Animal model
The human NCI-N87 cells (3x10 6/mouse) were subcutaneously injected into right dorsal flank of each BALB/c-nu/nu nude mouse. After 1–2 weeks of implantation with tumor cells, when tumors reached ~20-30 mm 3, the animals were randomized into control and treatment groups (24 animals per group). The 125I seeds (0.9 mCi) were injected into mice in treatment group through 18-gauge needles, while ghost seed were injected into the mice in control group.The tumor size was measured using calipers and the tumor volume was estimated by the formula: tumor volume (mm3) = (L x W 2) × 1/2, where L is the length and W is the width of the tumor. Tumor volumes and body weights were monitored every 3 days over the course of treatment. The tumor weight was measured when the mouse was sacrificed. Mice were sacrificed after 28 days of treatments and tumors were removed and fixed in 10% neutral buffered formalin for histologic and immunohistochemical analyses. All animal procedures were carried out with the approval of the Animal Ethics Committee of Kunming Medical College.
Histological analysis of tumors
Tumors were embedded in paraffin, sectioned at 5 μm, and stained with H&E (Sigma Aldrich, St. Louis, Missouri, USA). The mitotic index and apoptotic index were assessed by quantitative morphometric analysis of proliferating cell nuclear antigen (PCNA) expression and in situ terminal transferase-mediated fluorescein deoxy-UTP nick end labeling (TUNEL), two established markers of proliferation and apoptosis. For PCNA localization, formalin-fixed, paraffin embedded sections were incubated for 30 min with a mouse monoclonal anti-PCNA (Nova Castra Laboratories, Newcastle Upon Tyne, UK) at a 1:100 dilution. A peroxidase -conjugated antibody to mouse IgG (Abcam Inc., Cambridge, MA, USA) was applied followed by diaminobenzidine (Sigma Aldrich,St. Louis, Missouri, USA) to localize PCNA in the sections. DNA fragmentation was assessed by TUNEL, using the Apoptag Peroxidase In situ Apoptosis Detection Kit (Serologicals Corp., Norcross, Ga, USA). PCNA- or TUNEL-positive cells were quantified in 40 randomly selected high-power fields (x 200) of each tissue section.
RNA extraction
Total RNA was retracted from tumors using Trizol reagent (Life Technologies Inc., Gaithersburg, Maryland, USA) according to manufacturer’s instructions. Total RNA from each sample was quantified by the NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, DE, USA) and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA from one tumor from each mouse (6 tumors per group) was used for qRT-PCR analysis, whereas total RNA from tumors from four mice per group (12 tumors per group) was pooled for each microarray hybridization.
Microarray analysis
Microarray analysis of whole-genome gene expression profiling was performed using Human 12 x 135 K Gene Expression Array (Roche Applied Science, Indianapolis, IL, USA). Double-strand cDNA (ds-cDNA) was synthesized from 5 μg of total RNA using a SuperScript ds-cDNA synthesis kit (Life Technologies Inc., Gaithersburg, Maryland, USA) in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (Roche Applied Science, Indianapolis, IL, USA). Microarrays were then hybridized with Cy3 labeled ds-cDNA in a hybridization chamber (Roche Applied Science, Indianapolis, IL, USA). After hybridization and washing, the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Union City, CA, USA). Then, the data files were imported into Agilent GeneSpring Software (Agilent Technologies, Santa Clara, CA, USA) for analysis. NimbleScan software’s implementation of robust multichip average offers quantile normalization and background correction. The six gene summary files were imported into Agilent GeneSpring Software for further analysis. Genes that have values greater than or equal to lower cutoff of 50.0 in all samples were chosen for data analysis. The microarray experiment was independently repeated in triplicate for each sample group. Differentially expressed genes were identified through Fold-change and T-test screening. GO analysis and Pathway analysis were performed using the standard enrichment computation method.
Real-time polymerase chain reaction (PCR)
DNase-treated total RNA extracted from each tumor sample was reverse transcribed using the Transcriptor 1st Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). Real-time PCR was performed for quantitative analysis using SYBR green dye (TaKaRa, Tokyo, Japan) on the ABI-Prism 7900HT system (Applied Biosystems, Foster City, CA, USA) according to the protocols recommended by the manufacturer. Cycling parameters: pre-denaturation 1 min, 95°C; denaturation 15 s, 95°C; annealing 15 s, 60 °C; extension 45 s, 72°C, 40 cycles; final extension 5 min, 70°C. The fold change was calculated using the 2 -ΔΔCt method, presented as the fold-expression change in irradiated tumors relative to control tumors after normalization to the endogenous control, GAPDH. All experiments were carried out in triplicate technically. All primers are listed in Additional file 1: Table S1.
Methyl-DNA immunoprecipitation and microarray hybridization
Genomic DNA from tumors from six mice in the control group was pooled for Methyl-DNA immunoprecipitation (MeDIP) experiment. MeDIP was performed as described previously [12]. Briefly, Genomic DNA was sonicated to produce random fragments in size of 200–600 bp. Four micrograms of fragmented DNA was used for a standard MeDIP assay as described. After denaturation at 95°C for 10 min, immunoprecipitation was performed using 10 μg monoclonal antibody against 5-methylcytidine in a final volume of 500 μL IP buffer (10 mmol/L sodium phosphate, pH 7.0), 140 mmol/L NaCl, 0.05% Triton X-100) at 4°C for 2 h. Immunoprecipitated complexes were collected with Dynabeads Protein A and M-280 sheep anti-mouse IgG (Roche Diagnostics GmbH, Mannheim, Germany) at 4°C for 12 h, washed with 1 × IP buffer for 4 times, treated with Proteinase K at 50°C for 4 h, and purified by phenol-chloroform extraction and isopropanol precipitation. Immunoprecipitated methylated DNA was labeled with Cy5 fluorophere and the input genomic DNA was labeled with Cy3 fluorophere. Labeled DNA from the enriched and the input pools was combined (1–2 μg) and hybridized to a NimbleGen HG18 CpG promoter Array (Roche Diagnostics GmbH, Mannheim, Germany), which contained all well-characterized RefSeq promoter regions [from −800 bp to +200 bp transcription start sites (TSSs)]. Array was then washed and scanned with Axon GenePix 4000B microarray scanner. After normalization, raw data was input into SignalMap software (Roche Diagnostics GmbH, Mannheim, Germany) to observe and evaluate the methylation peaks. A customized peak-finding algorithm provided by NimbleGen was applied to analyze methylation data from MeDIP-microarray as previously described. The algorithm was used to perform the modified Kolmogorov-Smirnov test on several adjacent probes using sliding windows to predict enriched regions across the array.
MeDIP-quantitative PCR assay
A MeDIP assay, combined with qPCR, was used to evaluate quantitatively the methylation status of candidate genes in the tumors derived from the control and 125I treatment groups. MeDIP was performed as described above. Purified DNA from the immunoprecipitated DNA complexes and from input DNA was analyzed by qRT-PCR on an Applied Biosystems 7900 Real- Time PCR System. The experiment was performed in triplicate. The relative changes in the extent of gene methylation were determined by measuring the amount of detected genes in immunoprecipitated DNA after normalization to the input DNA. The primer sequences are listed in Additional file 1: Table S1.
Statistical analysis
The results of the animal experiments and real-time PCR were analyzed using SPSS 13.0 software. (SPSS Inc., Chicago, IL, USA) All data were plotted as mean ± standard deviation. Student’s t-test was used to compare values between two independent groups. Differences were considered to be significance when p < 0.05.
