Full length PR39 (RRRPR PPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP-NH₂ and FITC-PR39 (FITC-RRRPRPPYLPRPRPPPFFPPRLPPR IPPGFPPRFPPRFP-NH₂) were prepared by Shanghai Sangon Biological Engineering Technology and Services Corporation (Shanghai, China). The synthetic peptide PR39 was at least 95% pure by high performance liquid chromatography (HPLC). PR39 was dissolved in phosphate-buffered saline (PBS, pH 7.4) to appropriate concentrations (4 mM). The small interference RNA oligo used for Stat3 gene silencing (sense: 5′-GGACGACUUUGAUUUCAACtt-3′, antisense: 5′-GUU GAAAUCAAAGUCGUCCtg-3′) (24) and negative control (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). 5′Cy3-labeled siRNA was ordered from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). The siRNA was dissolved in sterilized distilled H₂O to the concentration of 20 μM.

The 4T1 cells supplied by our laboratory were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Gibco, USA) with 10% fetal bovine serum (HyClone, USA) at 37°C in a 5% CO₂ atmosphere (Heraeus, Germany). Anti-Stat3 was purchased from Becton-Dickinson, USA; anti-MMP-9 was obtained from Bioworld (USA); anti-β-actin was from Santa Cruz Biotechnology, Inc. (USA); HRP-conjugated secondary antibody was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (China).

The Stat3 scrambled siRNA was diluted with appropriate PBS to a concentration of 100 nM, and PR39 was added into PBS. To determine the optimal relative concentration at which the PR39/siRNA complex formation would be advanced, 100 nM of siRNA was incubated with different amounts of PR39 at 25°C for 15 min in PBS, with the siRNA/PR39 concentration ratio ranging from 0:1 to 1:90. The Scr siRNA/PR39 complexes were detected by electrophoresis on a 2% agarose gel (Invitrogen Life Technologies, USA) with GoldenView in TBE buffer at 180 V for 15 min. Then, stained bands were visualized under UV light and photographed using the Bio-Rad Chemi Doc XRS imaging system (Bio-Rad, USA).

4T1 cells were seeded on 10-mm cover slips with 5×10⁴ cells/cover slip 18 h before transfection with the 5′Cy3-siRNA. The 5′Cy3-siRNA (2.5 μl) was diluted into 50 μl serum-free DMEM. PR39 (1.125 μl) was diluted in 50 μl serum-free DMEM, mixed gently and incubated for 5 min at room temperature. 5′Cy3-siRNA was combined with the diluted PR39. They were mixed gently and incubated for 30 min at room temperature to form a complex. This complex was added to the cells with 400 μl serum-free DMEM. Then 4T1 cells were rinsed thrice with PBS after 6 h of transfection and fixed in methanol for 20 min at −20°C. The cells were washed twice with PBS and DAPI fluorescent stain (Beyotime, China) which was applied to the cells for 5 min at room temperature. The cells were rinsed three times with PBS and observed under a fluorescence microscope (Nikon, Japan).

Stat3 siRNA and scrambled control were transfected with PR39 (concentration ratio 1:90) or Lipofectamine 2000 reagent (Lipo 2000; Invitrogen, USA). All steps were performed following the manufacturer’s instructions. The 4T1 cells suspended in complete DMEM medium were seeded at 6×10⁵ cells/well in 6-well plates (Costar, USA) and allowed to attach overnight before transfection. The siRNA (10 μl) was diluted into 250 μl serum-free DMEM. PR39 (4.5 μl) or Lipo 2000 (5 μl) was diluted in 250 μl serum-free DMEM, mixed gently and incubated for 5 min at room temperature. Then, Stat3 siRNA was combined with the diluted PR39 or Lipo 2000. They were mixed gently and incubated to form a complex for 30 min at room temperature. Then the complex was added to the cells with 1500 μl serum-free DMEM. After 6 h transfections, cells were rinsed with PBS and the medium was replaced with fresh complete growth medium. The cells were incubated at 37°C in a 5% CO₂ atmosphere, and further analysis was performed 24, 48 or 72 h post-transfection.

