Patients and specimens
Cancerous tissues and surrounding non-cancerous hepatic parenchyma were obtained from 40 primary HCC Japanese patients who underwent curative resection surgery at Chiba University Hospital, Japan, from January 1995 to December 2001. They did not have any other malignancies. The ethics committee of Chiba University Hospital approved this study. Informed consent was obtained from every patient for the use of resected tissue before the study began. Samples were obtained from 31 men and 9 women aged 49-78 years. Specimens were histologically classified by the Japanese staging system of the Liver Cancer Study Group of Japan [30
]. In the corresponding noncancerous parenchyma, cirrhosis was found in 14 patients (35%).
The human HCC cell lines HuH7, HepG2, HLE, HLF, PLC/PRF/5, JHH1, JHH2, JHH5, JHH6, and JHH7 were obtained from the Health Science Research Resources Bank (Osaka, Japan). HuH7, HepG2, HLE, HLF, and PLC/PRF/5 were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum (Sigma-Aldrich Corp., St. Louis, MO). JHH1, JHH2, JHH5, JHH6, and JHH7 were cultured in William's Medium E with 10% fetal bovine serum. Primary cultures of human hepatocytes were prepared and cultured as described in previous papers [31
RNA extraction and cDNA synthesis
Total RNA from the cell lines and tissues was isolated using RNeasy Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Specimens were taken from viable and non-fibrotic areas of tumors and noncancerous tissues. They were quickly frozen in liquid nitrogen and then stored at -80°C until use. Synthesis of cDNA from total RNA was conducted using a Ready-to-Go® cDNA synthesis kit (Amersham Biosciences Corp., Piscataway, NJ) following the manufacturer's protocols.
Real-time quantitative RT-PCR analysis
The expressions of FGF19 and FGFR4 were examined by reverse transcription polymerase chain reaction (RT-PCR) with the following primers: (forward) (5'-TCT CCT CTG ACT TCA ACA GCG ACA C-3') and (reverse) (5'-TGT TGC TGT AGC CAA ATT CGT TGT C-3') for human GAPDH, (forward) (5'-CAG CTG TAC AAG AAC AGA GGC TTT C-3') and (reverse) (5'-AAA TGG GTC CAT GCT GTC GGT CTC C-3') for FGF19, and (forward) (5'-CAT CCG CTG GCT TAA GGA TGG AC-3') and (reverse) (5'-ATC ACG AGA CTC CAG TGC TGA TG-3') for FGFR4. All PCR reactions were performed using the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) under the following conditions: 1 cycle at 95°C for 10 min, 50 cycles at 95°C for 10 sec, 60°C for 5 sec, 72°C for 10 sec, and 80°C for 1 sec. Real-time detection of the SYBR Green emission intensity was conducted with a LightCycler® (Roche, Mannheim, Germany). An equivalent amount of cDNA sample, derived from 40 ng total RNA, was used for each PCR reaction. The mRNA in each sample was then automatically quantified with reference to the standard curve constructed with each use of the LightCycler®. Quantitative RT-PCR was performed at least three times per sample. To standardize the amount of RNA, we quantified the expression of GAPDH mRNA in each sample and then divided the amounts of expressed FGF19 and FGFR4 mRNA by that of GAPDH.
An immunohistochemical analysis was performed on paraffin-embedded sections using the Envision kit (Dako, Glostrup, Denmark) following the manufacturer's instructions. The sections were boiled in retrieval solution to expose antigens. Anti-FGF19 (R&D Systems, Inc., MN) monoclonal antibodies, anti-FGFR4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) monoclonal antibodies and control antibody (R&D Systems, Inc., MN) were applied as the primary antibodies to the sections at a dilution of 25 μg/mL and 1:50. The section slides were counterstained with hematoxylin, dehydrated, and mounted. The immunostaining was evaluated independently by two pathologists.
Cell fixation and staining
HuH7, HepG2, HLE, HLF, and JHH7 cells (1 × 104/well) were seeded and cultured at 37°C in a humidified 5% CO2 atmosphere for 24 h. Then, the cells were washed 3 times with phosphate-buffered saline (PBS) and fixed in 100% acetone for 10 min at 4°C. After fixation, cells were washed 3 times with PBS. Cells were blocked in blocking serum at room temperature for 1 h in a humidified chamber. Cells were washed 3 times with PBS and stained at the same dilution as paraffin-embedded sections.
Cell proliferation assay with FGF19 recombinant protein
Cells were seeded (6 × 103/well) with FGF19 recombinant protein (Cell Sciences, MA, USA) at concentrations of 0.1, 1, 10, or 100 ng/mL and cultured. After 48, 72, or 96 h, 20 μL of Cell Titer 96® AQueous (Promega, WI, USA) was added to the culture media then incubated for 1 h at 37°C in a humidified 5% CO2 atmosphere. Then, the absorbance of each of the plates at 490 nm was recorded using a 96-well plate reader (Bio-Rad, CA, USA). The proliferation index (PI) was defined as OD values of the recombinant protein-treated cells divided by those of the untreated control cells. We examined proliferation assay with 12 chambers in the same condition at the same time.
Cell apoptosis assay with FGF19 recombinant protein
Cells were harvested at 48 h after seeding and incubation with 1 ng/ml of FGF19 recombinant protein. To initiate apoptosis, 5-fluorouracil (5-FU) (Roche) was added at a final concentration of 10 μg/mL. After further incubation and gravity sedimentation for 24 h, the supernatant was removed carefully and the cell pellets were resuspended in 200 μL lysis buffer. Then, ELISA was performed using the Cell Death Detection Kit (Roche) according to the manufacturer's specifications. The apoptosis index (AI) was defined as OD values of recombinant protein-treated cells divided by those of the controls. We examined apoptosis assay with 12 chambers in the same condition at the same time.
