The siRNA sequences that target human VEGF were constructed according to established guidelines (29). The primer sequences were 5′-GGAGUACCCUGAUGAGAUCUU-3′ (forward) and 5′-GAUCUCAUCAGGGUACUCCUU-3′ (reverse). The negative control scramble siRNA (siRNA_SCR) primer sequences were 5′-GCGUAACGCGGGAAUUUACUU-3′ (forward) and 5′-GUAAAUUCCCGCGUUACGCUU-3′ (reverse). These sequences were verified by DNA sequencing according to the manufacturer’s instructions (Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China).

The MCF-7 human breast cancer cell line was kindly provided by the Cell Culture Center of the Peking Union Medical College of China and was cultured in RPMI-1640 medium containing 10% fetal calf serum in a 37°C humidified 5% CO₂ incubator. The medium was changed every two days. To maintain the cells at optimal proliferating conditions, they were passaged at 80% confluence and seeded at 20% confluence. Transfection was performed at ~90% confluence using Lipofectamine™ 2000 (Invitrogen Co., Carlsbad, CA, USA) following the manufacturer’s protocol. The cells were collected at 48 h following transfection and used for western blot analysis.

The VEGF concentration in the conditioned medium of MCF-7 cells was measured using a commercially available human VEGF ELISA kit (Chemicon, Millipore Corporation, Temecula, CA, USA). MCF-7 cells were plated on a 96-well plate at a density of 6×10³ cells/ml. After 24 h of culture, the cells were transfected with siRNA (25 nmol/l, 50 nmol/l, 100 nmol/l and 200 nmol/l), siRNA_SCR, or Lipofectamine reagent overnight at 37°C. The cultures were then washed twice with Hanks’ Balanced Salt solution and incubated with fresh medium for an additional 48 h. The supernatants were collected and VEGF concentration was quantified (pg/ml) by ELISA according to the manufacturer’s recommendations to determine the effect of specific downregulation and the optimal concentration of siRNA transfection.

Using a 96-well plate, a total of 8×10³ MCF-7 cells were seeded in each well and allowed to attach for 18 h. The cells were later treated with siRNA (25 nmol/l, 50 nmol/l, 100 nmol/l or 200 nmol/l) or siRNA_SCR. At 48 h following transfection, 20 μl MTT (5 mg/ml) was added to the cells in each well. The cells were subsequently incubated for 4 h, followed by the addition of 100 μl dimethyl sulfoxide and the cells were incubated for another 15 min. The optical density was determined at 492 nm with a microculture plate reader (SpectraMax M2; Molecular Devices, Sunnyvale, CA, USA). To determine the inhibition rate, the absorbance values were normalized to the values obtained from the blank control group of cells.

Following treatment with 100 nmol/l siRNA or siRNA_SCR for 48 h, the MCF-7 cells attached to culture dishes were trypsinized and washed in cold phosphate-buffered saline (PBS). Briefly, 100 μg protein samples were subjected to 10% standard SDS-PAGE, with prestained molecular weight markers being run in parallel to identify VEGF protein. Subsequently, the resolved proteins were transferred to polyvinylidene difluoride membranes. The membranes were then incubated with mouse monoclonal anti-VEGF antibody (cat. no. sc-7269; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and mouse monoclonal anti-β-actin antibody (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). antibodies. Following extensive washing, the membranes were incubated with anti-mouse IgG-horseradish peroxidase-conjugated antibody for 1 h at room temperature and developed with a Luminol chemiluminescence detection kit (Promega Corporation, Madison, WI, USA). Protein expression was quantified with a Gel EDAS analysis system and Gel-Pro Analyzer 3.1 software (Shenzhen Tianneng Corporation, Shenzhen, Guangdong, China).

Human PBMCs were obtained from heparinized blood of healthy volunteers by density gradient centrifugation following informed consent. The cells were cultured for 5–6 days in complete RPMI-1640 medium [GlutaMAX (Invitrogen Co.), supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 g/ml streptomycin] with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml interleukin-4 (IL-4). Maturation was induced for 48 h in the presence of 10 ng/ml tumor necrosis factor-α (TNF-α). The DCs were divided into the siRNA and control groups. The DCs of the siRNA group were cultured in RPMI-1640 medium with the addition of the culture supernatant of MCF-7 cells transfected with siRNA (optimal concentration of 100 nmol/l) according to the volume ratio of 1:2 (supernatant vs. RPMI-1640). The DCs of the control group were cultured in RPMI-1640 medium with the addition of the culture supernatant of MCF-7 cells transfected with siRNA_SCR (100 nmol/l) according to the volume ratio of 1:2 (supernatant vs. RPMI-1640).