4T1 cells were seeded at a density of 6×10³/well into 96-well plates (Costar, USA) and treated with various concentrations of PR39 or various treatment combinations for 24 h. The next day, cells were incubated in methylthiazole tetrazolium (MTT, Sigma, USA) solution for 4 h. The spectrophotometric absorbance was measured in a microplate reader (Bio-Rad) at 490 nm. Absorbance rates obtained by untreated cells were considered as 100% cell survival. The relative cell viability was calculated according to the equation (Abs_sample − Abs_blank)/(Abs_control − Abs_blank) × 100. Each assay was repeated at least three times, using three wells per drug concentration in each experimental condition.

About 5×10⁵ 4T1 cells/well were seeded in 6-well plate before various treatments. The Stat3-specific siRNA complex was added to the 4T1 cells as above. The 4T1 cells from the control or treated group were trypsinized and suspended in 70% ethanol at 4°C at least overnight. Then samples were kept on ice and analyzed on a FACSCalibur flow cytometer (Becton-Dickinson).

Total cellular RNA was extracted by the Trizol Reagent (Takara, Japan) according to the manufacturer’s instructions. Then, cDNA was obtained from the cellular RNA. For the reverse transcriptase reaction, 1 μg of total RNA was mixed with oligo(dt)15 primer and Maloney murine leukemia virus (M-MLV) reverse transcriptase (Takara). Then the mixture was incubated at 42°C for 60 min and 70°C for 15 min. The sequences of PCR primers (Invitrogen, China) were: Stat3 forward, 5′-AGAGAAGCAGCAGATGTTGG AGCA-3′, and reverse, 5′-ATCCTGCATGTCTCCTTGGCT CTT-3′ (PCR product, 148 bp) (25); MMP-9 forward, 5′-ACAG CCAACTATGACCAG-3′ and reverse; 5′-TGCCACCAGGA ACAGG-3′ (PCR product, 249 bp). β-actin forward, 5′-CCA CTGCCGCATCCTCTTCCTC-3′, and reverse, 5′-TCCTGCTT GCTGATCCACATCT-3′ (PCR product, 400 bp). The transcribed cDNA was mixed with each primer and Taq DNA polymerase (Takara, Japan), and then amplified. PCR was performed as follows: after incubation at 94°C for 5 min, Stat3, MMP-9 and β-actin underwent 35 cycles of reaction (94°C for 30 sec, 61°C of Stat3; 58°C of MMP-9 and β-actin for 30 sec, 72°C for 40 sec). After cycling, the samples were incubated at 72°C for 10 min. The PCR products were analyzed on 1.5% agarose gel containing GoldenView. Stained bands were visualized under UV light and were photographed.

4T1 cells were harvested from 6-well plates at 80% confluency. Cells were washed with cold PBS and lysed with cell lysis buffer containing protease inhibitor (Beyotime). Whole cell lysates were centrifuged at 13000 r for 30 min at 4°C. Protein concentrations were determined by the BCA reaction (Beytime Biotechnology, China). Proteins (50 μg of protein/lane) from control or treated cells were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) membranes with a Bio-Rad semi-dry transfer system (Bio-Rad). The membranes were blocked with 5% (v/v) non-fat milk overnight and then incubated with primary antibody for 12 h at 4°C. After washes, the membrane were incubated with appropriate antibody conjugated with horseradish peroxidase as secondary antibody for 1 h at 37°C, followed by three washes with TBST (Tris-buffer saline containing 0.1% Tween 20). Observations of signals were obtained by enhanced chemiluminescence (ECL reagent, Millipore, USA) according to the manufacturer’s instructions. Intensities of the bands obtained from the RT-PCR and western blot analysis were analyzed using the Quantity One software (Bio-Rad).

The 4T1 cell invasive ability with or without treatment was examined using the membrane Transwell culture system. Transwell membrane (8 μm pore size, 6.5 mm diameter; Sigma) coated with Matrigel (Sigma) overnight at 4°C were used for the invasion assay. The 4T1 cells from the control or treated groups after 48 h were trypsinized, centrifuged, and resuspended at 2.5×10⁵/ml in DMEM without FBS. Then, cells (2.5×10⁴/well) were seeded onto the upper chamber of pre-coated Transwell membranes. The lower chamber of the Transwells contained DMEM with 10% FBS. The cells on the upper chamber and Matrigel coating the membranes were swabbed with Q-tip after 18 h incubation. Then, the membranes were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells attaching to the lower surface of the polycarbonate filter were counted under light microscopy (magnification, ×200). The experiments were performed in thrice in triplicate. The 4T1 cells obtained from the Transwell assay were analyzed using the Image J software (NIH, USA).

