A total of 20 pairs of tumor tissues and adjacent normal tissues were obtained from patients who underwent surgery at the Department of Breast Cancer at Hubei Cancer Hospital (Wuhan, China). Written informed consent was obtained from each patient and the study was approved by the Department of Breast Cancer at Hubei Cancer Hospital institutional review board, and the Ethics Committee of Hubei Cancer Hospital.

Breast cancer cell lines (MCF-7, MDA-MB-453, MDA-MB-468 and ZR-75-1) and normal breast epithelial cells (HBL-100) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies). Cultures were maintained at 37°C in a humidified incubator containing 5% CO₂.

Total RNA from tissues or cells was harvested using an miRNA Isolation kit (Ambion, Austin, TX, USA). Expression of mature miRNAs was assayed using a Taqman MicroRNA assay (Applied Biosystems, Foster City, CA, USA) specific for human miR-802. qPCR was performed using a 7900 Real-time PCR system (Applied Biosystems). All samples were normalized to the internal control, U6 small nuclear RNA. All RNA samples were examined as to their concentration and purity. RNA purity was measured using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Based on the absorbance ratio at 260/280 nm (mean ± standard deviation =1.86±0.02), all RNA samples were pure and protein free. The reaction conditions were as follows: Initial holding period at 95°C for 5 min, then 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The primer sequences used were as follows: p27, forward 5′-ATGAGCCGCAAACTGGGT C-3′ and reverse 5′-AGAGCCGAACTCCACAAT CTC-3′; cyclin A, forward 5′-CGCTGGCGGTACTGAAGTC-3′ and reverse 5′-GAGGAACGGTGACATGCTCAT-3′; cyclin B1 forward 5′-AATAAGGCGAAGATCAACATGGC-3′ and reverse 5′-TTTGTTACCAATGTCCCCAAGAG-3′. Relative quantitation analysis of gene expression data was performed according to the 2^−ΔΔCt method.

Transfected cells were plated onto 12-well plates at a density of 1×10⁴ per well and cultured for 1–3 days. Cell counts were estimated by trypsinizing (Invitrogen Life Technologies) the cells and performing analysis using a Coulter counter (Beckman Coulter, Fullerton, CA, USA). For bromodeoxyuridine (BrdU; Beyotime Institute of Biotechnology, Shanghai, China) incorporation assays, a cell proliferation enzyme-linked immunosorbent assay (Beyotime Institute of Biotechnology) was used to analyze the incorporation of BrdU during DNA synthesis according to the manufacturer's instructions. Absorbance was measured at 450 nm using the Spectra Max 190 ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

For the miR-802 expression plasmid, human miR-802 precursor was cloned by PCR and inserted into the XbaI and XhoI sites of pSilencer (Ambion). The negative control plasmid consists of a scrambled sequence (Ambion). MCF-7 cells were transfected with the miR-802 precursor or the negative control using Lipofectamine 2000^® (Invitrogen Life Technologies) according to the manufacturer's instructions.

Cells were resuspended in 70% ethanol and fixed at room temperature for 30 min. Cells were then washed three times with phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology) and resuspended in propidium iodide (PI) stain solution, consisting of 50 µg/ml PI (Beyotime Institute of Biotechnology) and 100 µg/ml ribonuclease A (Sangon Biotech, Shanghai, China) in PBS. The cells were incubated in PI stain solution for 30 min and were then analyzed by flow cytometry using a FACScalibur (BD Biosciences, Oxford, UK). The flow cytometry data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR, USA).

Cells were harvested and lysed using ice-cold lysis buffer (50 mM Tris-HCl, pH 6.8; 802 mM 2-ME, 2% w/v SDS, and 10% glycerol). Following centrifugation at 20,000 × g for 10 min at 4°C, proteins in the supernatants were quantified and separated using 10% SDS-PAGE, then transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Following blocking with 10% non-fat milk in PBS, membranes were immunoblotted with the following primary antibodies: Anti-p27 (ab32034; rabbit monoclonal; 1:2,000), cyclin A (ab2097; rabbit polyclonal; 1:2,000), cyclin B1 (ab72; mouse monoclonal; 1:1,000) and Forkhead box protein M1 (FoxM1) (ab83097; rabbit polyclonal; 1:1,000) antibodies purchased from Abcam (Cambridge, MA, USA). Protein levels were normalized to that of GAPDH (sc-365062; mouse monoclonal, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The blots were then incubated with horseradish peroxidase-linked secondary antibodies (Cell Signaling Technologies, Inc., Danvers, MA, USA). Antibody signals were detected using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Inc., Rockford, IL, USA) according to manufacturer's instructions.

