Breast cancer cell lines MDA-MB-231 and MCF-7 and human normal breast cell line HS578Bst were acquired from the Cell Banks of the Chinese Academy of Sciences (Shanghai, China). The following reagents were used: Roswell Park Memorial Institute-1640 (RPMI-1640) medium and fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA); FBS (HyClone Laboratories, Logan, UT, USA); TRIzol kits, reverse transcription kits, and RT-PCR kits (Invitrogen, Carlsbad, CA, USA); MMP-2, MMP-9, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit anti-human primary polyclonal antibodies (cat. no. 10373-2-AP, 10375-2-AP and 10494-1-AP), and mouse anti-rabbit horse-radish peroxidase (HRP) secondary monoclonal antibodies (cat. no. HRP-60004; Proteintech Group, Inc., Wuhan, China); bicinchoninic acid (BCA) protein quantification kits (Beyotime Institute of Biotechnology, Jiangsu, China); and IHC kit SP-9001 (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). Primer synthesis was performed by Takara (Dalian, China).

A total of 80 female breast cancer patients accompanied by complete clinical data admitted to Yidu Central Hospital of Weifang for treatment between January, 2007 and December, 2010 were selected for this study. Patients were aged 24–78 years (median age, 55 years). All patients were clinically and pathologically diagnosed with breast cancer and received surgical treatment for the first time without any prior history of receiving radiotherapy and chemotherapy. Informed consents were signed by the patients and/or guardians. The study was approved by the Ethics Committee of Yidu Central Hospital of Weifang. Breast cancer tissue samples were extracted from tumors by excision. Tumor-adjacent normal tissues were extracted from an area 10 cm away from the tumor edge (no infiltration of tumor cells was observed by microscopy). Specimens were fixed with 10% formaldehyde and embedded in conventional paraffin after extraction. All patients had complete follow-up records, including age, tumor size, lymph node status, clinical staging and survival condition.

HS578Bst, MCF-7 and MDA-MB-231 cells were cultured in RPMI-1640 medium containing 10% FBS in an incubator at 37°C with 5% CO₂. The medium was changed every other day and cells were digested by Trypsin and then subcultured upon initiation of cell fusion.

Cells were collected during the logarithmic growth phase following digestion and centrifugation. Total RNA was extracted from the sample using TRIzol kits according to manufacturer's instructions. RNA concentration and purity were measured, with an A260/A280 value from 1.8–2.0 deemed acceptable. Reverse transcription was conducted according to the instructions provided in the reverse transcription kit, and mRNA expression was detected according to the methods provided in the RT-PCR kit using the RT-derived cDNA as the template (primer sequences are listed in Table I). GAPDH was selected as the internal reference. The reaction conditions were as follows: 94°C for 5 min; 94°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec for 30 cycles; 72°C for 5 min. Ct values were generated by instrument software (ABI7300; Thermo Fisher Scientific, Waltham, MA, USA). Relative expression levels were calculated using the 2^−ΔCt method according to the following formula: ΔCt (target gene) = Ct (target gene)-Ct (control gene).

Digestion and centrifugation were used to collect cells during the logarithmic growth phase. Cell lysates were generated and protein concentrations were determined by BCA assay. Protein samples (50 µg) were then loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Isolated proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% skim milk powder at room temperature for 2 h. Rabbit anti-human MMP-2, MMP-9, and GAPDH primary polyclonal antibodies (1:1,000) were added, respectively, and membranes were incubated overnight at 4°C. Membranes were then completely washed by Tris-buffered saline with Tween-20 (TTBS) and mouse anti-rabbit secondary monoclonal antibodies (1:2,000) were added, with the membrane incubated for 1 h at room temperature. Enhanced chemiluminescence (ECL) development was conducted in the dark, with a gel imager (Bio-Rad Laboratories, Hercules, CA, USA) used to obtain results. GADPH was used as an internal reference and gray scale analysis was performed.