Invasion assay with FGF19 recombinant protein
Double-chamber transwell plates with 8-μm pore-size polycarbonate membrane inserts at the base of the upper chamber (Cell Biolabs, Inc., San Diego, CA) were used. Here, 300 μL of warm, serum-free media was added to each upper chamber and incubated for 1 h at 37°C. Upper chambers containing the reconstituted Matrigel membranes were set into the lower chambers of the 24-well cluster plates. JHH7 cells were trypsinized, counted, and resuspended with fresh medium before being plated into the upper chambers of the assay wells. Approximately 1 × 106 cells were plated in 300 μL of defined medium into each upper chamber. Then, 500 μL of medium was added to each lower chamber. Both the media in the upper and lower chambers contained 1 ng/ml of human recombinant FGF19. JHH7 were cultured in this way for 48 h in a humidified 5% CO2 atmosphere. The medium in the upper chamber was then aspirated, and cells from its inner surface were wiped off using a cotton swab. The membranes were stained with 400 μL of Cell Stain Solution and incubated for 10 min at room temperature. Stained inserts were gently washed several times with water. Then, the stained inserts were transferred to an empty well containing 200 μL of extraction solution and incubated for 10 min on an orbital shaker. Subsequently, 100 μL from each sample was transferred to a 96-well microtiter plate, and the OD 560 nm of each sample was measured in a 96-well plate reader (Bio-Rad, Hercules, CA). We examined invasion assay with 6 chambers in the same condition at the same time.
Migration assay with FGF19 recombinant protein
Migration assay was performed by using transwell plates with 8 μm pore (BD Biosciences). JHH7 cells (1 × 105) suspended in 500 μl serum-free Williams medium were seeded into the upper part, whereas the lower compartment was filled with 1 ml medium with 1 ng/ml of the human FGF19 recombinant protein. After incubation for 24 h at 37°C in 5% CO2, non-migrating cells were removed from the upper surface of the membrane by scrub. Cells on the reverse side were stained with 0.1% crystal violet, and counted under a microscope at x100 magnification. We examined migration assay with 6 chambers in the same condition at the same time.
Gene knockdown using small interfering RNA transfection
Transfections of FGF19, FGFR4 and non-targeting negative control small interfering RNA (siRNA) (AMBION, Austin, TX) were conducted with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 96-well plates according to the manufacturer's specifications. The day before transfection, the JHH7 cells were trypsinized, counted, and seeded at 6 × 103 cells per well into 96-well plates. Lipofectamine 2000 diluted in Opti-MEM (Invitrogen) was supplemented to the siRNA mixture. The mixture was incubated for 20 min at room temperature. The mixture and Opti-MEM were added to the plate to a final siRNA concentration of 20 nM.
Control and FGFR4-specific siRNA were transfected into JHH7 as described above. At 72 h after transfection, the cells were collected and stained with either a phycoerythrin-conjugated anti-FGFR4 antibody (BioLegend, San Diego, CA) or an isotype-matched control (R&D systems, Minneapolis, MN). Flow cytometric analysis of cell surface FGFR4 was performed using a FACSCalibur analyzer (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Cell proliferation assay after FGF19 and FGFR4 siRNA transfection
Cells (6 × 103 per well) were seeded into 96-well plates, cultured for 24 h, and transfected with control, FGF19, or FGFR4 siRNA. At 72 h after transfection, cells were harvested. Then, 20 μL of Cell Titer 96® AQueous (Promega) was added to the culture media and incubated for 2 h at 37°C in a humidified 5% CO2 atmosphere. The absorbance of the plates at 490 nm was recorded using a 96-well plate reader (Bio-Rad).
Apoptosis assay after FGF19 and FGFR4 siRNA transfection
Cells (6 × 103 per well) were seeded into 96-well plates, cultured for 24 h, and transfected with control, FGF19, or FGFR4 siRNA. Cells were harvested at 72 h after transfection. To initiate apoptosis, 5-FU (Roche) at a final concentration of 10 μg/mL was added. After further incubation and gravity sedimentation for 24 h, the supernatant was carefully removed, and the cell pellets were resuspended in 200 μL lysis buffer. Then, ELISA was performed using the Cell Death Detection Kit (Cell Biolabs, Inc.) according to the manufacturer's specifications.
Changes in serum FGF19 levels in patients with HCC after hepatectomy
Blood serum was obtained from 10 healthy subject and 29 primary HCC patients who underwent curative resection at Chiba University Hospital, Japan, from 2005 to 2007 (22 men, 7 women). The samples were obtained on the day before and the day after surgery. A sandwich ELISA kit was used for colorimetric detection of FGF19 in serum (FGF19 Quantikine ELISA kit, Minneapolis, MN) following the manufacturer's instructions.
The relative mRNA expression levels (FGF19/GAPDH and FGFR4/GAPDH) were calculated from the quantified data. Mann-Whitney's U test was used to analyze the differences in the FGF19 and FGFR4 expression levels between HCCs and the corresponding noncancerous hepatic tissues. To analyze the correlation between FGF19, FGFR4, and clinicopathological parameters, differences in the numerical data between the two groups were evaluated using the Kruskal-Wallis test. Overall and disease-free survival rates were then calculated using the Kaplan-Meier method, and the differences in survival curves were analyzed using the log-rank test. Survival was counted if the patient was still alive or had died of other causes. Independent prognostic factors were analyzed by the Cox proportional hazards regression model in a stepwise manner. All the statistical analyses were performed using Stat View software (Version 5.0, Abacus Concepts, Berkeley, CA). Data are expressed as mean ± SE. P < 0.05 denoted the presence of a statistically significant difference.