Animals and Sampling
Female TA2 mice (five month-old TA2 mice and spontaneous breast cancer-bearing TA2 mice) were purchased from the Experimental Animal Center of Tianjin Medical University. The Animal Ethics Committee of National Research Institute for Family Planning Beijing approved the animal experimentation protocols and all animal experiments were performed according to guidelines (Guidelines for the Care and Use of Laboratory Animals) established by the Chinese Council on Animal Care.
A total of 12 five month-old mice and 28 cancer-bearing mice were used in this study. As for the 28 cancer-bearing mice, spontaneous breast cancer was found with an average of 307 days after birth (213 days to 408 days). After euthanasia, mammary glands and spontaneous breast cancer tissues were collected from each cancer-bearing animal. Two abdominal mammary glands were collected from the five month-old mice (Group A). One was immediately frozen in liquid nitrogen and stored at -70°C, and the other was fixed in 4% formalin and embedded in paraffin. For cancer-bearing mice, spontaneous breast cancer tissues (Group C) and normal mammary glands located on the side opposite to the tumor (Group B) were collected. Portions of the tumor tissues and one of the normal mammary glands were immediately frozen in liquid nitrogen and stored at -70°C; the remaining tumor tissues and mammary glands were routinely fixed in 4% formalin and embedded in paraffin.
RNA Extraction
Total RNA was extracted from frozen tissue using Trizol reagent (Invitrogen, Paisley, UK) and isolated using the RNeasy extraction protocol from Qiagen (Valencia, CA, USA). The integrity of total RNA for each sample was determined by denaturing gel electrophoresis (1.2% methyl aldehyde running gel), and the purity of RNA was checked by spectrophotometry. The O.D. 260/280 nm ratio was between 2.05-2.15 for each RNA sample.
Microarray hybridization and data analysis
Total RNA from six samples harvested from three mice (three samples each for Groups B and C) was used for microarray hybridization. Microarray analysis was conducted using Affymetrix (Santa Clara, CA) Mouse Genome 430 2.0 Arrays (over 39,000 transcripts and variants from over 34,000 well characterized mouse genes). The procedure was conducted according to the manufacturer's instructions (Affymetrix) using T7-(dT)24-oligonucleotide primers for cDNA synthesis (Affymetrix), the cDNA Cleanup Module (Affymetrix) for purification and the IVT Labeling Kit (Affymetrix) for making biotin-labeled cRNA. Clean-up of cRNA using RNeasy columns (Qiagen, Crawley, Sussex, UK) was performed to remove unincorporated ribonucleotides prior to quantification by spectrophotometry. The cRNA was fragmented by metal-induced hydrolysis at 94°C for 35 min in a 40 mM final concentration Tris buffer. The length of the fragmented cRNA was between 25 bp and 200 bp. Adequacy of cRNA fragmentation was determined by 1.2% denaturing gel electrophoresis, and a hybridization control was prepared in hybridization buffer. The Affymetrix GeneChip system was used for hybridization, staining, and imaging of the arrays according to the standard Affymetrix protocol. Hybridization cocktails were hybridized to Mouse Genome 430 2.0 Arrays, after which the arrays were washed using a Fluidics Station 450 (FS450, Affymetrix) and then scanned using a Scanner 3000 7G 4C (Affymetrix). Microarray Suite 5.0 software (MAS5.0, Affymetrix) was used to process images and estimate transcript expression levels. The expression data were analyzed by MAS5.0, BGX and Arrary2BIO methods.
Real-time PCR
The relative expression levels of decorin, EGFR, cyclin D1 and PCNA were determined by quantitative PCR using SYBR Premix Ex Taq(TaKaRa Code: DRR041A) purchased from TaKaRa Biotechnology (Dalian) Co., Ltd with β-actin as a reference (TaKaRa Code: D3751). Samples were run in separate tubes on an ABI Prism 7500 Sequence Detection System according to the manufacturer's suggested protocols. In brief, the 50 μl samples were treated at 50°C for 2 min and 95°C for 10 s followed by 40 cycles of 95°C for 5 s and 64.2°C (for EGFR and PCNA), 65.0°C (for cyclin D1), or 60°C (for decorin) for 33 s. Relative quantitation using the comparative CT method was performed for each sample. Primers were synthesized by TaKaRa Biotechnology (Dalian) Co., Ltd. with the following sequences: decorin (GenBank accession no. NM_007833), forward 5'-TGATGCACCCAGCCTGAAAG-3', reverse 5'-TCCATAACGGTGATGCTGTTGAA-3'; EGFR (GenBank accession no. NM_207655), forward 5'-AGGACTGGGCAATCTGTTGGA-3', reverse 5'-GAAGATCGAAGACCTGGTGCTGTAA-3'; PCNA (GenBank accession no. NM_011045), forward5'-GGACTTAGATGTGGAGCAACTTGGA-3'; reverse 5'-AATTCACCCGACGGCATCTTTA-3'; cyclin D1 (GenBank accession no. NM_007631), forward 5'-AGTCAGGGCACCTGGATTGTTC-3', reverse 5'-AACAGATTAAATGATGCACCGGAGA-3'. Experiments were performed in triplicate for each sample.
