The human hepatoma-derived cell line HCCLM6, with a high metastatic ability as evaluated by xenograft models, was established by the Liver Cancer Institute (Fudan University, Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). All cells were maintained at 37°C in a humidified incubator containing 5% CO₂. Five-week-old male nude mice with an average weight of 20±5 g were purchased from Institute of Materia Medica (CAS, Shanghai, China). The nude mice were kept in specific pathogen-free conditions with lamina flow devices in accordance with related regulations. The study was approved by the ethics committee of Zhongshan Hospital (Fudan University, China).

The RNAi candidate sequences for human NRP-1 were designed according to a previous study (13) and the detailed sequences were as follows: sense, 5′-GAGAGGTCCTGAATGTTCC-3′; anti-sense, 5′-GGAACATTCAGGACCTCTC-3′. Stem-loop DNA oligonucleotides were synthesized and cloned into the lentivirus-based RNAi vector pLVTHM. The negative control plasmid was formed by an empty vector pLVTHM. Lentiviral particles were prepared as described previously (21). HCCLM6 cells were seeded in six-well plates at a concentration of 5×10⁵ per well. Lentivirus transfection was conducted when the cells reached 70–80% confluence. Cells were divided into three groups as follows: the knockdown (KD) cells were transfected with NRP-1 shRNA lentivirus (MOI 30); the negative control (NC) cells were transfected with empty lentivirus (MOI 30) and the blank control (BC) cells were not transfected. After 48 h, transfection efficiency was detected using flow cytometry (BD Pharmingen, San Diego, CA, USA).

Total RNA extraction was carried out using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality was assured by the A260/280 absorbance ratio and 0.5 μg total RNA was reverse transcribed into single-strand cDNA using PrimeScript™ RT Enzyme Mix I (Takara, Kyoto, Japan). RT-PCR was carried out for 15 min at 37°C and 5 sec at 85°C in a thermocycler. For the NRP-1 gene, 2.0 μl cDNA template was used for routine PCR in a final volume of 20 μl. The forward primer 5′-TCCCGCCTGAACTACCCTTGAGA-3′ and the reverse primer 5′-TTTGAAATGGCGCCCTGTGTCC-3′ were used and amplified in one cycle at 95°C for 10 sec, then 40 cycles at 95°C for 5 sec and 60°C for 30 sec. To measure the relative amount of the NRP-1 gene in the different samples, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level was determined and employed as a control. The PCR was performed in a DNA Engine Opticon system (MJ Research, Reno, NV, USA) by using SYBR^®-Green PCR Master mix (Takara, Kyoto, Japan). The ΔΔCt method was used for relative quantitative comparison among samples (22).

Approximately 20 mg total protein extracted from groups of cells were separated on 6% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking the membranes, the diluted primary antibodies against NRP-1 and GAPDH (Santa Cruz Biotechnology, CA, USA) were incubated for 24 h at 4°C. After extensive washing in Tris-buffered saline (TBS) buffer, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody for 1 h at room temperature. Labeled proteins on western blots were visualized using the Chemiluminescence Reagent Plus detection system (New England Nuclear, Boston, MA, USA). Quantification of bands was carried out using an Alpha Imager (Alpha Innotech, San Leandro, CA, USA).

Three groups of cells were individually seeded into 96-well plates at a concentration of 7×10³ per well filled with DMEM supplemented with 10% FBS. After 24 h, the medium was removed and replaced with fresh medium. One plate was examined immediately after the medium change and other plates were analyzed every 24 h for 3 days. Assays were initiated by adding 20 μl MTT (5 mg/ml) to each well and incubating the cells for an additional 4 h. Finally, the medium was removed and 150 μl dimethylsulphoxide (DMSO) was added to each well. Plates were read at a wavelength of 570 nm.

Eighteen nude mice were randomly divided into 3 groups and each was subcutaneously injected with 1×10⁷ KD cells, NC cells or BC cells, respectively, in the right upper flank region, for the establishment of subcutaneous xenograft models. Every 5 days after the injection, tumors in the mice were observed visually. Mice were sacrificed at 50 days and tumor mass and volume were recorded. Volume was calculated as (length/2) × (width²). Then NRP-1 protein expression in tumors was detected by western blot analysis as described above.

One xenograft from each group was formalin-fixed, paraffin-embedded and sectioned into 4-μm-thick sections. Then, sections were deparaffinized in xylene and treated with a graded series of alcohol [100, 95 and 80% (V/V) ethanol in double-distilled water] and rehydrated in phosphate-buffered saline (pH 7.5). For MVD assessment, the slides were microwaved for 5 min for antigen retrieval. Then rat monoclonal anti-CD34 antibody (Abcam, Cambridgeshire, UK) was added and incubated at room temperature for 2 h. Afterwards, horseradish peroxidase-conjugated goat anti-rat secondary antibody (Beyotime, Shanghai, China) was added and incubated for a further 1 h at room temperature. Slides were incubated with stable 3,3′-diaminobenzidine (DAB) for 10–15 min, and then rinsed with distilled water and counter-stained with Gill’s hematoxylin (Sigma, St. Louis, MO, USA) for 1 min. Then slides were observed under a Leica CTR 5000 microscope system (Leica Microsystems, Hong Kong, China) to count MVD as described previously (23).

Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) using a Student’s t-test throughout the present study. P<0.05 was considered to indicate a statistically significant difference.

After 48 h transfection, green fluorescent protein (GFP) expression rates of the KD and NC cells were 97.71 and 98.96%, respectively (Fig. 1). The real-time PCR results demonstrated that NRP-1 shRNA had a significant suppressive effect. The NRP-1 mRNA level of the KD group decreased by 81.5% compared to the BC group. In the NC group cells transfected with empty lentivirus, the expression of NRP-1 mRNA was barely affected (Fig. 2A). In the western blot assay, NRP-1 protein expression was inhibited by 74.9% in the KD group compared to the BC group (P<0.05). No obvious variance was found between the NC and BC group (P>0.05; Fig. 2B).

For the MTT assay, the growth rate of the KD group was significantly lower than that of the BC and NC groups (P<0.05). There was no significant difference between the NC and BC group (P>0.05; Fig. 3).

As shown in Fig. 4, NRP-1 shRNA also caused significant inhibition of NRP-1 protein expression in xenograft tumor tissue. NRP-1 protein expression was inhibited by 52.2% in the KD group compared to the BC group (P<0.05) while little variance was found between the NC and BC group (P>0.05). Additionally, we found that tumors in the KD group were smaller in size and lighter in weight than those in the BC group (P<0.05), while no obvious difference was found between the NC and BC group (P>0.05; Table I).

As the results of the immunohistochemical analysis showed, NRP-1 shRNA had a suppressive effect on the neovascularization and angiogenesis of tumors. The MVD of the KD group was 17±6, lower than that of the BC group (38±8; P<0.05) while no significant difference was found between the NC group (34±7) and the BC group (P>0.05; Fig. 5).