Firstly, the basal migratory ability of these two cell lines was examined using a Transwell assay. The results revealed that SKBR3 cells possessed substantially increased migratory properties compared with MDA-MB-231 cells (Fig. 2A). Subsequently, MDA-MB-231 or SKBR3 cells were treated with miR-27a mimics or inhibitors. The miR-27a levels were determined by RT-qPCR analysis followed by a Transwell assay to validate the migration capacity of treated cancer cells (Fig. 2B and C). The data from the Transwell assays indicated decreased miR-27a expression significantly inhibited MDA-MB-231 and SKBR3 cell migration (Fig. 2D), while miR-27a overexpression promoted MDA-MB-231 and SKBR3 cell migration (Fig. 2E). These data demonstrated that miR-27a positively regulated breast cancer cell migration.

To define the underlying mechanism of miR-27a in regulating cell migration in breast cancer cells, breast cancer cells were treated with a miR-27a inhibitor to detect the expression of EMT-associated proteins. Inhibition of miR-27a suppressed the expression of the EMT-associated transcription factors Snail and ZEB1, in addition to the mesenchymal markers N-cadherin and Vimentin, while increasing the E-cadherin protein expression levels in MDA-MB-231 cells (Fig. 3A). All these findings indicated that the EMT of MDA-MB-231 cells was reversed when miR-27a was inhibited (27). However, only the mRNA expression levels of N-cadherin, Vimentin and E-cadherin were significantly increased compared with their respective control, consistent with their protein expression profiles, while the mRNA expression levels of Snail and ZEB1 were not significantly increased.

To further elucidate the underlying mechanism, the mRNA expression profiles in MDA-MB-231 cells treated with miR-27a inhibitor or NC were analyzed by next-generation sequencing (data not shown). Among the mRNAs most notably upregulated by miR-27a inhibition, it was hypothesized that FBXW7 may be a novel target gene, according to the prediction results from the database TargetScan. In the present study, the mRNA and protein expression of FBXW7 in MDA-MB-231 cells transfected with miR-27 inhibitor was determined. The results revealed that FBXW7 was effectively upregulated at the protein and mRNA levels when miR-27a was inhibited (Fig. 3A and B). Therefore, one explanation for the inconsistency between the mRNA and protein expression levels of Snail and ZEB1 is that these two EMT-associated transcription factors may be regulated at the post-transcriptional level by FBXW7 (22). In addition, to determine whether suppressing FBXW7 may induce EMT alone, the present study produced an ectopic expression model by transfecting the FBXW7 plasmid into MDA-MB-231 cells. As presented in Fig. 3C, the ectopic expression of FBXW7 reduced the expression of Snail, ZEB1, N-cadherin and Vimentin in treated MDA-MB-231 cells. Combined results suggested that the downregulation of FBXW7 by miR-27a may promote human breast cancer cell metastatic capacity, potentially by triggering EMT.

It has been reported that FBXW7 is a target gene of miR-27a and serves a negative role in EMT regulation in esophageal cancer cells (28). However, whether FBXW7 was also regulated by miR-27a directly in human breast cancer remained to be confirmed. In the present study, luciferase reporter plasmids containing FBXW7 3′-UTR sequences with either a wild-type miR-27a binding site or the mutant version were constructed. A luciferase assay revealed that the miR-27a mimics were able to effectively repress the transcriptional activity of the wild-type plasmid, although not that of the mutant type. All results suggested that FBXW7 was also a downstream target gene of miR-27a in human breast cancer (Fig. 3D).

The present study initially detected the expression levels of FBXW7 protein in human breast cancer cell lines and revealed that FBXW7 expression was lower in MDA-MB-231 and SKBR3 cells compared with MCF-12A cells (Fig. 4A). Similarly, FBXW7 expression was significantly lower in breast cancer tissues compared with paracancerous tissues, as analyzed by western blotting (P<0.01; Fig. 4B). These results supported the idea that FBXW7 was suppressed in human breast cancer, which was negatively associated with the expression profile of miR-27a. More importantly, FBXW7 levels were significantly positively associated with the overall survival rate of patients with breast cancer, according to public datasets (log-rank P<0.05; Fig. 4C).

Furthermore, the function of FBXW7 in breast cancer migration was also illustrated. As expected, the ectopic expression of FBXW7 significantly inhibited MDA-MB-231 cell migration (P<0.01; Fig. 4D). To determine whether miR-27a-mediated breast cancer cell migration was FBXW7-dependent, the MDA-MB-231 cells were transfected with miR-27a mimics or an FBXW7 overexpression vector alone or in combination. A Transwell assay revealed that the accelerated migration induced by miR-27a was able to be significantly reversed by the ectopic expression of FBXW7 (P<0.01; Fig. 4E). Consequently, the results illustrated that FBXW7 was negatively regulated by miR-27a in human breast cancer tissues and cell lines. Notably, miR-27a may promote breast cancer cell migration by targeting FBXW7.