Study design and experimental grouping design
The study was concentrated on the following three parts. Firstly, the expression of TRAF6 was compared between group of HCC and non-HCC liver tissues. Groups of non-HCC liver tissues included group of para-tumor liver tissue, group of cirrhosis liver tissue and group of normal liver tissue. Secondly, the relationship between TRAF6 and clinicopathological data of HCC patients was analyzed. Subgroup classification was based on different status of each clinicopathological data, including age, gender, tumor differentiation, tumor size, number of tumor nodes, metastasis, clinical TNM stages, status of portal vein tumor embolus, vaso-invasion condition, capsular infiltration, hepatitis C virus (HCV) and hepatitis B virus (HBV) infection status, AFP expression, cirrhosis, as well as nm23, p53, p21, vascular endothelial growth factor (VEGF), Ki-67 expression and micro-vessel density (MVD) level as stained by CD34. Thirdly, in vitro experiments on HCC cell lines, including HepG2 and Hep3B, were performed to validate the biological function of TRAF6 on HCC cells. The cell viability, cell proliferation, cell apoptosis and Caspase-3/7 activity were compared between the group of negative controls and the group transfected with TRAF6 siRNA.
HCC tissues (n = 171) and their corresponding para-cancer tissues, 37 cirrhosis liver tissues and 33 normal liver tissues were collected from patients or autopsies admitted to the First Affiliated Hospital, Guangxi Medical University from March 2010 to December 2012. All these HCC tissues were taken from patients with primary HCC, who had not received any other treatment before surgery. The clinicopathological data of 171 HCC patients were listed above. All the formalin-fixed, paraffin embedded (FFPE) liver tissues were evaluated retrospectively, and their diagnoses were confirmed by two experienced pathologists independently. Besides, enzyme-linked immunosorbent assay (ELISA) was used to measure AFP level in sera and IHC was applied to detect nm23, p53, p21, VEGF, Ki-67 and MVD. All the features above were recorded. The study was approved by the Ethical Committee of the First Affiliated Hospital of Guangxi Medical University. Informed consent was signed by all of the patients who participated in the study. Related research procedure complied with the Helsinki Declaration.
Immunohistochemical technique was used to detect the expression of TRAF6. The TRAF6 antibody obtained from Santa Cruz Biotechnology, lnc. (D-10, sc-8409, 1:300 dilution, Heidelberg, Germany) was applied to antigen–antibody interaction. TRAF6 Antibody (D-10) is a mouse monoclonal IgG1 provided at 200 µg/ml and it is raised against amino acids 1–274 mapping at the N-terminus of TRAF6 of human origin. All the experimental operations were completed strictly according following instructions. Two pathologists reviewed the H&E sections and scored the staining independently, without knowing the status of the samples. The scoring standards were defined as follows : (1) the staining intensity was marked from 0 to 3 points, 0 for no staining, 1 for weak, 2 for moderate and 3 for strong. (2) The positive expression rate ranged from 0 to 4 points, 0 stands for no staining, 1 for <25 %; 2 for 26 to 50 %, 3 for 51 to 75 %, and 4 for >75 %. (3) The points of staining intensity plus the points of positive expression rate resulted in the final expression score.
Culture and transfection of the cell lines
Human HCC cell lines HepG2 and Hep3B were purchased from Shanghai Institute of Cell Biology of Chinese Academy of Sciences. Cells were cultured in the RPMI1640 medium with 10 % fetal bovine serum at 37 ℃ in a 5 % CO2 incubator. HepG2 and Hep3B cells were inoculated in 96 or 24-well plates and cultured for 24 h. Cell confluence was controlled at 50 to 60 % when cells were transfected with TRAF6 siRNA with Lipofectamine 2000 reagent (Invitrogen). After the transfection, we chose 0, 5 and 10 days as time points to detect the effects of TRAF6 siRNA on the malignant phenotypes of HCC cells.
Real time RT-qPCR and Western blot
TRAF6 expression of the HepG2 and Hep3B cell lines transfected with TRAF6 siRNA was measured with real time RT-qPCR and western blot.
