For the current study, 30 paired FFPE breast cancer and non-cancerous samples were obtained from patients who underwent surgical treatment for breast cancer between January 2004 and December 2005 at Sun Yat-sen University Cancer Center (Guangzhou, Guangdong, China). In addition, 161 FFPE breast cancer samples were obtained from patients who underwent radical mastectomy between June 2002 and December 2006 at the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, Guangdong, China). All breast cancer samples were pathologically diagnosed as invasive ductal carcinoma by two experienced pathologists. None of the 161 patients had distant metastasis at diagnosis. The percentage of tumor cells in the FFPE breast cancer tissues was >70%. The 161 patients had not received radiotherapy or chemotherapy prior to mastectomy. Clinical staging was based on the American Joint Committee on Cancer staging manual (25). The clinical characteristics of the 161 patients are summarized in Table I. The median follow-up time for patients from the First Affiliated Hospital of Guangzhou Medical University was 75.2 months (range, 6.7–115.8). Overall survival (OS) time was calculated between the date of surgery and the date of mortality due to any cause or of last follow-up, and the disease-free survival (DFS) time was calculated between the date of surgery and the date of the first distant metastasis, relapse or mortality due to any cause. The study was reviewed and approved by the Ethics Committees of Sun Yat-sen University Cancer Center and the First Affiliated Hospital of Guangzhou Medical University.

The breast cancer and non-cancerous tissues were cut into 8–10 µm sections, deparaffinized and rehydrated. Total RNA was extracted from the tissue sections using a modified phenol/chloroform extraction method, as previously described (26). Total RNA from cell lines was extracted using Invitrogen TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. A NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.) was used to estimate the concentration and quality of total RNA.

Reverse transcription was performed on the 30 paired tissue samples using 10 ng of total RNA, 1X miRNA-specific reverse transcription primers (Thermo Fisher Scientific, Inc.), 100 µM nucleoside triphosphates (Thermo Fisher Scientific, Inc.), 3.33 U/µl MultiScribe™ Reverse Transcriptase (Thermo Fisher Scientific, Inc.), 1X Reverse Transcription Buffer (Thermo Fisher Scientific, Inc.) and 1.33 U/µl RNase inhibitor (Thermo Fisher Scientific, Inc.) in a final volume of 15 µl. The reaction was conducted at 16°C for 30 min, followed by 30 min at 42°C and 5 min at 85°C. The qPCR reaction was performed using a 20 µl volume containing 1.33 µl reverse transcription products, 1X TaqMan® Small RNA Assay solution (including specific primers and probes; Applied Biosystems; Thermo Fisher Scientific, Inc.) and 1X Universal PCR Master Mix II (no UNG; Thermo Fisher Scientific, Inc.). The RT-qPCR was performed in triplicate for each sample using an Applied Biosystems PRISM 7900HT System (Thermo Fisher Scientific, Inc.) with the following conditions: 50°C for 2 min; 95°C for 10 min; and 45 cycles of 95°C for 15 sec and 60°C for 60 sec. The primer sequences used were as follows: MiR-711 forward, 5′-ACACTCCAGCTGGGGGGACCCAGGGAGAGA-3′; and reverse, 5′-TGGTGTCGTGGAGTCG-3′. Small nuclear RNA U6 was used as a normalization control. The 2^−ΔΔCq equation was used to represent the relative expression of miRNA (27) and Student's t-test was used to analyze the results.

MCF-7, MDA-MB-231 and ZR-75-30 human breast cancer cell lines were cultured in Gibco Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% Gibco fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C with an atmosphere of 5% CO₂. The miR-711 mimic, inhibitor and miRNA negative control (NC) were designed and synthesized by Shanghai GenePharma, Ltd. (Shanghai, China). When the cells reached 60–70% confluence, Invitrogen Lipofectamine® 2000 RNAiMAX reagent (Thermo Fisher Scientific, Inc.) was used to perform the transfection of cells with 100 nM miR-711 mimic or inhibitor, or NC, according to the manufacturer's protocol.

Following transfection for 36 h, the cells were cultured in 96-well plates (1,000 cells/well) for an additional 7 days. The growth of the cells was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At a specific time every day for 7 days, 20 µl MTT (dilution, 0.5 mg/ml; Weijia Biology Science and Technology Co., Ltd, Guangzhou, China) was added, followed by additional incubation for 4 h at 37°C. Following the removal of the supernatant, 150 µl dimethyl sulfoxide (Weijia Biology Science and Technology Co., Ltd) was added to each well. The cells in the 96-well plate were gently agitated for 10 min and the absorbance was measured at 490 nm (SpectraMax® M5 Multi-Mode Microplate Reader; Molecular Devices LLC, Sunnyvale, CA, USA) to calculate the cell growth rate.

At 36 h subsequent to transfection, MCF-7 cells were seeded on a 6-well plate (1,000 cells/well) and incubated at 37°C in an atmosphere of 5% CO₂ for 2 weeks. The surviving colonies (>50 cells/colony) were counted using crystal violet staining.

Following transfection with miR-711 inhibitors for 48 h, the cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 400 µl 1X Binding Buffer (Bestbio Co., Shanghai, China). The cells were stained with Annexin V-fluorescein isothiocyanate (Bestbio Co.) for 15 min at 4°C. Subsequently, propidium iodide was added to the cells for 5 min at 4°C. The percentage of apoptotic cells was analyzed using flow cytometry (FC 500; Beckman Coulter, Inc., Brea, CA, USA).

