The Ethics Committee of Nanjing Medical University Affiliated Wuxi Second Hospital (Wuxi, Jiangsu, China) approved the current protocols, according to the 1975 Declaration of Helsinki. Informed consent form was signed by each patient.

The 82 female patients enrolled in the present study underwent curative surgery for breast cancer without radiation or chemotherapy prior to surgical treatment at the Nanjing Medical University Affiliated Wuxi Second Hospital, between January 2008 and December 2009. The mean age of the patients was 54.8 years (standard error of the mean, 3.2; range, 29–73 years). Tissue specimens were confirmed separately by two experienced pathologists under double-blinded conditions, and none of the patients received any therapy prior to operation. The demographic features and clinicopathological data were reviewed in the patients' medical records. The specimens were collected and immediately stored in 4% paraformaldehyde for immunohistochemistry (IHC), or in liquid nitrogen for western blotting. The prognosis concerning disease-specific survival and overall survival, which were defined as the time from surgery to first recurrence or mortality, respectively, were analyzed. A total of 60 months of clinical follow-up data were available.

The human breast cancer cell lines MCF7 and SKBR3 were cultured in complete Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), while the human breast cancer cell line MDA-MB-231 was cultured in L15 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS, in a humidified 5% CO₂ incubator at 37°C. The cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).

Paraformaldehyde-fixed, paraffin-embedded tissue sections were used for IHC with an anti-SREBP-1 (1:500; catalog no. sc-8984; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibody, according to the streptavidin-peroxidase method (15). The staining results for the SREBP-1 protein were evaluated by the staining intensity and the percentage of positive cells. The staining intensity was scored as follows: 0=none; 1=weak; 2=moderate; and 3=strong. The percentage of positive cells was scored as follows: 0, <5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, >75%. A total of 10 independent, high-magnification (×400) fields were observed to calculate a mean score. A total score >1 was defined as positive staining.

siRNA specific against SREBP-1 and non-silencing scrambled sequence siRNA (used as the negative control) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection in accordance with the manufacturer's protocol. Further experiments were performed after 48 h. The specific SREBP-1 siRNA sequence was: Sense 5′-GGAAGAGUCAGUGCCACUGTT-3′ and anti-sense 5′-CAGUGGCACUGACUCUUCCTT-3′.

Total RNA was extracted from breast cancer tissues and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA concentration was quantified by a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Complementary (c)DNA was synthesized using a first-strand cDNA synthesis kit (GE Healthcare Life Sciences; Chalfont, UK). cDNA (2 µl) was amplified and quantified by RT-qPCR using SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd., Dalian, China) with the following cycling conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec and 60°C for 30 sec. The human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was regarded as the internal control. The primers used for PCR were synthesized by Shanghai GenePharma Co., Ltd., and their sequences were as follows: 5′-CAGTCCAGCCTTTGAGGATA-3′ (forward) and 5′-CAAAGGATTGCAGGTCAGAC-3′ (reverse) for SREBP-1, and 5′-CAAGCTCATTTCCTGGTATGAC-3′ (forward) and 5′-CAGTGAGGGTCTCTCTCTTCCT-3′ (reverse) for GAPDH. All samples were normalized to GAPDH and calculated with the relative 2^−ΔΔCq method (17).

A modified radioimmunoprecipitation assay buffer with protease inhibitor was used for protein isolation (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Total proteins (50 µg) were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA), which were blocked with 5% non-fat dry milk (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in phosphate-buffered saline (PBS) with 0.1% Tween 20. Next, the membranes were incubated with primary anti-SREBP-1 (1:300; catalog no. sc-365513; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin (1:5,000; catalog no. 3700; Cell Signaling Technology, Inc.) antibodies at 4°C overnight, followed by horseradish peroxidase-conjugated secondary antibodies (1:5,000; catalog no. bs12471; Bioworld Technology, Inc., St. Louis Park, MN, USA) for 2 h at room temperature. Bands were detected with an enhanced chemiluminescence reagent (EMD Millipore).

Cells (5×10⁵ cells/well) were seeded into a 6-well plate and incubated to a confluent monolayer. Scratch wounds were created using a pipette tip, and washed twice with sterile PBS. Cells were then cultured in serum-free medium in a humidified 5% CO₂ incubator at 37°C for 48 h, and visualized under an inverted microscope.

