Patients and specimens
In our study, a total of 191 HCC specimens were collected randomly from patients who underwent radical resection of pathologically confirmed tumors from 1999 to 2006 at the Liver Cancer Institute and Zhongshan Hospital (Fudan University, Shanghai, China). No patients received any preoperative anticancer treatment. The ethics committee of Zhongshan Hospital approved this research. All patients provided written informed consent to participate in this study.
Tissue microarray (TMA) and Immunohistochemistry (IHC)
A tissue microarray (TMA) was constructed as described previously[25]. Briefly, two cores were taken from each representative tumor tissue and from peritumoral liver tissue (in collaboration with Shanghai Biochip Company Ltd, Shanghai, China). Duplicate cylinders from two different areas, intratumoral and peritumoral (a total of four punches for each patient), were obtained. The rabbit anti-plakoglobin (Cat. No. A303-718A, CST) was used in the immunochemistry to investigate the expression. The density of positive staining was measured with the use of a computerized image system composed of a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd, Cambridge, United Kingdom).
Cell lines and Culture Conditions
Four HCC cell lines (HepG2, HCCLM3, MHCC97L and MHCC97H) and human hepatic cell line L02 were used in cell experiments. HCCLM3, MHCC97L and MHCC97H are human HCC cell lines with high metastatic potential[26] (established at the liver cancer institute, Zhongshan Hospital, Fudan University, Shanghai, China) and HepG2 is HCC cell line with low metastatic potential (provided by the liver cancer institute, Zhongshan Hospital, Fudan University, Shanghai, China). L02 is normal liver cell line (obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China). Cells were incubated at 37℃ in a humidified atmosphere of 5% CO2 maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen GIBCO), 100U ml-1 penicillin and 50 μg ml-1 streptomycin.
RNA Isolation and qRT-PCR
Total RNA was extracted from the four HCC cell lines (Hep G2, MHCC97L, MHCC97H and HCCLM3) and one normal liver cell line (L02) using Trizol reagent (Invitrogen) according to the manufacturer's instructions. qRT-PCR was performed using a ABI SYBR Green qPCR super Mix Kit according to the manufacturer's instructions. GAPDH was used as an internal control. The primers were as follows: plakoglobin forward primer 5'- ATGGAGGTGATGAACCTGATGGAG-3' and reverse primer 5'- TTGAGCGTGTACTGGCGCCC-3' and GAPDH forward primer 5' -TTCACCACCATGGAGAAGG -3' and reverse primer 5' -GGCATGGACTGTGGTCATGA-3'. Relative mRNA levels were calculated based on Ct values, corrected for GAPDH expression, according to the equation: K×2-ΔCt [ΔCt=Ct (plakoglobin)-Ct (GAPDH)], while K is a constant. All the experiments were performed in triplicate.
Western Blot
Cells were lysed using cell lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100) containing protease and phosphatase inhibitors. Equivalent amounts of whole cell extracts were subjected to SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with respective primary antibody overnight at 4°C, followed by the incubation of the appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Blots were visualized with an ECL detection kit (Pierce, IL) and analyzed using Quantity One 1-D Analysis Software (Bio-Rad, San Francisco, CA).
Statistical analysis
Data analysis was performed with SPSS 20.0 for Windows (SPSS Inc. Chicago, IL, USA). Unpaired Student’s t-test or Pearson’s χ² test was used to compare quantitative variables. Kaplan-Meier analysis was used to determine the survival and recurrence, the Cox regression model was used to perform multivariate analysis. P<0.05 was considered statistically significant.