Immunohistochemistry
Formalin-fixed and paraffin-embedded mammary gland and spontaneous breast cancer specimens were used for immunohistochemical detection of decorin, EGFR, cyclin D1 and PCNA. Sections 4 μm in thickness were deparaffinized and rehydrated with xylene and graded alcohol solutions. After washing with PBS, endogenous peroxidase activity was quenched by 3% hydrogen peroxide, and sections were boiled in 10 mM citrate buffer (pH 6.0) for 3 min in an autoclave sterilizer followed by cooling at room temperature for more than 20 min. After rinsing with PBS, sections were incubated with primary antibodies (1:100 dilution in antibody diluent, Zhongshan Goldbridge Biotechnology CO., Ltd, Beijing, China) for 18 hr at 4°C. Sections were stained with anti-decorin (SC-73896, Santa Cruz Biotechnology, Inc), anti-EGFR (BA0843, Boster Biological Technology, Ltd, Wuhan, China), anti-cyclin D1 (Cat. #RM-9104-S1, Neomarker Labvision, USA), or anti-PCNA (BM0104, Boster Biological Technology, Ltd, Wuhan, China) antibodies. After rinsing with PBS, sections were incubated with PV6001 or PV6002 (Zhongshan Goldbridge Biotechnology CO., Ltd, Beijing, China) for 30 min at 37°C and stained with DAB (AR1022, Boster Biological Technology, Ltd, Wuhan, China) for 1 to 2 min. The slides were counterstained with hematoxylin, dehydrated with ethanol, cleared with xylene, and mounted in neutral gum. Control sections were incubated with PBS instead of a primary antibody. All slides were analyzed by two independent observers.
Immunohistochemical staining evaluation
For cyclin D1 and PCNA, only the percentage of immunoreactive epithelial cells and breast cancer cells was considered (labeling index). Briefly, the areas of high percentage of cyclin D1 positive cells ('hot spots') were identified at low magnification (×10 ocular and ×10 objective) as the "hot spots". Then, ten hot spot areas per section were selected and were observed at a higher magnification (×10 ocular and ×40 objective, high power field) with a grid (OLYMPUS 100×) in the ocular lens. All epithelial cells or cancer cells and immunohistochemistry positive cells in the grid were counted in every high power field, respectively. The mean percentage of positive cells (labeling index) was used to evaluate the expression of the protein in a section. For EGFR, both the percentage and intensity of EGFR-positive epithelial cells and breast cancer cells were considered in a semi-quantitative assessment [17]. The percentage of EGFR-positive cells was scored as 0 (0% positive cells), 1 (1-25% positive cells), 2 (26-50% positive cells), 3 (50-75% positive cells), or 4 (>75% positive cells). The intensity of EGFR immunostaining was also scored as 0 (negative), 1 (weak), 2 (intermediate) and 3 (strong). The intensity score (0-3) was multiplied by the percentage score (0-4) and a final score was assigned 0 (negative), 1-4 (weak expression), 5-8 (moderate expression), and 8-12 (strong expression). Samples with scores of 0-4 were considered to show low expression, while those with scores of 5-12 were considered to show high expression. For decorin, the percentage of decorin-positive cells or decorin-positive areas located around the terminal duct and gland alveolus was scored as 0 (0% positive cells or substance), 1 (1-25% terminal duct and gland alveolus), 2 (26-50% terminal duct and gland alveolus), or 3 (>50% terminal duct and gland alveolus), and samples with scores of 3 were considered to show high expression. In tumor tissues, the distribution of decorin-positive cells or decorin-positive areas was recorded.
Statistical Analysis
All data were analyzed using SPSS statistical software (version 11.5 for Windows). The Kruskal-Wallis and Mann-Whitney tests were used to evaluate statistical significance of differences, and the Spearman rank test was used to assess the correlation between the expression of EGFR and cyclin D1 or PCNA. Differences were considered statistically significant at P < 0.05.