A Boyden chamber Transwell assay (24-well Transwell plate; BD Biosciences, Franklin Lakes, NJ, USA) was used to measure cell migration and invasion, and was performed according to the manufacturer's protocol. For Transwell migration, 5×10⁴ MDA-MB-231 cells were plated into the top chamber of the Transwell plate in DMEM without FBS. The bottom chamber of the Transwell plate was filled with DMEM supplemented with 10% FBS to stimulate migration. Following an incubation period of 16 h, the cells in the membrane of the bottom chamber were fixed with 100% methanol, stained with 0.1% crystal violet (Weijia Biology Science and Technology Co., Ltd) and counted under a microscope (BX53; Olympus Corp., Tokyo, Japan). For Transwell invasion, the membrane of the top chamber of the Transwell plate was coated with Matrigel (BD Biosciences) and 2×10⁵ cells were plated into the top chamber. Following an incubation period of 24 h, the fixed and stained cells in the bottom chamber were counted.

The Kaplan-Meier method and the log rank test were used to analyze OS time and DFS using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Univariate and multivariate Cox regression analysis were performed to identify the independent prognostic factors for survival. The association between miR-711 and clinical characteristics of the patients was analyzed using Student's t-test, χ² test or Fisher's exact test. SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for all analysis. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether miR-711 is dysregulated in breast cancer, 30 paired FFPE breast cancer and non-cancer tissue samples were collected, and miR-711 expression was assessed in the samples using TaqMan RT-qPCR. The findings demonstrated that the expression level of miR-711 in breast cancer tissues was significantly increased compared with the paired non-cancerous breast tissues (Fig. 1A). This suggests that miR-711 may be a potential oncogene in breast cancer.

To additionally investigate the clinical significance of overexpressed miR-711, RT-qPCR analysis was conducted in 161 FFPE breast cancer samples. Based on the miR-711 expression levels measured by RT-qPCR, the 161 patients were divided into a high or low miR-711 expression group using the median expression level as the cut-off point (0.57; range, 0.23 to 1.21). The associations between miR-711 expression level and clinical characteristics were evaluated; however, no significant associations were observed (Table I). By contrast, a Kaplan-Meier survival analysis indicated that patients in the high miR-711 expression group demonstrated significantly poorer OS and DFS times compared with patients in the low-expression group (P=0.002 and P=0.004, respectively; Fig. 1B and C).

Based on the findings that miR-711 expression is significantly associated with survival time of patients with breast cancer, the present study investigated whether miR-711 is an independent prognostic factor for breast cancer. The effects of miR-711 expression and various clinical characteristics on the survival time of 161 patients was analyzed using univariate and multivariate Cox regression models. The results demonstrated that miR-711 expression was an independent prognostic factor for OS time [hazard ratio (HR), 2.549; 95% CI, 1.303–4.988; P=0.006] and DFS time (HR, 2.873; 95% CI, 1.392–5.929; P=0.004) in patients with breast cancer (Table II). Tumor-node-metastasis stage was also found to be an independent prognostic factor for OS (HR, 1.928; 95% CI, 1.162–3.198; P=0.011) and DFS (HR, 2.068; 95% CI, 1.209–3.536; P=0.008). Furthermore, miR-711 was not associated with other clinical characteristics, suggesting that this miRNA independently affects the survival time of patients with breast cancer. Overall, the results indicate that miR-711 is an independent prognostic factor in breast cancer.

In order to understand how miR-711 may affect the survival time of breast cancer patients, the present study investigated the mechanisms by which miR-711 promotes the development and progression of breast cancer. MCF-7, MDA-MB-231 and ZR-75-30 human breast cancer cell lines were transiently transfected with a miR-711 mimic and the cell proliferation was observed. MTT assay demonstrated that the growth rate of the cells transfected with miR-711 mimic was significantly increased compared with cells transfected with the NC miRNA (P=0.002; Fig. 2A). Subsequently, cells transfected with a miR-711 inhibitor were analyzed. The findings demonstrated that the proliferation of the cells transfected with the miR-711 inhibitor were markedly decreased compared with cells transfected with NC (P=0.001; Fig. 2B). A colony formation assay was performed on MCF-7 cells transfected with a miR-711 mimic or inhibitor. As expected, the colony numbers of MCF-7 cells transiently transfected with miR-711 mimics was significantly increased compared with MCF-7 cells transfected with NC (P=0.007; Fig. 3A), and the colony numbers were significantly decreased in cells transfected with a miR-711 inhibitor compared with cells transfected with NC (P=0.008; Fig. 3B). These findings indicate that miR-711 promotes the oncogenic growth of breast cancer cells.

The present study aimed to identify whether apoptosis affects the growth of breast cancer cells induced by miR-711. In the apoptosis assay, MCF-7 cells were transiently transfected with a miR-711 inhibitor or NC, and flow cytometry was used to evaluate the results. The findings demonstrated that the apoptotic rate of MCF-7 cells transfected with the miR-711 inhibitor was markedly increased compared with cells transfected with NC (Fig. 4). This suggests that downregulation of miR-711 may induce apoptosis in breast cancer cells. Therefore, miR-711 may inhibit cell apoptosis, leading to the proliferation of breast cancer cells.

To additionally investigate miR-711 in the progression of breast cancer, migration and invasion assays were conducted using Transwell plates. MDA-MB-231 cells were transiently transfected with miR-711 mimic, inhibitor or NC and were seeded in the wells of the Transwell plate. The ectopic expression of miR-711 significantly promoted the migration and invasion of MDA-MB-231 cells (Fig. 5A and C), and the downregulation of miR-711 using a miR-711 inhibitor resulted in marked inhibition of migration and invasion of the cells (Fig. 5B and D). Therefore, the present results indicate that miR-711 serves a functional role in tumor progression and metastasis, which is consistent with the poor survival time observed in patients with breast cancer who have an increased expression of miR-711.