This study was approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical College. Tissue samples and clinical data were collected after the patients or their families signed the informed consent documents. All 62 breast cancer and paracancerous tissue samples (tissues located >2 cm away from the tumour resection margins) examined in the present study were derived from the surgical resection specimens preserved during the 2007–2009 period at the Department of Breast Surgery, Affiliated Hospital of Guilin Medical College. The tissue samples were subjected to pathological examination and associated with complete clinical information. All surgical resection specimens were separately packed and stored in liquid nitrogen within 30 min of resection. None of the patients received palliative surgery, radiotherapy, chemotherapy or other related treatments prior to the surgical resection.

The patients were comparable in terms of age, menopausal status, family history of cancer, ethnicity, pathological type and grade, tumour size, axillary lymph node metastasis and clinical stage. The differences in these parameters were not statistically significant. The histological grade of the breast tumours were assessed according to the World Health Organization (WHO) standards and were classified into grades I–III. Tumour stage was determined based on the seventh edition of the tumour-node-metastasis (TNM) classification of malignant tumours published by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC). The 62 patients were followed up for five consecutive years and were required to undergo a computed tomography (CT) scan and ultrasound once every 6 months.

MCF-10F human breast epithelial cells and MCF-7, MDA-MB-231 and SKBR-3 human breast cancer cells were purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The human breast epithelial cells and breast cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute (RPMI)-1640 medium, respectively. All media were supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 U/ml of streptomycin. The cells were cultured at 37°C in a humidified 5% CO₂ incubator.

Breast cancer cells were maintained at a logarithmic growth phase until 75% confluency. The cells were then transduced with 5 µl of virus (titre, 1.0×10⁹ TU/ml). In addition, polybrene was added to each well of cells. After 12 h of viral infection, the virus-containing medium was replaced with fresh conventional culture medium, and the cultured cells remained at 37°C in a 5% CO₂ incubator. At 4 days after viral infection, fluorescence within the cells was observed and photographed. In addition, the mRNA or total protein was extracted based on the goals of each experiment.

The total RNA was extracted from breast cancer tissues and cells using the TRIzol method. After quantification, total RNA was reverse transcribed into total complementary DNA (cDNA) using the Takara RT kit in accordance with the manufacturer's instructions. The PCR was performed using the cDNA as a template and the appropriate proportion of pre-synthesized PCR primers. The sequences of the DOR primers were 5′-ACCAAGATCTGCGTGTTCCT-3′ for the upstream primer and 5′-CGATGACGAAGATGTGGATG-3′ for the downstream primer. The sequences of the β-actin primers were 5′-CTGGGACGACATGGAGAAAA-3′ for the upstream primer and 5′-AAGGAAGGCTGGAAGAGTGC-3′ for the downstream primer. The volume of the PCR reaction was 50 µl. The PCR reaction conditions consisted of 31 cycles of 94°C for 2 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec. The obtained PCR products were subjected to agarose gel electrophoresis (1.5% gel). The gel was scanned and analysed using a gel imaging system.

The total protein was extracted from tissues and cells and quantified using the bicinchoninic acid (BCA) assay. The protein samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membrane was blocked at 4°C overnight in a Tris-buffered saline/Tween-20 (TBST) blocking solution containing 5% non-fat dry milk. After blocking, the membrane was incubated with the primary antibody at 37°C for 1 h. The membrane was washed three times for 10 min each wash with TBST and then incubated with the secondary antibody at 37°C for 1 h. The membrane was washed three more times for 10 min each wash with TBST. Subsequently, the proteins were visualized using enhanced chemiluminescence (ECL) reagents. The images were scanned and analysed.

Logarithmically growing cells were selected. Each group of cells was treated for 48 h. Subsequently, the cells were incubated in the presence of 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) for 4 h at 37°C in a 5% CO₂ incubator. The spent solution was discarded and 150 µl of dimethyl sulphoxide was added to each well of cells. The cells were then agitated at low speed for 10 min on a shaker at room temperature. The optical density (OD₄₉₀) value was determined using a microplate reader.

The cells were collected and fixed with pre-cooled 70% ethanol at 4°C overnight. The cell density was adjusted to 1×10⁶ cells/ml. Subsequently, 50 mg/l of RNase, 100 mg/l of propidium iodide (PI) and 1 ml/l of Triton X-100 were added to the cells. The cells were incubated in the dark for 15 min, and the labelled cells were examined by flow cytometry.

The cells were digested with trypsin and then resuspended at a density of 1×10⁶ cells/ml. After the addition of 5 µl of Annexin V-fluorescein isothiocyanate (FITC) and PI, the cells were incubated at 37°C for 15 min in the dark. Subsequently, the cells were analysed by flow cytometry.

The animal experiments were conducted after receiving approval from the Medical Ethics Committee of Guilin Medical College. All the mice used in the present study were purchased from the Experimental Animal Centre of Guilin Medical College. The mice were male, aged 6–8 weeks, and weighed 20±0.5 g. The mice were randomly divided into two groups, and each group contained five mice. Xenograft tumours were established using the conventional method of subcutaneous inoculation. Four weeks later, the mice were euthanized by cervical dislocation. The xenograft tumours were collected for subsequent experiments.

All experimental data were subjected to statistical analysis using SPSS 16.0 statistical software. The quantitative data were expressed as the means ± standard deviation. Comparisons between two groups were conducted using t-tests. The relationship between DOR and clinical pathology data was assessed using the Chi-square test. The differences in survival rate were assessed using the Kaplan-Meier survival analysis. In addition, survival rates were compared between the groups using the log-rank test. P<0.05 indicated a statistically significant difference between two groups.