Transwell chambers (8 µM pore-sized; Nalge Nunc International; Thermo Fisher Scientific, Inc.) were coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at 1 mg/ml on the inner layer. Cells transfected with control siRNA or SREBP-1 siRNA were seeded in the upper chamber at a density of 1×10⁵ cells/chamber in 100 µl serum-free medium. The lower chamber was filled with 600 µl DMEM containing 10% FBS. Plates were incubated for 24 h at 37°C, and cells in the top surface of the matrigel membrane were swabbed carefully. The adherent cells on the undersurface of the insert were stained with 0.3% crystal violet and counted under a light microscope. A total of four fields were randomly selected to calculate the mean cell number.

All data are presented as the mean ± standard deviation. SPSS version 13 (SPSS, Inc., Chicago, IL, USA) was used for Pearson χ² test and multivariate Cox regression analysis. Two-tailed Student's t test or Kaplan-Meier method was used to calculate the survival, and log-rank test or analysis of variance was used to analyze the difference in multivariate Cox regression using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the potential role of SREBP-1 in human breast cancer, RT-qPCR, IHC staining and western blotting were performed to investigate SREBP-1 messenger (m)RNA and protein expression in tissue samples of 82 patients with primary breast cancer, together with matched para-cancerous tissue samples. Notably, the SREBP-1 mRNA and protein level was robustly increased in the majority of the cancer samples compared with para-cancerous tissues (P<0.01; Fig. 1A and B). SREBP-1 was positively stained in both the nucleus and the cytoplasm. Compared with malignant cells, SREBP-1 immunostaining in benign cells was negative or relatively weak (Fig. 1A). According to the criteria of semi-quantitative assessment employed, SREBP-1 was highly expressed in 58 (70.7%) of 82 breast cancers and in 24 (29.3%) of 82 para-cancerous tissues. Statistical analysis of SREBP-1 staining scores confirmed increased staining in malignant cells compared with benign cells (P<0.01; Fig. 1A). Western blotting further confirmed the IHC results (P<0.01; Fig. 1C).

Next, the association between the expression of SREBP-1 protein and clinicopathological features was analyzed (Table I). Pearson χ² test suggested that high SREBP-1 expression was strongly correlated with tumor differentiation, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.01). However, there was no significant association between SREBP-1 expression and other clinicopathological variables. Taken together, these data reveal that the expression of SREBP-1 in breast cancer is elevated, and increased SREBP-1 expression is correlated with poor clinicopathological features in breast cancer.

To determine the role of SREBP-1 in predicting the prognosis of patients, Kaplan-Meier survival curves were constructed using 5-year overall and disease-specific survival to analyze cases with high and low SREBP-1 expression (n=82; follow-up time, 60 months). The results indicate that high SREBP-1 staining predicts a poor overall and disease-specific patient survival (P<0.01, log-rank test; Fig. 2A and B).

In addition, SREBP-1 was an independent prognostic marker for both 5-year overall survival [hazard ratio, 2.976; 95% confidence interval (CI), 1.109–7.987; P=0.030; Table II) and disease-specific survival (hazard ratio, 2.327; 95% CI, 1.093–4.955; P=0.029; Table II) according to multivariate Cox regression analysis. These results definitely confirmed that high SREBP-1 expression is associated with poor prognosis, suggesting that SREBP-1 may function as a prognostic marker for breast cancer.

In order to identify the function of SREBP-1, the SREBP-1 mRNA and protein levels were analyzed in three different breast cancer cell lines. Consistent with the clinical results, the expression levels of SREBP-1 were significantly higher in MCF7 and MDA-MB-231 cells than in SKBR3 cells (Fig. 3A and B). Based on such difference in SREBP-1 expression, MCF7 and MDA-MB-231 cells were selected, and SREBP-1 was knocked down by transfecting specific SREBP-1 siRNA into these cells. The RT-qPCR and western blotting results demonstrated that SREBP-1 was markedly downregulated by SREBP-1 siRNA in these cell lines (Fig. 3C and D).

To elucidate effect of SREBP-1 knockdown on breast cancer cells migration and invasion, mechanical scrape wound healing and transwell assays were performed. The wound healing assay revealed that the loss of SREBP-1 expression could significantly reduce the migration rate in both MDA-MB-231 and MCF7 breast cancer cells (P<0.01; Fig. 4A and B, respectively). Consistently, the transwell assay also confirmed that the cell migration rate was significantly decreased by SREBP-1 knockdown in these cells (P<0.01; Fig. 5A). Furthermore, compared with those in the control groups, the number of invaded cells in the SREBP-1 siRNA group was significantly reduced (P<0.01; Fig. 5B). These data, therefore, indicate that SREBP-1 has an impact on the migration and invasion of breast cancer cells.