The potential targets of miR-802 were analyzed by TargetScan (www.targetscan.org) and miRWalk (www.umm.uni-heidelberg.de/apps/zmf/mirwalk) softwares. Wild-type and mutant 3′-untranslated region (UTR) fragments of the FoxM1 gene were cloned into pMir-Report (Ambion), yielding pMir-Report-FoxM1. Mutations were introduced into potential miR-802 binding sites using a QuikChange site-directed mutagenesis kit (Stratagene Inc., La Jolla, CA, USA). For luciferase assays, cells were seeded in 24-well plates and transfection efficiency was normalized by cotransfecting the Simian virus 40 (SV40) plasmid. Values were determined using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA).

Male BALB/c nude mice (age, five weeks) were purchased from the Experimental Animal Center of the Third Military Medical University (Chongqing, China). A total of 8×10⁵ MCF-7 cells stably expressing miR-802 or negative control were injected subcutaneously into the skin under the front legs of the mice. Mice were observed over four weeks for tumor formation. At four weeks, the mice were sacrificed via cervical dislocation, tumors were recovered and the wet weights of each tumor were determined. Experiments were performed using six mice per group (two groups; MCF-7 cells with stable overexpression of miR-802 precursors and negative controls).

Values are presented as the mean ± standard error of the mean (n>3). Differences between groups were analyzed using Student's t-test using GraphPad Prism software, version 6.0.1 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05, P<0.01 and P<0.001 were considered to indicate statistically significant differences.

In order to explore the role of miR-802 in breast cancer carcinogenesis, microRNAs were extracted from malignant and normal breast tissue biopsies and then analyzed using qPCR. As shown in Fig. 1A, miR-802 expression was significantly downregulated in cancer tissues compared with that of the adjacent non-cancerous tissues. In addition, miR-802 expression was significantly decreased in the breast cancer cell lines compared with that of the normal breast epithelial cells (Fig. 1B).

In order to determine the effect of miR-802 on breast cancer cell growth, MCF-7 cells overexpressing miR-802 precursor were constructed, which led to a significant increase in miR-802 expression (Fig. 2A). As a result, miR-802 overexpression significantly inhibited the proliferative ability of MCF-7 cells post-transfection compared with that of the negative control (Fig. 2B–D). Furthermore, cell cycle analysis revealed that a significantly higher percentage of cells overexpressing miR-802 were in G1/G0 phase and a decreased percentage of these cells were in S phase, compared with that of the negative control-transfected cells (Fig. 2E).

MCF-7 cells with stable overexpression of miR-802 were then evaluated for tumorigenic potential in vivo. Cells were injected subcutaneously into the skin under the front legs of nude mice and tumor growth was closely monitored for four weeks. As a result, the tumor size and weight was markedly reduced in miR-802-overexpressing tumors compared with that of the control tumors (Fig. 2F), suggesting that miR-802 suppressed breast cancer growth in vivo.

In order to understand the underlying mechanisms of miR-802-induced growth inhibition in MCF-7 cells, potential targets of miR-802 were searched using TargetScan and miRWalk software. The results showed that FoxM1, an oncogene in human cancer, harbored a potential miR-802 binding site (Fig. 3A). In order to verify whether FoxM1 is a direct target of miR-802, a luciferase reporter vector was constructed, which contained the putative miR-802 binding sites within the FoxM1 3′-UTR. As shown in Fig. 3B, miR-802 overexpression significantly repressed luciferase activity when the reporter construct contained the FoxM1 3′UTR in MCF-7 cells (Fig. 3B). However, mutation of the miR-802 binding site from the FoxM1 3′-UTR abolished the effect of miR-802, suggesting that miR-802 directly inhibited FoxM1 expression through targeting its 3′-UTR (Fig. 3B).

A shown in Fig. 3C, western blot analysis revealed that the miR-802 precursor significantly decreased the protein expression of FoxM1, while its mRNA levels remained unchanged (Fig. 3D). Therefore, the results suggested that miR-802 negatively regulated FoxM1 expression at the translational level.

FoxM1 was previously reported to transcriptionally downregulate the expression of p27, while upregulating Cyclin A and Cyclin B1, key regulators of cell-cycle progression (11). In concurrence with this previous study, the present study observed the enhanced expression of p27 and downregulation of Cyclin A and Cyclin B1 in MCF-7 cells overexpressing miR-802 (Fig. 3E and F). Therefore, these results further indicated that FoxM1 was an important target gene of miR-802 in breast cancer cells.

MCF-7 cells were transfected with miR-802 antisense in order to block the functions of endogenous miR-802. As a result, ectopic expression of the miR-802 antisense led to the increased proliferative ability of MCF-7 cells, compared with that of the negative control-transfected cells (Fig. 4A–C). In addition, the inhibition of miR-802 significantly reduced the percentage of cells in G0/G1 phase and increased the percentage of cells in S phase (Fig. 4D). Furthermore, protein levels of FoxM1 were upregulated in MCF-7 cells transfected with miR-802 antisense (Fig. 4E). The downregulation of p27 and upregulation of Cyclin A and Cyclin B1 was also observed following miR-802 antisense transfection (Fig. 4F and G). These results therefore supported the conclusion that miR-802 regulated FoxM1 expression in breast cancer cells.