RT-PCR and western blot analysis were completed to analyse DOR expression in breast cancer tissues and cells. The examination of the 62 breast cancer and paracancerous tissues revealed that DOR mRNA was highly expressed in cancer tissues. In contrast, the corresponding paracancerous tissues either expressed a low level of DOR mRNA or showed no DOR mRNA expression (p<0.05). Among the examined cell lines, MCF-7 and SKBR-3 cells highly expressed DOR, and MDA-MB-231 and MCF-10F cells showed low or no DOR expression (p<0.05) (Fig. 1A). To further verify the RT-PCR results, a western blot analysis was performed. The results showed that the expression level of DOR protein was significantly higher in breast cancer tissues than in the corresponding paracancerous tissues (p<0.05). In addition, the expression level of DOR protein was higher in various types of breast cancer cells than in normal breast epithelial cells (p<0.05) (Fig. 1B).

The relationship between DOR expression in breast cancer and clinical pathology data was examined. Among the 62 breast cancer patients, 54 patients (83.1%) showed high DOR expression in cancer tissues, whereas only 19 patients (30.6%) showed DOR expression in paracancerous tissues. This difference was statistically significant (p<0.05). DOR expression was positively correlated with tumour metastasis (p<0.05) and clinical stage (p<0.05, Table I). In contrast, no correlation existed between DOR expression and other clinical parameters, including age, tumour size, tumour location and the presence/absence of tumour recurrence.

All of the patients were subjected to regular follow-ups. The Kaplan-Meier survival curves were analysed, and the results showed that the overall survival rate was significantly lower in patients from the DOR-positive group when compared with the DOR-negative group (Fig. 2). The survival rates were compared between the groups using a log-rank test, and the results revealed that high DOR expression was related to a poor prognosis in breast cancer patients (p<0.05). The results indicate that high DOR expression exerted an adverse effect on the prognosis of patients with breast cancer.

Based on the expression status of DOR in breast cancer cells, we selected two cell lines, MCF-7 and SKBR-3, to investigate the effect of DOR on the proliferation of breast cancer cells. [D-Ala², D-Leu⁵]enkephalin (DADLE) is a specific DOR agonist. The proliferation rates of the breast cancer cells after DADLE treatment were determined using an MTT assay. The OD₄₉₀ value of the breast cancer cells showed a gradual dose-dependent increase at DADLE concentrations between 10 nM and 1.0 µM. In contrast, the OD₄₉₀ value of the control group did not significantly change (p<0.05). However, when the concentration of DADLE exceeded 1.0 µM, the OD₄₉₀ value of the breast cancer cells did not show a further increase (Fig. 3). The results demonstrate that activation of DOR promoted the proliferation of human breast cancer cells. In addition, this DOR effect was concentration-dependent.

The RNA interference (RNAi) technique was employed to silence DOR expression in the breast cancer cell line MCF-7. A 48-h siDOR transfection led to the successful silencing of DOR expression. The OD₄₉₀ value was significantly lower in the siDOR-transfected group when compared with the control group and the group transfected with a negative control oligonucleotide (p<0.05) (Fig. 4A). The results indicate that DOR is capable of inhibiting breast cancer cell proliferation. To further clarify the mechanism used to affect the proliferation of human breast cancer cells, flow cytometry was performed to examine apoptosis and the cell cycle. The apoptotic rate was significantly increased in breast cancer cells after RNAi-mediated silencing of DOR when compared with the control group and the negative control oligonucleotide-transfected group (p<0.05). The expression levels of caspase-3, caspase-9 and cytochrome C protein were also elevated in breast cancer cells after DOR silencing when compared with the control group and the negative control oligonucleotide-transfected group. The expression levels of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL) were reduced in breast cancer cells after DOR silencing compared to those in the control group and the negative control oligonucleotide-transfected group. The majority of the tumour cells were arrested in the G1 phase after silencing of DOR (p<0.05). In addition, the levels of cyclin-dependent kinase 4 (CDK4) and cyclin D1 were decreased and the levels of p15 and p21 were increased in these cells (Fig. 4B–E). These results indicate that the inhibitory effect of DOR on breast cancer progression is closely related to the promotion of apoptosis and the blockage of cell cycle progression.

Four weeks after the subcutaneous inoculation of tumour cells, the rate of tumour formation reached 100% in the si-Control group. The rate of tumour formation was 80% in the si-DOR group. The xenograft tumour grew more rapidly in the si-Control group than in the si-DOR group. In addition, the volume and weight of the xenograft tumours were significantly reduced in the si-DOR group compared to those in the si-Control group at all time points examined (p<0.05) (Fig. 5A–D). The results indicate that DOR inhibits tumourigenesis in nude mice.

The phosphorylation levels of PKC and ERK proteins were examined to verify the specific role of the PKC/ERK signalling pathway in the DOR-mediated effects on human breast cancer progression. The results showed that activation of DOR led to significantly increased phosphorylation of the PKC protein in the cytoplasm (p<0.05). In addition, the intracellular phosphorylation level of the ERK proteins increased accordingly (p<0.05). The results demonstrate that DOR induced the phosphorylation of PKC and ERK proteins. However, treatment of the tumour cells with PKC-specific antagonist GF109203X (10 µM) significantly inhibited the proliferation of tumour cells, regardless of DOR activation. Moreover, blocking the PKC pathway resulted in a decline in ERK phosphorylation levels (Fig. 6). The results demonstrate that the PKC pathway plays a critical role in the DOR-mediated effects on the progression of human breast cancer.