The clinical samples used in the present study were all from surgical specimens of patients with breast cancer admitted to the Henan Province People's Hospital between June 2012 and April 2014. The patients were aged between 25 and 56 years, and the average age was 45–23.4 years. Normal women were aged 26–58 years, and the average age was 43.3–23.9 years. The study patients were randomly divided into the control and observation groups. The control group included 27 normal women, and the observation group included 27 women with breast cancer.

The breast cancer detection kit used in this study was purchased from Roche Diagnostics (Basel, Switzerland). Other relevant drugs were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Relevant fluorescence quantitative primers were produced by Takara Bio (Dalian, China). Primary mouse monoclonal MEG-A3 antibody was provided by PeproTech (Rocky Hill, NJ, USA; cat. no. 60054-1; dilution, 1:500). Peroxidase-conjugated secondary polyclonal goat-anti-mouse antibody (1:1,000) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; cat. no. sc-395763).

Tissue (0.1 g) was dispensed in 0.45 ml of RNA Plus and homogenized in the precooling mortar. Subsequently, the contents were transferred to the 1.5-ml EP tube and 0.45 ml of RNAPlus was added. Chloroform (200 µl) was added to the tube, followed by centrifugation at 10,000 × g, at 4°C for 15 min. The supernatant was transferred into the EP tube containing dimethyl carbinol and the contents were homogenized on ice for 10 min. The solution was centrifuged at 12,000 rpm, at 4°C for 10 min, and then 750 µl of 75% ethanol was added. The tube contents were centrifuged at 12,000 rpm, 4°C for 10 min. The supernatant was discarded and RNase-free water was added to resuspend the pellet. The extracted RNA was quantified for purity and concentration with a UV-visible spectrophotometer (BioSharp, Hefei, China).

The procedure was conducted according to the protocol of the kit (Takara fluorescence quantitative PCR specification).

The procedure was conducted according to the specification of the ELISA kit (8). The standards were diluted according to the proportion of 1:25 with assay buffer, and the standard curve was designed. The sample to be tested was first diluted at a 1:100 ratio and then TMB chromogenic substrate was added to each well. After incubation for 2 h at 20°C, the absorbance was determined at 495 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The content and concentration of MEG-A3 in each sample were calculated according to the standard curve.

The control and test samples were incubated using streptavidin-peroxidase (SP) as previously described (8). The criteria for the evaluation of the immune system were as follows (9): If capsule staining was <10% or tumor cells exhibited a negative reaction after staining, it was determined as negative. If only the cell membrane was stained or capsule staining showed >10% content of tumor cells, it was determined as positive. Over 10% of tumor cells showing a weak or medium complete stain was considered as strongly positive (++), whereas >10% of tumor cells showing strong complete membrane staining was determined as strongly positive (++).

A Roche animal cell protein extraction kit was used to extract the total protein of samples (10). Western blotting was carried out as previously described (10).

SPSS 20.0 statistical software (Chicago, IL, USA) was used for data analysis. The correlative measurement results were indicated as mean ± SD. P<0.05 was considered statistically significant.

The expression of MEG-A3 mRNA in patients with breast cancer was higher than that in controls, and the expression level was different in patients with a different course of disease (Fig. 1). Comparison of the MEG-A3 mRNA levels for normal and observation group showed that the average expression level of MEG-A3 gene in patients with breast cancer was 6- to 7-fold significantly higher than that for controls (p<0.05). Thus, there is a certain correlation between MEG-A3 gene and breast cancer.

The ELISA results showed that the average expression of MEG-A3 in serum of healthy women was ~7.14–5.76 ng/ml. The results of MEG-A3 in serum of 27 female patients with breast cancer showed that its average level of MEG-A3 in serum was 12.47–10.17 ng/ml. It indicated that levels in serum of female patients with breast cancer was significantly higher than those in normal controls (Table I).

The immunohistochemical staining of breast cancer tissues for MEG-A3 revealed that the positive staining of MEG-A3 expression was mainly concentrated in the breast cancer cell membrane (A/B) (Fig. 2). The main feature was yellow brown small particles of non-uniform sizes, which was not identified in the breast tissue of normal women.

The result of MEG-A3 mRNA expression levels of patients with different stages of breast cancer is shown in Fig. 3. The data analysis showed that MEG-A3 mRNA expression levels of patients was increased with the prolongation of the disease. The increase had a downward-ascend-downward trend, especially between 8 and 18 months of disease. The mRNA expression level was significantly increased, which indicated that there was an interrelation between MEG-A3 gene and breast cancer. Thus, there is a positive correlation between the level of MEG-A3 gene expression and the severity of breast cancer patients.

MEG-A3 protein expression was detected in the patients with breast cancer at different time points using western blotting (Fig. 4). It was identified that MEG-A3 protein expression level in the serum of patients increased with the prolongation of the disease, and the increments increased apparently since six months after diagnosis with disease. Thus, there was a positive correlation between the level of MEG-A3 in serum and the severity of breast cancer patients.

The detection result of MEG-A3 expression level in the tissue of 27 patients with breast cancer showed that the level was 24.62–9.24 ng/ml. The MEG-A3 content in tissue of normal women was 11.7–5.22 ng/ml, and significant differences were detected (P<0.05). The detection of MEG-A3 expression level in serum of 27 patients with breast cancer showed that the level was 12.47–10.17 ng/ml. The MEG-A3 content in tissue of normal women was 7.14–5.76 ng/ml, and significant differences were observed (P<0.05). There was a positive correlation between the content of MEG-A3 in tissue and serum (r=0.401). The serological detection method was consistent with the histological detection method (κ consistency check κ=0.392).