Intrahepatic cholangiocarcinoma patient samples
Intrahepatic cholangiocarcinoma tissues were collected from five patients who underwent hepatectomy in our Hospital. None of the patients had received preoperative chemotherapy or radiotherapy. The five cholangiocarcinoma patients included 3 cases with infiltration of the surrounding tissue (such as the liver, portal vein, nerve, and pancreas) and 2 cases with regional lymph node metastasis. The specimens were obtained with written informed consent from all patients. The study was approved by the Committees for Ethical Review of Research involving Human Subjects in our Hospital.
The human normal biliary epithelial cells established from histologically normal liver tissues obtained from five patients who underwent liver transection for metastatic tumors were gifts from Dr. Ludwik K Trejdosiewicz (University of Leeds, UK) . The human cholangiocarcinoma cell lines QBC939, RBE, and ICC-9810 were obtained from ATCC and cultured in Ham’s F12 Medium supplemented with 10% FBS at 37°C in a humidified chamber containing 5% CO2.
Antibodies against Notch-1, E-cadherin, Vimentin, F-actin and α-SMA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The GAPDH antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).
RNA extraction and reverse transcription-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized using TaqMan RT reagents (Applied Biosystems) following the manufacturer’s instructions. The primers for Notch1 and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control were synthesized by Invitrogen (Carlsbad, CA, USA). The upstream Notch1 primer was 5′-GCAAGAAGAAGCGGAGAG-3′, and the downstream primer was 5′- AGCTGGCACCCTGATAGATG -3′; the Notch1 PCR product length was 423 bp. The upstream control GAPDH primer was 5′-AGATCCACAACGGATACATT-3′, and the downstream primer was 5′-TCCCTCAAGATTGTCAGCAA-3′; the GAPDH PCR product length was 308 bp. The PCR conditions were as follows: predenaturing at 94°C for 2 min, denaturing at 94°C for 30 s, reannealing at 53°C for 45 s, and elongation at 72°C for 30 s, for 30 cycles; and final elongation at 72°C for 10 min. The PCR products underwent 1.5% agarose gel electrophoresis.
Western blot analysis
Protein was quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA), and equal amounts of protein were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). After blocking in 5% skim milk for 1 h at room temperature, the membranes were incubated with the indicated primary antibody at 4°C overnight, followed by a horseradish peroxidase-conjugated secondary antibody. The proteins were detected by chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The Western blot data were quantified by measuring the intensity of the hybridization signals using an image analysis program (Fluor-ChemTM 8900, Alpha Inotech).
Plasmid constructs and siRNA transfection
The full-length Notch1 cDNA was amplified and cloned into the pReciever M68 expression vector (FulenGen, Guangzhou, China). The expression plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Oligonucleotide siRNA duplexes were synthesized by Shanghai Gene Pharma (Shanghai, China). The following siRNA sequences for Notch1 were used: 5′- UGGCGGGAAGUGUGAAGCG-3′ and 5′- CGCUUCACACUUCCCGCCA-3′. The siRNAs were transfected into ICC-9810 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to manufacturer’s instructions.
BrdU incorporation analysis
Ten micrograms per milliliter of BrdU were added to the culture medium for 24 h. The cells were fixed with 100% ethanol for 10 min, then incubated with 2 ml HCl for 45 min and 0.1 ml sodium tetraborate for 15 min at room temperature. The cells were then incubated with a mouse monoclonal anti-BrdU antibody overnight at 4°C and incubated with fluoresce in isothiocyanate-conjugated goat anti-mouse IgG for 1 h at room temperature. Hoechst 33342 was used to label nuclei.
Rac activation assay
Rac1 intracellular activity was examined using Rac1 activation assay kits (Upstate Biotechnology, Lake Placid, NY, USA) according to the manufacturer’s protocols. Briefly, cells were lysed with Mg2+ lis buffer. After clarifying the cell lysates with glutathione agarose and quantifying the protein concentrations, aliquots with equal amounts of proteins were incubated with the Rac assay reagent (PAK-1 PBD, agarose) at 4°C for 1 h, using the GTPgS-pretreated lysates as positive controls. The precipitated GTP-bound Rac1 was then eluted in Laemmli reducing sample buffer, resolved by 12% SDS-PAGE, and immunoblotted with a monoclonal anti-Rac1 antibody. Five percent of the cell lysate was resolved by 10% SDS-PAGE and immunoblotted with a Rac1 antibody to measure the total amount of Rac1.
Immunohistochemical staining was performed on 4-μm paraffin-embedded kidney sections. Antigen retrieval was performed by microwave treatment. The sections were exposed to 3% H2O2 for 20 min, blocked with 10% sheep serum in PBS at 37°C for 40 min, then incubated with the indicated antibodies at 4°C overnight. After rinsing three times with PBS, the sections were incubated with ChemMate™ EnVision/HRP Rabbit/Mouse secondary antibody (Dako, Copenhagen, Denmark) for 1 h. The degree of immunostaining was reviewed and independently scored by two observers based on the proportion of positively stained tumor cells and intensity of staining.
Cells (1 × 105) were suspended in 200 μl of serum-free DMEM medium and seeded on the upper side of the invasion chamber (Millipore, Billerica, USA). The lower side of the chamber was filled with DMEM supplemented with 10% fetal bovine serum. After incubation at 37°C for 18 h, cells that had penetrated through the chamber were fixed with methanol for 15 min at room temperature and stained with 0.1% crystal violet for another 15 min. The upper surface of the chamber was carefully wiped with a cotton-tipped applicator. Cells that had passed through the pores were counted in five non-overlapping fields (×40 magnification) and photographed.
Cell morphology examination and immunofluorescence
Cell morphology was monitored on a phase contrast microscope equipped with a video camera. Cells grown on glass coverslips were fixed with 3.7% formaldehyde solution in PBS for 10 min at room temperature. Following three extensive washes with PBS, the cells were permeabilized in PBS containing 0.1% Triton X-100 for 3 min and blocked with PBS containing 5% BSA for 1 h at room temperature. The cells were incubated overnight at 4°C with primary antibodies diluted in PBS containing 3% BSA, followed by incubation with Alexa Fluor 488-conjugated goat anti–rabbit secondary antibody (1:1000; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature for detection. Actin filaments were visualized by staining the cells with Alexa Fluor 633-conjugated Phalloidin (1:1000; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. To identify nuclei, the cells were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 3 min. The coverslips were mounted in fixation medium (Biomeda, Foster City, CA, USA). Images were collected and analyzed using the Zeiss LSM 510 Confocal Imaging System (Zeiss, Germany).
The statistical analyses were performed using SPSS 13.0 statistical software (Chicago, IL, USA). Significant differences between two groups were determined by Student’s t-test. P < 0.05 was considered statistically significant. The results are expressed as the mean ± SD from at least three experiments.