The TRAF6 mRNA level was determined after 5 and 10 days post-transfection by RT-qPCR. Briefly, total cellular RNA isolation was performed with RNeasy Mini kit (Qiagen, Hilden, Germany). RNA concentrations were evaluated with a ND-2000 NanoDrop (Thermo Fisher Scientific, Wilmington, Delaware USA). Two hundred nanogram of cellular RNA was converted to cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a total volume of 10 μl. The primers for TRAF6 were: forward 5′- AGGGACCCAGCTTTCTTTGT-3′ and reverse 5′- GCCAAGTGATTCCTCTGCAT-3′. The primers for GAPDH were: forward 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and reverse 5′-CATGTGGGCCAT GAGGTCCACCAC-3′. RT-qPCR analysis was carried out on a LightCycler 1.5 using the Faststart DNA master SYBR green mastermix. Quantitative values were obtained from the PCR quantification cycle number (Cq) at which point the increase in signal for the PCR product was exponential. The target mRNA abundance in each sample was normalized to its GAPDH mRNA level as ΔCq = CqTRAF6−CqGAPDH. The value ΔΔCq was defined as the difference with a mock transfected control. Experiments were performed in triplicate. The knockdown ratio of TRAF6 mRNA expression was calculated with the formula: (1−1/2ΔΔCq) × 100 %.
For western blot, totally, 25 μg proteins obtained from the cell lines were added in 8 % SDS-PAGE separation gel. Then the mixture was transferred to nitrocellulose membranes and probed with primary antibodies against TRAF6 (D-10, sc-8409, dilution: 1:500, Santa Cruz Biotechnology). After incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies (D-3004), enhanced chemiluminescence detection was performed to identify the expression levels of TRAF6 protein.
Cell viability assay
The assay was conducted with Cell Titer-Blue kit (G8080, Promega, USA). The detection time points were set on 0th, 5th and 10th day after transfection. The cell viability was determined with FL600 fluorescent detector (Bio-Tek, USA) at 560 nm or 590 nm.
Cell proliferation assay
Cell proliferation was measured with MTS kit according to manufacturer’s instructions on 0th, 5th and 10th day post transfection. The medium was cleared out and washed with phosphate-buffered saline twice. Fetal bovine serum of 10 % and MTS reagents were added to subset of wells. After the cells were incubated for 2 h, absorbance at 490 nm was measured with a microplate reader (Thermo).
Cells were washed with cold phosphate-buffered saline and stained with 1 mg/ml Hoechst 33342 and 1 mg/ml PI for 15 min, fluorescence microscope (ZEISS Axiovert 25) was then used to observe the cellular morphology and staining changes. Ten different photos were taken under 200 times visual sight. The judging criterion was as follows: (1) Positive Hoechst 33342 staining and negative PI staining was found in living cells with complete shape. (2) Early apoptotic cells were stained with positive Hoechst and negative PI, containing blue apoptotic bodies, while apoptotic cells in the late phase were stained with negative Hoechst and positive PI, containing red apoptotic bodies. (3) Necrotic cells were those cells with negative PI staining but with none apoptotic bodies.
Caspase-3/7 activity assay
After the cell viability assay was completed, we carried out the experiments on the same 96-well were to detect caspase-3/7 activity by adding caspase-3/7 reagents. Then the cells were incubated for an hour. Fluorescence intensity was measured at 499 nm or 512 nm with FL600 fluorescent detector (Bio-Tek, USA).
SPSS21.0 was employed for statistical analysis. Kruskal–Wallis H tests were applied to analyze the status of TRAF6 protein expression among normal, cirrhosis, para-cancer and HCC tissues as well as their pathological grades. Mann–Whitney U tests were performed to identify the expression of TRAF6 in different subgroups with other clinicopathological features, including age, gender, tumor size, tumor nodes, metastasis, clinical TNM staging and so on. Correlation between the TRAF6 expression and clinicopathological features was detected with Spearman analysis. Furthermore, we performed receiver operating characteristic (ROC) curve to analyze the diagnostic ability of TRAF6 expression. Comparisons among groups of blank control, siRNA control and TRAF6 siRNA were performed with One-Way ANOVA or Kruskal–Wallis H tests. A P value less than 0.05 was considered statistically significant.