The 4T1 cells from the control or treated group were trypsinized and suspended in DMEM medium without FBS. Transwell membranes were used. Then, cells (2×10⁴/well) were seeded onto the upper chamber of Transwells. Lower wells of the Transwell chamber contained DMEM with 10% FBS as chemotactic medium. Then, the chamber was incubated in a humidified incubator for 3 h. The medium was removed from the upper chamber and the filtered cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The migrated 4T1 cells were evaluated under the microscope, and random fields were scanned (five fields per filter) for the cells obtained at the lower membrane side only. The number of 4T1 cells obtained from the Transwell assay were analyzed using the Image J software.

The repeated-measures ANOVA test was used between mutiple comparisons analysis with the SPSS17.0 software. P-values <0.05 were taken to denote significant differences.

In order to ascertain whether PR39 and siRNA could form a complex, 100 nM scrambled siRNA was mixed with PR39 at different concentration ratios. As shown by a gel-shift assay PR39 was able to bind siRNA in a dose-dependent manner (Fig. 1A), and the 1:90 molar ratio of siRNA to PR39 was an optimal condition. We measured the cytotoxicity of PR39 usig the MTT assay (Fig. 1B), and observed that PR39 at 3 μM to 36 μM had no effect on the cell viability of 4T1 cells. These results indicate that PR39 could interact with siRNA and form complexes, and it was barely nontoxic to cells.

To determine whether PR39 could deliver siRNA into the cytoplasm, 4T1 cells were transfected with Cy3-labeled siRNA complexed to FITC-labeled PR39. After 6 h treatment, the intracellular colocalization of the double-labeled PR39/siRNA complex in 4T1 cells was assessed by fluorescence microscopy (Fig. 1C).

To detect the gene-silencing effect of the Stat3 siRNA delivered by PR39, 4T1 cells stably expressing high levels of Stat3 were transfected with Stat3-siRNA/PR39. Stat3 mRNA expression was determined after 24–72 h of incubation with an increasing molar ratio from 0:1 to 1:180 Stat3-siRNA/PR39. A maximum gene silencing effect assessed by mRNA and protein levels was observed after 48 h of treatment, and the optimal ratio of siRNA to PR39 was 1:90 (Fig. 2A). Lipo 2000/Stat3 siRNA was used as a positive control, and it seemed more potent than PR39/siRNA in silencing Stat3 expression (Fig. 2B and D).

Cell proliferation was measured by the MTT and FCM assays. The results suggest that the proliferation and cell cycle of 4T1 cells were not affected by Stat3 knockdown (Fig. 3A and B). Cyclin D1 protein levels were not influenced by the Stat3 reduction (data not shown).

The effect of Stat3 siRNA on the invasiveness and motility of 4T1 cells was determined using the Transwell assay. The cells treated with PR39/Scr siRNA, PR39/Stat3 siRNA were significantly fewer compared to the naked Stat3 siRNA and the untreated group (Fig. 3C-F). More interesting, although Lipo 2000/Stat3 siRNA displayed more potent activity than PR39/siRNA in silencing Stat3 expression, the invasive and migration capacity of the cells after treatment with Lipo 2000/Stat3 siRNA was not less than that of the PR39/siRNA group.

In addition, matrix matalloproteinase-9 (MMP-9) has been shown to be one of the Stat3 downstream target genes, which plays an important role in tumor cells invasion and migration. Therefore, MMP-9 expression was examined after 48 h transfection with PR39/Stat3 siRNA and Lipo 2000/Stat3 siRNA. These observations suggested that MMP-9 expression was significantly inhibited by the PR39/siRNA group compared to the Lipo 2000/Stat3 siRNA group (Fig. 2C and D). Given that previous reports demonstrated that PR39 gene transduction altered the invasive activity and actin structure in human hepatocellular carcinoma cells (26), we investigated the effect of PR39 alone on cell invasion and migration. As shown in Fig. 3G and J, PR39 (9 or 18 μM) could significantly inhibit 4T1 cell invasion and migration, and it was estimated that PR39 and Stat3 siRNA could have a synergistic effect in the invasion and migration of 4T1 cells.