The MCF7, T47D, BT474, BT549, MDA-MB-468 and MDA-MB-231 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured as previously described (20).

Breast cancer specimens were obtained from Tianjin Medical University Cancer Institute and Hospital. A total of 30 primary breast cancer tissues and paired adjacent normal breast tissue specimens were included in this study. All tumors were from patients with a newly diagnosed breast cancer who had received no therapy before sample collection. After mastectomy surgery, the primary breast cancer tissues and the adjacent normal tissues were flash-frozen in liquid nitrogen and stored at −80°C. This study was approved by the Institutional Review Board of the Tianjin Medical University Cancer Institute and Hospital, and written consent was obtained from all participants.

To further validate the expression of miR-410-3p in breast cancer, we analyzed miR-410-3p expression profiling data set from The Cancer Genome Atlas (TCGA) including 683 cases of breast cancer tissues and 87 cases of normal breast tissues.

In pcDNA3.1-HA, annealed oligonucleotides encoding the HA tag were ligated into the HindIII and BamHI sites of pcDNA3.1 (Invitrogen). The ORF of human Snail was generated from MDA-MB-231 cells, the resultant PCR product of which was connected together with pcDNA3.1 tagged HA (Snail-HA), the Snail 3′-UTR containing the miR-410-3p binding site or the miR-410-3p binding site mutated fragments were cloned into the pGL3-Control vector (Snail-3′UTR-wt and Snail-3′UTR-mu; Promega, Madison, WI, USA) and the resulting constructs were confirmed by DNA sequencing. miRNAs were purchased from RiboBio (Guangzhou, China). Antibodies against Snail (Abcam, Cambridge, MA, USA) and β-actin (Cell Signaling Technology, Beverly, MA, USA) were used. Recombinant human TGFβ1 was purchased from R&D Systems (Redmond, WA, USA).

Total RNA from the cultured cells and surgically resected fresh breast tissues was extracted using mirVana PARIS kit (Life Technologies) according to the manufacturer's instructions. For miRNA detection, miRNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription kit and real-time quantitative PCR was performed using TaqMan miR-410-3p and U6 RNA (used as a normalizer) assays (Life Technologies) following the manufacturer's instructions.

Cells were lysed in RIPA buffer protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). Samples were denatured for 5 min at 95°C and subjected to 10% SDS/PAGE. The separated proteins were transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked in 5% (w/v) skim milk-TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.3) solution, followed by incubation with the primary antibodies diluted in skim milk-TBST solution overnight at 4°C. Then the membrane was incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) for 1 h at room temperature, and the immunoreactive protein bands were visualized by enhanced chemiluminescence reagents (Millipore).

For transfection, the cells were plated at a density of 2×10⁵ cells/well in 6-well plates. When the cells reached 60% confluency, 50 nmol/l miRNA or 4 µg Snail-HA was transfected into the cells using Lipofectamine 3000 (Invitrogen) for 48 h, according to the manufacturer's recommendations. After transfection, the RNA and protein were extracted after 24 and 48 h, respectively.

Luciferase assay was carried out on extracts from the different breast cancer cells co-transfected for 24/48 h with the corresponding plasmids or miRNAs using a dual-luciferase assay kit (Promega) according to the manufacturer's recommendations. The results were normalized against Renilla luciferase activity. All transfections were performed in triplicate.

MTT, plate colony formation and EdU assays were used to evaluate the ability of cell proliferation.

For the MTT assay, the cells were seeded in 96-well plates (5×10³/well). Cell viability was examined during the following 5 days. After incubation for the indicated time, the cells were incubated with 20 µl MTT (5 mg/ml in PBS; Sigma-Aldrich) at 37°C for 4 h. Then, the medium was removed and the formazan was dissolved in 150 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich). The absorbance was measured at 570 nm using a microplate auto-reader (Bio-Rad).

For the colony formation assay, 24 h after transfection, the cells were seeded into 6-well plates at a density of 500 cells/well. After ~15 days, the cells grew to visible colonies and were stained with crystal violet. The colonies were counted and compared with the control cells.

The EdU assay was performed using the EdU labeling/detection kit (RiboBio) according to the manufacturer's protocol. Briefly, after transfection for 48 h, the cells were incubated with 25 µM EdU for 12 h. The cells were fixed with 4% formaldehyde for 30 min at room temperature and treated with 0.5% Triton X-100 for 15 min at room temperature for permeabilization. After washing with PBS, the cells were reacted with Apollo reaction cocktail for 30 min. Times before fixation, permeabilization, and EdU staining. Subsequently, cell nuclei were stained with Hoechst 33342 at a concentration of 5 µg/ml for 30 min. Then the cells were observed under a fluorescence microscope. The pecentage of EdU-positive cells was examined by fluorescence microscopy.

The invasive ability of the breast cancer cells in vitro was evaluated using Matrigel-coated Transwell inserts (BD Biosciences, San Diego, CA, USA), respectively. Briefly, 5×10⁴ cells in 500 µl serum-free medium were added to the upper chamber, and medium containing 20% FBS was added into the lower chamber. Twenty-four hours later, the migrating cells that had attached to the lower surface were fixed with 20% methanol and stained for 20 min with crystal violet. The membranes were then carved and embedded under coverslips with the cells on the top. The number of migrating cells was counted under a microscope in five predetermined fields.

