HCC specimens and cell lines
Forty-three HCC specimens from archives of paraffin embedded tissues were collected at the Sun Yat-sen University Cancer Center (Guangzhou, China). Among them, 22 HCCs with metastasis including 8 portal vein metastases, 9 intra-hepatic metastasis and 5 extra-hepatic metastases (4 in lung and 1 in centrum). Another 50 pairs of frozen HCC specimens (tumor and adjacent non-tumorous tissues) were collected at the Sun Yat-sen University Cancer Center (Guangzhou, China) for RNA isolation. Samples used in this study were reviewed and approved by the Committees for Ethical Review of Research involving Human Subjects at Sun Yat-Sen University Cancer Center. Human HCC cell lines QGY-7703 and PLC8024 were obtained from the Institute of Virology of the Chinese Academy of Medical Sciences (Beijing, China). MHCC-97L was obtained from Liver Cancer Institute, Fudan University (Shanghai, China). All cell lines were cultured in high-glucose DMEM (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum.
Immunohistochemistry (IHC) Staining
Paraffin-embedded, formalin fixed liver tissue sections (5 µm in thick) were deparaffinized and rehydrated. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H2
) for 30 min. For antigen retrieval, slides were immersed in 10 mM citrate buffer (pH 6.0) and boiled for 10 min in microwave oven. Non-specific binding was blocked by 5% BSA in PBS for 30 min. The slides were incubated with a 1
300 dilution of antibody against human IL-17A (R&D Systems, Minneapolis, MN) at 4°C overnight in a moist chamber. Diaminobenzidine tetrahydrochloride was used as the visualization substrate followed by counterstaining with hematoxylin. Positively stained cells were counted under microscope by two independent investigators.
Cell growth assay and focus formation assay
Cell growth rate was determined by XTT assay. Briefly, cells were seeded in a 96-well plate at a density of 1×103 cells and incubated at 37°C in a humidified atmosphere containing 5% CO2. After 24 hr, cultured cells were treated with or without 50 ng/mL recombinant human IL-17A (rhIL-17A) (R&D System, Minneapolis, MN), and 10 ng/mL TNF-α (R&D System, Minneapolis, MN)was used as positive control. XTT assay using Cell Proliferation Kit II (Roche Molecular Biochemicals, Germany) was performed according to the manufacturer's instructions. Triplicate independent experiments were done and data were expressed as mean±SD. For focus formation assay, 1×103 cells were seeded onto a 6-well plate and stimulated with or without 50 ng/ml rhIL-17A for 1 week. Surviving colonies were fixed and stained with 1% crystal violet. Triplicate independent experiments were performed.
Cell migration and invasion assay
Cell migration and invasion ability were studied by wound healing and invasion assays. A series concentration of rhIL-17A (10 ng/ml, 50 ng/ml and 100 ng/ml) was tested and the result showed that 50 ng/ml rhIL-17A had the best effect (data did not shown), so 50 ng/ml rhIL-17A was used in this study. Cell migration was assessed by a scratch wound-healing assay. Cells were cultured in 6-well plate until confluent and then treated with or without rhIL-17A (50 ng/mL). The cell layer was wounded using a sterile tip and the spread of wound closure was observed and photographed under a microscope until healed area was found. Invasion assay was performed with 24-well BioCoat Matrigel Invasion Chambers (Becton Dicknson, Bedford, MA) according to the manufacturer's instructions. After cultured in medium with or without rhIL-17A (50 ng/mL), 5×104 cells were seeded onto inner well and number of cells that invaded through the Matrigel was counted under 20× objective lens.
Western Blot Analysis
Whole cell lysates from HCC cells were harvested with cell lysis buffer. Nuclear lysates from cultured PLC8024 and MHCC-97L cells were harvested with NucBuster™ ProteinExtraction Kit (Novagen, Germany) according to manufacturer's instructions. Western blotting analyses were performed with the standard protocol using antibodies against β-actin, E-cadherin, α-SMA, vimentin, P-p65 (Santa Cruz Biotechnology, Santa Cruz, CA), histone H3, fibronectin and MMP9, (Abcam,UK), N-cadherin, α-catenin and β-catenin (Cell Signalling Technology, Beverly, MA).
Quantitative real-time PCR (qPCR)
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an Advantage® RT for PCR Kit (Clontech, Mountain View, CA) according the manufacturer's instructions. For qPCR analysis, aliquot of double-stranded cDNA was amplified with primers (Table S1
) using a SYBR Green PCR Kit (Applied Biosystems, Carlsbad, CA) and an ABI PRISM 7900 Sequence Detector. 18s rRNA was used as internal control. The threshold cycle (CT
) was measured during the exponential amplification phase, and the amplification plots were analyzed using SDS 1.9.1 software (Applied Biosystems). The relative expression level of target genes (IL17A, MMP2 and MMP9) is given by 2−ΔΔCT
ΔCT (target gene)
−ΔCT(average of target gene in non-tumor tissue)
). All reactions were performed in duplicate.
All data were analyzed with SPSS software (version 16.0) for statistical analysis. Comparisons between groups were analyzed by Student's t-test. Correlations between variables were determined by linear regression analysis. Survival was estimated by the Kaplan–Meier method and compared by the log-rank test. Univatiate and multivariate analysis of prognostic factor was performed with Cox progression model. Value of P<0.05 (two-tailed) was considered statistically significant.