HCC patients
This study was performed according to the guidelines of the Helsinki Declaration. It was registered and approved by the ethics committee at The First Affiliated Hospital of Sun Yat-sen University. All patients provided written informed consent before treatment. The serum samples were collected from 57 primary HCC patients who underwent RFA treatment at the First Affiliated Hospital of Sun Yat-sen University between January 2012 and March 2013. All patients underwent abdominal ultrasound and computed tomography (CT) or magnetic resonance imaging (MRI), chest X-ray or CT before RFA. All of these patients were more than 18 years of age with complete clinical and laboratory data. Patients who received preoperative chemotherapy or radiotherapy were excluded. In addition, no patient had coexistent haematologic disorders or known active infection before treatment, ensuring that the serum parameters tested were representative of the normal baseline value. Specimens were obtained with informed consent from all patients. Sera were collected before RFA and 7 days after RFA from each patient. Fine needle biopsy was carried out on each patient before RFA. All patients were histologically diagnosed with HCC. Patients with mixed HCC and cholangiocarcinoma or without full follow-up data were excluded.
Sample preparation for proteomic analysis
The blood samples of 5 of the 57 HCC patients were collected into clean glass tubes without an additive and were allowed to clot at room temperature for 60 min. Serum was separated by centrifugation at 1000 x g for 30 min to remove the insoluble solids. Aliquots of serum were then stored at − 80 °C until use. The removal of albumin and IgG was performed using the ProteoPrep Blue Albumin Depletion kit (Sigma, St. Louis, MO, USA), according to the manufacturer’s instructions. The 2-D cleanup kit (GE Healthcare, UK) was used to remove impurities from the protein extraction prior to the determination of the sample concentration using the 2-D Quant kit (GE Healthcare).
2-de
Proteins derived from 5 samples before and after RFA were pooled separately, and 2-DE was performed three times per sample to minimize gel-to-gel variations. The Immobiline Dry strip (pH 4–7 L, length 18 cm; GE Healthcare) was immersed with 120 μg of proteins in 350 μl of rehydration buffer containing 5 M urea, 1 M thiourea, 4% CHAPS, 65 mM dithiothreitol, 5 mM tributylphosphine, 1% IPG buffer, and 1 mM phenylmethylsulphonyl fluoride. Isoelectric focusing (IEF) was performed using an IPGphor IEF apparatus with 0.002% bromophenol blue for 14 h at room temperature (GE Healthcare) at 70 kVh. The strip was then subjected to two-step equilibration in equilibration buffer containing 6 M urea, 30% glycerol, 2% SDS and 50 mM Tris-HCl (pH 6.8) with 2% dithiothreitol (w/v) for the first step and 2.5% (w/v) iodoacetamide for the second step. The two-dimensional SDS-PAGE gel (12.5% T, 18× 16 × 0.015 cm) was run at 7 W for 30 min followed by 17 W for 4 h. Separated proteins were stained with Deep Purple fluorescence dye (GE Healthcare; 1:200 diluted in 100 mM borate buffer) at room temperature for 1.5 h and then were rinsed 3 times (5 min each) with deionized water. The resolved protein spots in individual stained 2-D gels were visualized using a Typhoon 9200 laser scanner (GE Healthcare).
In-gel enzymatic digestion
ImageMaster 2-D Elite software 5.0 (GE Healthcare) was used for image analysis, which included spot detection, quantification and normalization. The intensity volume of each spot was normalized with the total intensity volume (summation of the intensity volumes obtained from all spots within the same 2-D gel) and was expressed as the relative intensity. In-Gel Enzymatic Digestion Protein spots were excised from the gel with an Ettan Spot Picker (version 1.0, GE Healthcare), destained twice with 30 mM potassium ferricyanide and 100 mM sodium thiosulphate (1:1, v/v) and then equilibrated in 50 mM NH4HCO3 to pH 8.0. After dehydration with acetonitrile (ACN) and drying in a speed vacuum concentrator for 20 min, the gel pieces were rehydrated in a minimal volume of sequencing grade porcine trypsin (Promega) solution (20 μg/ml in 25 mM NH4HCO3) and were incubated at 37 °C overnight. The peptides were extracted twice using 0.1% TFA in 50% ACN and were completely dried in a speed vacuum concentrator.
Protein identification and database searching
MALDI-TOF-MS/MS identification and database searching protein identification were performed using an Ultraflex III mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in the reflectron mode at an accelerating voltage of 20 kV. A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% ACN and 0.1% TFA was used as the matrix. Peptide mass fingerprints and MS/MS analysis were searched using BioTools software (version 3.0, Bruker Daltonics, Germany) against the SwissProt protein database. Protein identification was accepted when the peptide score was higher than the threshold value (P < 0.05), and manual interpretation had to confirm the agreement between the spectra and peptide sequence.
Enzyme-linked immunosorbent assay (ELISA) analysis
The levels of Clusterin (CLU) (E91180Hu; Cloud Clone Co.), Ficolin-3 (FCN3) (E91903Hu; USCN), and retinol binding protein 4 (RBP4) (E90929Hu; Cloud Clone Co.) in serum were measured using ELISA according to the manufacturer’s instructions. After development with a chromogen-substrate solution, the reaction was terminated by adding 100 μl of stop solution. Optical density values were read at 450 nm, and the concentrations were automatically calculated according to the standard curve.
Follow-up
Patients were regularly followed up at outpatient clinics every month for the first half year, every 3 months for the next one and a half years, and once annually thereafter. Patients received a physical examination, liver ultrasound, chest X-ray and serum alpha foetal protein (AFP) test at each follow-up. Abdominal computed tomography (CT) was performed every 6–12 months or when recurrence was suspected. Recurrence was defined as the emergence of clinical, radiological, and/or pathologic diagnosis of tumours from a previous origin locally or distantly. Once recurrence was confirmed, salvage treatments, including percutaneous ablation, surgery, or transcatheter arterial chemoembolization (TACE) were selected as needed.
Statistical analysis
Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA, version 16.0 for Windows). Student’s t-test and one-way analysis of variance (ANOVA) were used to analyse differences between groups. Disease-free survival (DFS) was calculated from the date of RFA to the date of recurrence. Survival curves were plotted using the Kaplan-Meier method and were compared using the log-rank test. A P-value < 0.05 was considered statistically significant.