All the experiments were performed at least twice independently, and data are presented as mean ± standard error mean. All statistical analyses were performed using SPSS18.0 software system for Windows (SPSS Inc.). Statistical significance of difference was calculated using the Student's t-test with significant differences defined as at least a P-value of <0.05.

The expression of miR-410-3p in 30 breast cancer tissues and paired adjacent normal tissues was detected by RT-qPCR. The expression of miR-410-3p was downregulated in 23 (76.7%) of the 30 breast cancer samples (Fig. 1A). To further validate the expression of miR-410-3p in breast cancer, we analyzed miR-410-3p expression profiling data set from The Cancer Genome Atlas (TCGA) including 683 cases of breast cancer tissues and 87 cases of normal breast tissues. The validation data confirmed that the miR-410-3p expression was downregulated in breast cancer tissues (Fig. 1B). Moreover, we compared the cumulative disease-free survival (cum DFS) between patients with high miR-410-3p expression and low miR-410-3p expression and found that the cum DFS of patients with high miR-410-3p expression was higher (n=268) than that of patients with low miR-410-3p expression (n=260) according to the TCGA data (Fig. 1C). Taken together, these results indicate that miR-410-3p is downregulated in breast cancer.

Next, we assessed the miR-410-3p expression levels in various breast cancer cell lines by RT-qPCR. We observed that the miR-410-3p expression was highly expressed in the MCF7 cells and lowly expressed in the MDA-MB-231 cells by RT-qPCR (Fig. 2A). Next, we investigated the influence of miR-410-3p on cell proliferation by transiently transfecting the miR-410-3p mimic or inhibitor, as well as their corresponding controls in the MDA-MB-231 and MCF7 cell lines (Fig. 2B). miR-410-3p overexpression reduced cell growth, colony formation and the number of EdU-positive cells in the MDA-MB-231 cells (Fig. 2C–E; left panels). In contrast, inhibition of miR-410-3p in the MCF7 cells resulted in a higher proliferation rate as assessed by MTT assay, plate colony formation and EdU assays (Fig. 2C–E; right panels). To investigate the role of miR-410-3p in cell invasion we used a Transwell assay. miR-410-3p overexpression reduced cell invasion in the MDA-MB-231 cells, while its inhibition enhanced cell invasion in the MCF7 cells as compared to the control cells (Fig. 2F). These results indicated that miR-410-3p inhibits breast cancer cell proliferation and invasion, suggesting that miR-410-3p is a tumor suppressor in breast cancer.

To elucidate the biological mechanisms underlying the role of miR-410-3p in the inhibition of breast cancer progression, we investigated the potential targets of miR-410-3p. Target prediction programs, miRanda and TargetScan, were applied to identify Snail as a putative miR-410-3p target (Fig. 3A). To further confirm this regulation, Snail 3′-UTR and its mutant containing the putative miR-410-3p binding sites were cloned into the downstream of the luciferase ORF (Fig. 3A). These reporter constructs were co-transfected into MDA-MB-231 cells with the miR-410-3p mimic. Overexpression of miR-410-3p significantly suppressed luciferase activity with inhibition rates of 40% compared to that of the control MDA-MB-231 cells (Fig. 3B; left panel). These effects were abolished when mutated Snail 3′-UTR, in which the binding sites for miR-410-3p were inactivated by site-directed mutagenesis (Fig. 3B; right panel). Functional regulation of Snail expression by miR-410-3p was analyzed by modulating miR-410-3p levels via overexpression in MDA-MB-231 and depletion in MCF7 cells. The Snail mRNA level was decreased in the miR-410-3p-overexpressing MDA-MB-231 cells compared with that in the control cells (Fig. 3C; left panel). Meanwhile, the protein level of Snail was also reduced in the miR-410-3p-overexpressing MDA-MB-231 cells (Fig. 3D; left panel). On the other hand, depletion of miR-410-3p in MCF7 cells resulted in elevated mRNA and protein levels of Snail (Fig. 3C and D; right panels). Collectively, these data support the bioinformatic prediction of Snail as a direct target of miR-410-3p.

To ascertain whether miR-410-3p regulates breast cancer progression through its interaction with Snail, we performed a rescue experiment. We overexpressed Snail in the miR-410-3p-overexpressing MDA-MB-231 cells (Fig. 4A and B) and observed that Snail overexpression greatly impaired the anti-proliferative properties of miR-410-3p, as documented by MTT, colony formation and EdU assays (Fig. 4C–E). Similarly, the ability of invasion was rescued by Snail overexpression (Fig. 4F). To address whether the expression of miR-410-3p is associated with its target, Snail mRNA expression was examined in 30 cases of primary breast cancer tissues by RT-qPCR. There was a significant inverse correlation between miR-410-3p and Snail expression in the breast cancer tissues (Fig. 4G). miR-410-3p-overexpressing MDA-MB-231 cells exhibited a significant upregulation of β-catenin and E-cadherin, while mesenchymal marker vimentin was dramatically downregulated as determined by RT-PCR (Fig. 4H) and western blot analysis (Fig. 4I). Furthermore, overexpression of Snail rescued the effects of miR-410-3p overxpression on the expresssion of β-catenin and vimentin (Fig. 4H and I). Together, these data indicate that miR-410-3p regulates the EMT phenotype by targeting Snail in breast cancer cells.