The study protocol was approved by the Ethics Committees at the Chinese PLA General Hospital, and informed consent was obtained from each patient. The study was carried out in accordance with the guidelines of the 1975 Declaration of Helsinki and was consistent with good clinical practice guidelines and local regulatory requirements.
Cell Culture, Treatment, Reporter Genes, Expression Vectors, Plasmid Construction, and DNA Transfection
Human liver cancer cell lines (PLC/PRF/5, SK-Hep1, HepG2, SMMC-7721, and Bel-7402) were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen, Melbourne, Australia) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT). HCC cell lines were split to low density (30% confluence) 24 hours before treatment. Conditioned with 5-aza-2′-deoxycytidine (5-AZA) (Sigma-Aldrich, St. Louis, MO) at 2 μM, the growth medium was changed every 24 hours for a total 96-hour treatment. The cells were serum-starved for 36 hours for detection of TGF-β and then stimulated by TGF-β1 (Peprotech, Rocky Hill, NJ) at a concentration of 5 ng/mL for 1 hour to determine the activity of phosphorylated Smad2/3. The cells were further starved for 48 hours to investigate the expression of c-Myc, and for another 96 hours to perform the cell viability assay with or without pretreatment with 5-AZA. For chemosensitivity assay, the cells were treated with 5-fluorouracil (5-FU) (Sigma-Aldrich) of eight concentrations from a highest concentration of 500 μg/mL diluted serially 2-fold for 72 hours. The vector expressing DACH1 was a gift from Dr. Cvekl and the reporter plasmids SBE4-Luc were described previously. The cells (2.5 × 103) were seeded in 96-well plates, incubated for 24 hours, and transfected with an appropriate combination of the reporter, expression plasmid, and control vector using Fugene HD (Roche, Indianapolis, IN). They were serum-starved for 36 hours and then stimulated with or without TGF-β1 for 12 hours before luciferase assay. The transfection efficiency was normalized by cotransfection with 2 ng of pRL-TK plasmid (Promega, Madison, WI) and subsequently measured with the Promega dual-GLO reporter assay system. DACH1 was subcloned into lentivirus expression vector and expression of DACH1 was determined by immunofluorescence staining with anti-DACH1 antibody.
Patients and Collection of Tissue Samples
Postoperative cases of HCC were randomly recruited between January 2010 and November 2011. None of these patients had received preoperative anticancer treatment. The tumor sample and adjacent tissue (i.e., 1 cm zone of peritumoral parenchyma) were collected in pairs and tumor staging was determined according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 2010 (7th Ed.) (Table 1). Normal hepatic tissue was obtained from the edge of resected hemangioma of the liver. Prior to molecular genetic analysis, the presence of neoplastic tissue was confirmed by histopathological examination of hematoxylin and eosin (H&E)-stained sections.
Table 1. Clinicopathologic Features of the Study Patients
|Age, years|| || |
|Gender|| || |
|Preoperative ALT, U/L|| || |
|Preoperative AST, U/L|| |
|α-Fetoprotein, μg/L|| || |
|Liver cirrhosis|| || |
|HBsAg|| || |
|Tumor diameter, cm|| || |
|Tumor differentiation|| || |
|Multicentric occurrence|| || |
|Intrahepatic metastasis|| || |
|UICC TNM stage|| || |
Methylation-Specific Polymerase Chain Reaction (PCR) (MSP)
Genomic DNA from HCC cell lines and primary HCC tissues were prepared as described. The following MSP primers were designed according to genomic sequences flanking the transcription start sites: DACH1-M-sense, 5′-GGA AAA AAT TAT TAG TTT TCG CGG AC-3′; DACH1-M-antisense, 5′-AAA CCG AAA ACA CAA AAA TAA CGA TCG-3′; DACH1-U-sense, 5′-TTT GGA AAA AAT TAT TAG TTT TTG TGG AT-3′ and DACH1-U-antisense, 5′-AAA AAA CCA AAA ACA CAA AAA TAA CAA TCA-3′. The primer sequences were oligo-synthesized (Invitrogen, Beijing, China) for MSP to detect bisulfate-induced changes affecting unmethylated (U) and methylated (M) alleles.