The procedure was carried out according to the instructions of the IHC kit. Briefly, after paraffin sections were dewaxed, endogenous peroxidases were inactivated with 3% H₂O₂. Citrate buffer was used for thermal remediation and proteins were blocked with 10% goat serum. MMP-2 and MMP-9 primary antibodies (1:100 dilution) were applied, respectively, and samples were incubated at 4°C overnight. After washing with phosphate-buffered saline (PBS) three times, biotin-labeled secondary antibodies were added, with samples allowed to incubate for 15 min. After that, proteins were washed with PBS three times and diaminobenzidine (DAB) solution was applied for color development in the dark. Hematoxylin was used for restaining, and gum was used for mounting. Finally, pictures were taken under a microscope (TE2000-U; Nikon, Tokyo, Japan).

MMP-2 and MMP-9 protein expression was noted in the cytoplasm by the presence of a brown color. The proportion of positive cells among total cells in the entire visual field were recorded and divided into four categories: <10% (−); 10–25% (+); 26–75% (++); >75% (+++). Categories - and + represent low expression while ++ and +++ represent high expression. Scores were calculated and analyzed.

Patients were divided into MMP-2 low expression and high expression groups as well as MMP-9 low and high expression groups according to the IHC results. There were no statistically significant differences in terms of gender, smoking history, alcohol history, and family history among these groups. Regular follow-ups were conducted after surgery for 5 years in the form of phone calls or outpatient reviews. Survival duration was recorded from the first day after surgery to the patient death or the follow-up conclusion deadline. MMP-2 and MMP-9 expression effects on breast cancer patient survival conditions were analyzed statistically based on follow-up results.

Statistical Product and Service Solutions (SPSS) 17.0 (International Business Machines Corp., Armonk, NY, USA) was used in this study. Measurement data were analyzed by using one-way analysis of variance (ANOVA). The data were compared between the two groups using χ² analysis. Clinical prognostic data were analyzed by Kaplan-Meier survival analysis. p≤0.05 was interpreted as a statistically significant difference.

RT-PCR results showed that MMP-2 and MMP-9 mRNA expression levels in MDA-MB-231 and MCF-7 cells were significantly higher than in HS578Bst cells (p<0.01) (Fig. 1).

Western blot results showed that MMP-2 and MMP-9 protein expression in MDA-MB-231 and MCF-7 cells were significantly higher than those in HS578Bst cells (p<0.01) (Fig. 2). Representative western blot analyses for MMP-2 and MMP-9 protein expression (Fig. 2A). Gray-scale densitometry analysis results for MMP-2 and MMP-9 protein expression (Fig. 2B). MMP-2 and MMP-9 protein expression in MDA-MB-231 and MCF-7 cells were significantly higher than in HS578Bst cells, p<0.01.

IHC results showed that the positive expression of MMP-2 and MMP-9 appeared as brown particles in the cytoplasm (Fig. 3). Through statistical analysis, it was found that high expression rates of MMP-2 and MMP-9 were found in the tumors of 83.75% (67/80) and 78.75% (63/80) of the patients enrolled in this study, respectively. Differences in MMP expression between breast cancer tissues and tumor-adjacent normal tissues were statistically significant (p<0.01) (Table II).

Table III shows the analysis results of the relationship between breast cancer clinicopathological indices and MMP-2 and MMP-9 expression. Abnormal expression levels of MMP-2 and MMP-9 were correlated with the occurrence of lymph node metastasis and tumor staging (p<0.05), but not correlated with patient age and tumor size (p>0.05).

A total of 80 patients with breast cancer were followed up, in which 51 patients survived and 29 patients died (Table IV). Kaplan-Meier single factor analysis showed that MMP-2 and MMP-9 expression were associated with a significant effect on patient prognosis (p<0.05). Low MMP-2 and MMP-9 expression indicated a relatively good patient prognosis (Table IV and Fig. 4).