The DCs in the two groups were observed daily under an inverted Olympus microscope (Olympus Optical Co., Ltd., Tokyo, Japan) with a green light filter. Following 8 days of culture, the DCs were stained by standard direct procedure using phycoerythrin-conjugated mouse monoclonal antibodies (mAbs) against CD1a (cat. no. 555807), CD80 (cat. no. 557227), CD83 (cat. no. 556855), CD86 (cat. no. 555658) or HLA-DR (cat. no. 555812; all from BD Biosciences, Franklin Lakes, NJ, USA). The cells were incubated with mAbs for 20–30 min at ambient temperature, washed twice with PBS and resuspended in 100 μl PBS. Cell fluorescence was analyzed by the Epics XL flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

Responder T cells were purified from the PBMCs of healthy volunteers. T cells (1×10⁶/ml) were co-cultured with DCs loaded with MCF-7 antigen for 72 h to induce cytotoxic T lymphocytes (CTLs). The CTLs were then collected and used as the effector cells in CTL assays. As the target cells, MCF-7 cells were placed in 96-well culture plates at lx10⁴ cells per well and co-cultured with the effector cells at varied effector/target cell ratios (E/T) of l:10, 1:25 and l:50 for 48 h. The cytotoxic activity was detected with the MTT assay. The tests were performed in triplicate and the results are expressed as mean counts per minute with standard deviation.

Statistical analyses were performed using SPSS software, version 11.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the means ± SD. The results were considered statistically significant if P<0.05 was obtained by the appropriate ANOVA procedure and the Student’s t-test.

In order to test whether siRNAs affect the expression of VEGF, we measured the VEGF concentration in the culture supernatants of MCF-7 cells transfected with siRNA (25 nmol/l, 50 nmol/l, 100 nmol/l or 200 nmol/l), siRNA_SCR or Lipofectamine reagent using ELISA and analyzed the proteins by western blotting. The ELISA data demonstrated that the VEGF expression and protein production in the supernatants of MCF-7 cells transfected with siRNA (100 nmol/l and 200 nmol/l) were significantly decreased compared to that in the controls; however, the level of VEGF protein was not significantly different between the 100 and the 200 nmol/l siRNA groups, or among the siRNA_SCR, Lipofectamine control and the blank control groups (Fig. 1). The western blot analysis demonstrated that VEGF expression was significantly reduced in cells transfected with siRNA compared to those transfected with siRNA_SCR or Lipofectamine (P<0.05) (Fig. 1). There was no significant difference among the siRNA_SCR, Lipofectamine and non-transfected groups (Fig. 2). These data suggested that the constructed VEGF-targeted siRNA inhibited the expression of VEGF.

Optical microscopy was employed to investigate the effect of siRNA on DC morphology. Representative photographs taken on day 8 demonstrated that DCs generated from adherent human PBMCs of healthy donors in the presence of GM-CSF, IL-4 and TNF-α exhibited significant differences in morphology between the siRNA and siRNA_SCR groups (Fig. 3). The DCs in the blank control group exhibited typical arborizations (Fig. 3A). The PBMCs cultured with the culture supernatants from MCF-7 cells treated with siRNA started to elongate into irregular shapes on the 2nd or 3rd day and exhibited the morphological characteristics of DCs on the 8th day (Fig. 3B). The DCs in the siRNA_SCR group, cultured with the culture supernatants from MCF-7 cells transfected with siRNA_SCR, started to elongate marginally on the 3rd or 4th day, but exhibited no distinct DC characteristics until the 8th day (Fig. 3C). These data demonstrated that DC morphology was affected by cultivation with the culture supernatants from MCF-7 cells treated with siRNA.

In order to investigate the effects of VEGF on DC differentiation in the culture supernatants from MCF-7 cells and determine how this effect is affected by VEGF-targeted siRNA, DCs were labeled with a mixture of phycoerythrin-conjugated lineage-specific antibodies (anti-HLA-DR, CD80, CD83, CD1a, CD86) and analyzed directly using flow cytometry. The expression of HLA-DR on DCs in the siRNA group was higher compared to that in the control group that was transfected with siRNA_SCR (Table I). Folllowing siRNA transfection, the percentage of cells expressing CD80, CD86 and CD83 was increased (Table I). However, the expression of CD1a in the siRNA group was lower compared to that in the control group (Table I). These data demonstrated that siRNA altered the effect of VEGF on DC surface phenotypes.

In order to detect the inhibition rates of tumor-specific CTLs against MCF-7 cells mediated by DCs cultured in different media, the MTT assay was used. The results of the MTT assay demonstrated that the inducing activity of DCs in the siRNA group was enhanced in comparison to DCs in the control group (siRNA_SCR). The cytotoxic activity of CTLs was most significant at E/T 1:50 (P<0.01) (Table II). These data indicated that the cytotoxic activity of CTLs mediated by DCs was significantly increased by transfection with siRNA.