Methylation-Sensitive Restriction Enzyme Digestion and Real-Time Quantitative PCR (MSRED-qPCR)
The quantitative method MSRED-qPCR was employed to evaluate the degree of DACH1 promoter region methylation in HCC cell lines and patients' specimens as described[16, 17] with modification. In brief, each sample was subsequently divided into two equal aliquots. The first aliquot was digested with 10 U Hha I (Takara, Dalian, China) at 37°C overnight in a final reaction volume of 20 μL. To maintain the homogeneity of the digestion between the test and control samples, Hha I was replaced with 50% glycerol in the sham-treated control reaction. After incubation, digested samples were incubated at 65°C for 15 minutes to inactivate the enzyme. To ensure complete enzyme digestion, it was run in parallel with a positive and a negative control digestion in which 200 ng of completely methylated or unmethylated control DNA (EpiTect Control DNA, Qiagen, Valencia, CA) was digested. After digestion, the same amount of digested or undigested plasma DNA along with control digestion was subjected to quantitative PCR with the Lightcycler 480II Real-Time PCR Detection System (Roche). Each reaction was performed in a final volume of 20 μL containing digested (1 μL) or undigested (1 μL) DNA, 5 μM each primer and 1 × QuantiFast SYBR Green RT-PCR Kit (Qiagen). The PCR reaction was performed at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 68°C for 60 seconds, and 74°C for 60 seconds. At the end of the PCR cycles, melting curve analyses were performed to validate the specific PCR product. Primers used for this qPCR were: Forward, 5′-CGTGAAGTGGGGGCTGTGTTTTCGT and Reverse, 5′-CAGAGACTCCGAGAGCGCGAGACAC. The degree of DACH1 promoter methylation was expressed as 2-(Ctdigestion- Ctsham). Each sample was run in duplicate for analysis. For 100% digestion efficiency, the relative expression level of completely unmethylated control DNA must be close to zero, whereas the level of completely methylated control must be 1.
RNA Isolation and Semiquantitative Reverse Transcription-PCR
Total RNA was isolated using Trizol reagent (Life Technologies, Gaithersburg, MD) and chloroform/isopropanol precipitation. Reverse transcription of RNA (5 μg) was carried out using random 6-mer primers included in the Superscript III-reverse transcriptase kit (Invitrogen, Carlsbad, CA). The primer sets for DACH1 gene were: 5′-CGT GAA CAA GCA GAA CAG ACG-3′; 5′-CCC ATG ACG AAT GTC TGA CTG-3′.
The sections of neoplastic tissue were subjected to IHC for detecting DACH1. The staining was assessed by two independent pathologists blinded to the origin of samples. The staining intensity and extent of the stained area were graded according to the German semiquantitative scoring system: staining intensity of the nucleus, cytoplasm and/or membrane (no staining = 0; weak staining = 1; moderate staining = 2; strong staining = 3); the extent of stained cells (0% = 0, 1%-24% = 1, 25%-49% = 2, 50%-74% = 3, 75%-100% = 4). The final immunoreactive score (0 to 12) was determined by multiplying the intensity score with the extent of stained cells.
Soluble proteins were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gel and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The primary antibodies were: DACH1 (Abcam, Cambridge, UK); c-Myc, Smad2 (P459), and Smad3 (P209) (BioWorld, Minneapolis, MN); rabbit monoclonal antibodies against phospho-Smad3 (Ser423/425), phospho-Smad2 (Ser465/467), and p53 (Cell Signaling Technology, Danvers, MA); p21 (Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (Beyotime, Nanjing, China). The data were normalized to β-actin.
Cell Growth Assay
The cells (5 × 103) were seeded in 96-well plates, incubated for 24 hours, and treated as above. After treatment, the cell proliferation was determined by the Cell Counting Kit 8 (CCK-8) (Beyotime). Absorbance at 450 nm and 630 nm were measured with the EXL800 microimmunoanalyzer (BioTek, Burlington, VT). The results were plotted as means ± SD. The data are expressed as the percentage of viable cells: relative viability (%) = [A450-630 (treated) − A450-630 (blank)] / [A450-630 (control) − A450-630 (blank)] × 100%. IC50 was defined as the concentration required for 50% inhibition of cell growth. The values were calculated by nonlinear regression analysis using SPSS 11.5 (Chicago, IL). For cell proliferation assay, growth was measured daily by CCK-8.
Cell Cycle and Apoptosis Analysis
Cells were processed by standard methods using propidium iodide (Sigma-Aldrich) staining of cellular DNA. Each sample was analyzed by flow cytometry with a FACScan Flow Cytometer (Becton-Dickinson Biosciences, Mansfield, MA) using a 488 nm laser. Histograms were analyzed for cell cycle compartments using ModFit v. 2.0 (Verity Software House, Topsham, ME). Annexin-V (Keygen Biotech, Nanjing, China) staining was conducted as per the manufacturer's protocol.
2.0 × 103 cells were plated in triplicate in 2 mL complete growth medium. After 2 weeks incubation, colonies more than 50 μm in diameter were counted using an Omnicon 3600 image analysis system. The colonies were visualized after staining with 0.04% crystal violet in methanol for 1-2 hours.
Analysis was performed with SPSS v. 11.5. Data collected from multiple independent experiments are presented as mean ± standard error using Student t distribution with a 95% confidence interval. Parameters from multiple groups such as different histopathological grades of carcinoma were compared using one-way analysis of variance (ANOVA) with Student-Newman-Keuls (SNK)-q test. P < 0.05 was considered statistically significant.