Tumor tissues and matched adjacent normal tissues (20 breast cancer tissue samples and 20 normal adjacent tissue samples) were obtained from breast cancer patients at the Department of General Surgery, Sun Yat-Sen University Cancer Center, from 2010 to 2013. All of the patients had an accurate histological diagnosis according to the clinicopathological criteria of the International Union for Cancer Control (UICC). The median age of the patients was 49.6 years (range, 34–72 years). All of the malignant tissues were from stage II-III tumors, according to the International Federation of Gynecology and Obstetrics (FIGO) classification. All patients provided consent for the use of their specimens in research, and this use was approved by the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Center.

Homo sapiens breast cancer SKBR-3, MDA-MB-435S and MCF-7 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The breast cancer cells were maintained according to the vendor's instructions. In brief, SKBR-3 cells were cultured in McCoy's 5A medium (modified) (ATCC) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin). MDA-MB-435S cells were cultured in Leibovitz's L-15 medium (ATCC) with 10% FBS, 0.01 mg/ml bovine insulin, 0.01 mg/ml glutathione and 1% penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin). MCF-7 cells were cultured in Eagle's minimal essential medium (EMEM) (ATCC) with 10% FBS and 1% penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin). All cells were cultured and maintained in a humidified incubator under standard conditions.

AMOT 3′-UTR containing putative binding sites for miR-205 were amplified from the normal human genome DNA and cloned into downstream of the psi-CHECK2 vector (Promega, Madison, WI, USA), and named AMOT-3′-UTR-WT. AMOT mutant 3′-UTR recombi-nant plasmid was generated by the QuikChange Site-Directed Mutagenesis kit (Stratagene, USA), which generated a mutation of 7 bp from ATGAAGG to TACGGTC in the predicted miR-205 target binding site, named as AMOT-3′-UTR-MUT. The full length cDNA encoding human AMOT was amplified and the recombinant plasmid, pcDNA3.1/AMOT was constructed. All plasmids were confirmed by DNA sequencing.

The miR-205 mimics, and the scrambled sequence pre-miR negative control (NC) were purchased from a commercial manufacturer (RiboBio, China) and small interfering RNA (siRNA) targeting AMOT was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells (1×10⁵/well) were seeded in 24-well plates and incubated for 24 h, and then the cells were transfected with miRNA mimics (50 nM) or miR-NC (50 nM) or siRNA (50 nM) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in serum-free medium in accordance with the manufacturer's instructions.

Total RNA and miRNA were extracted from the tissues or the cultured cells using TRIzol reagent (Invitrogen). The relative expression of miRNA-205 was quantitated using a SYBR PrimeScript miRNA RT-PCR kit (Takara, China) in accordance with the manufacturer's instructions, and the relative amount of miRNA was normalized to U6 using the comparative threshold cycle method. For quantitative analysis of mRNA expression, 2 µg of total RNA was used to synthesize complementary DNA (cDNA) using M-MLV reverse transcriptase (Promega, USA), and the corresponding cDNA was used for quantitative PCR using SYBR-Green Real-Time Master Mix (Toyobo, Japan), and a constitutive expression gene, GAPDH, was used as an internal control to verify the fluorescent real-time RT-PCR reaction. The sequence-specific primers were synthesized by Sangon (Shanghai, China) (Table I). qPCR was performed using the Applied Biosystems 7500 Sequence Detection system (ABI, USA).

Total protein was extracted with SDS lysis buffer (Beyotime, China) and the concentration of total proteins was determined with the BCA protein assay kit (Pierce, USA). Equal amounts of total proteins were separated on 10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were subjected to overnight blocking in Tris-buffered saline (TBS) containing 5% non-fat dried milk and incubated with a primary antibody [AMOT, 1:1,000; and GAPDH, 1:3,000 (both from Abcam)] for 1 h at 37°C, and then the membranes were washed and subsequently treated with the secondary antibody (goat anti-rabbit IgG; Boster) at a 15,000 dilution for 1 h at room temperature and visualized by chemiluminescence.

For the reporter assay, the MCF-7 cells were cultured in 24-well plates one day before transfection. AMOT-3′-UTR-WT or -MUT vectors (100 ng) were co-transfected with 100 nM miR-205 mimics or negative control into MCF-7 cells using Lipofectamine 2000 reagent as previously described. After forty-eight hours of transfection, luciferase activities were measured using a Dual-Luciferase Reporter assay system (Promega) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. Three independent experiments were performed, and the data are presented as the mean ± SD.

The cell proliferation activities of the MCF-7 cells with miRNA mimics or siRNA duplexes or miR-205 and AMOT were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) assay at 0, 24, 48, 72 and 96 h. After a 24-h transfection, the MCF-7 cells were seeded into 96-well culture plates at 1×10⁴/well in a final volume of 100 µl. At the indicated time points, 20 µl of MTT (5 mg/ml) was added into each well for a 4-h incubation at 37°C. Following removal of the culture medium, the remaining crystals were dissolved in 200 µl dimethylsulfoxide (DMSO) (Sigma) and the absorbance at 450 nm was measured. Three independent experiments were performed.

Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC/PI apoptosis detection kit (BestBio, China) in accordance with the manufacturer's instructions. Briefly, the cells were harvested with 0.5% trypsin, and washed with cold PBS, and then centrifuged at 200 × g for 5 min. The cells (1×10⁶) were resuspended and stained using the Annexin V-FITC/PI apoptosis detection kit according to the manufacturer's instructions. Then, samples were analyzed using a Becton-Dickinson flow cytometer. Each experiment was performed three times, and data are shown as mean ± SD.

For the cell invasion assay, 24-well Transwell chambers (Millipore) were used according to the manufacturer's protocol. In brief, 1×10⁵ cells in 200 µl serum-free EMEM were added into the upper chamber of the Matrigel-coated inserts, and 500 µl of EMEM containing 10% FBS was added to the lower chamber for culture. The cells were cultured at 37°C in 5% CO₂ in a humidified atmosphere for 36 h, and the non-invading cells were removed. Then, the cells were fixed, and stained using 0.5% crystal violet (Sigma), and the invasive cells were counted under an inverted microscope (Olympus IX71; Japan).

For quantitative data, all experiments were performed at least three times, and all samples were tested in triplicate. All data are expressed as the mean ± SD. Statistical significance between groups was determined using one-way analysis of variance (ANOVA) or an unpaired Student's t-test using SPSS 18.0 (SPSS, Inc., USA). A P-value of <0.05 was considered to indicate a statistically significant result.

We first detected the expression level of AMOT by real-time PCR and western blotting. As shown in Fig. 1, the expression of AMOT was significantly upregulated in the tumor tissues when compared with that of the matched normal tissues both at the mRNA (Fig. 1A) and protein levels (Fig. 1B). The data revealed that AMOT is overexpressed in breast cancer tissues, suggesting that increased AMOT protein expression contributes to breast cancer development.

In order to determine whether miR-205 regulates AMOT expression in breast cancer, miRNA target predication databases were used for computational analyses, including TargetScan (www.targetscan.org), miRDB (http://mirdb.org/miRDB) and microRNA (www.microrna.org). miR-205 was found to have one predictive target site in the human AMOT 3′-UTR (Fig. 2A). The expression of mature miR-205 in the tumor and matched normal tissues was examined by real-time PCR. As shown in Fig. 2B, miR-205 was significantly down-regulated in the cancer tissues when compared with that in the matched normal tissues. The results showed that the expression of miR-205 and AMOT in the tumor tissues is inversely correlated. Therefore, miR-205 was selected to analyze the role of AMOT in breast cancer.

To investigate whether or not miR-205 inhibits the expression of the endogenous AMOT protein, the expression level of miR-205 and AMOT in three breast cancer cell lines were examined by real-time PCR and western blotting, respectively. miR-205 was significantly expressed at a low level in the MCF-7 cells (Fig. 3A), and the expression level of AMOT was significantly higher in the MCF-7 cells (Fig. 3B). Thus, the MCF-7 cell line was chosen to be used in the present study. The dual-luciferase reporter vectors were constructed containing wild-type or mutant-type seed sequences in the 3′-UTR of AMOT (Fig. 3C). Overexpression of miR-205 markedly reduced the expression of AMOT at the protein level in the MCF-7 cells (Fig. 3D), yet did not affect the AMOT mRNA level (Fig. 3E). The MCF-7 cells were co-transfected with AMOT-3′UTR-WT and miR-205 mimics which resulted in a significantly reduced activity of the luciferase reporter gene, yet the luciferase activity was not significantly attenuated in the target region of the mutated AMOT-3′UTR-MUT construct (Fig. 3F). Based on the results, miR-205 directly targets the 3′UTR of AMOT and downregulates AMOT expression.

To evaluate the role of miR-205 in tumor cell proliferation and invasion, miR-205 was overexpressed in the MCF-7 cells by transfection with miR-205 mimics (Fig. 4A). Overexpression of miR-205 as well as transfection with AMOT-siRNA markedly reduced the expression level of the AMOT protein (Fig. 4B). As shown in Fig. 4C, the growth of miR-205- and AMOT-siRNA-transfected cells was significantly suppressed compared with the NC-transfected MCF-7 cells (P<0.05 at 72 and 96 h). The role of miR-205 in tumor cell invasion was detected using Transwell assay. miR-205- and AMOT-siRNA-transfected cells exhibited much less invasive ability when compared with the negative control cells (Fig. 4D and E). When MCF-7 cells were transfected with miR-205 inhibitors, cell proliferation and invasion were not affected (data not shown). These results indicate that the miR-205-mediated growth and inhibition of invasion occur in an AMOT-dependent manner in MCF-7 cells.

In order to evaluate the effect of miR-205 on cell apoptosis, the Annexin V-FITC/PI staining method was used to perform the apoptosis assays. The data demonstrated that overexpression of miR-205 as well as AMOT-siRNA transfection did not affect cell apoptosis in the MCF-7 breast cancer cell line when compared with the negative controls (Fig. 5).

For further study, AMOT was over-expressed in miR-205-overexpressing MCF-7 cells as shown by western blotting (Fig. 6A). Moreover, overexpression of AMOT in the MCF-7-overexpressing miR-205 cells significantly decreased the inhibitory effect of miR-205 overexpression on breast cancer cell proliferation and invasion (Fig. 6B–D), but did not affect cell apoptosis (Fig. 6E and F).