Clinical characteristics of patients and samples
A total of 123 tissue samples were analyzed, taken from 113 patients of varying background: 48 male, 75 female, average age 63 years (range, 15–89 years), 10 black, 99 white, 14 unspecified. Sample set consisted of 71 malignant adenocarcinomas and 52 non-tumor controls including 11 patient-matched pairs. All adenocarcinomas (56 Grade II and 15 Grade III) were confirmed by pathological evaluation and contained a minimum density of 51 % lesion/49 % stroma. Stromal tissue does not appear to contribute significantly to a tissue’s 14-3-3 expression when analyzed by immunohistochemistry [23]. The dataset included patients with varying stages of colorectal cancer (14 Stage I, 28 Stage II, 28 Stage III, and 4 Stage IV). The control group included various diagnoses: 12 diverticulosis, 5 polyps, and 32 non-tumor. Deidentified tissue samples were provided by the Cooperative Human Tissue Network (CHTN Western Division, Vanderbilt University, Nashville, TN) which is funded by the National Cancer Institute. Other investigators may have received specimens from the same subjects. Samples were kept frozen at −70 °C until needed. These studies were reviewed by and designated as exempt by the University of Arizona Human Subjects Protection Program.
RNA extraction and purification
Total RNA was extracted from tissue samples using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines. To preserve RNA integrity, all tissue samples were maintained over dry ice prior to sample disruption using a VDI 12 Tissue Homogenizer (VWR, Radnor, PA). RNA concentration and quality were determined using a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).
Real-time reverse transcription analysis
cDNA was synthesized from 1 μg total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA), diluted into 15 μL with nuclease-free water. PCR products were detected with ROX using iTaq™ Supermix (Bio-Rad) and TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA) for 14-3-3 gamma (YWHAG, Hs01113553_mH), beta (YWHAB, Hs00268732_m1), epsilon (YWHAE, Hs00356749_g1), zeta (WYHAZ, Hs01122445_g1), theta (YWHAQ, Hs00863277_g1), eta (YWHAH, Hs00607046_m1), and sigma (SFN, Hs00968567_s1). Cycle threshold (Ct) values for all seven 14-3-3 genes were normalized to GAPDH (Hs02758991_g1, Applied Biosystems).
DNA extraction and purification
Genomic DNA was extracted from fresh-frozen tissue samples using the QIAamp® DNA Mini Kit (Qiagen), according to manufacturer’s guidelines. Samples were digested with 25 units each of bovine pancreas ribonuclease A (Sigma, St. Louis, MO) for five minutes to remove residual RNA. DNA concentration and quality were determined using a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific).
Bisulfite treatment of methylated human DNA
Four matched pairs of tumor and non-tumor genomic DNA (eight samples total) were subjected to bisulfite treatment using EpiTect® Bisulfite Kit (Qiagen), according to the manufacturer’s guidelines. Following column purification, samples were eluted into 10 μL buffer TE (10 mM Tris, pH 8.0; 1 mM EDTA).
Amplification of 14-3-3 promoter regions from bisulfite-treated DNA
Promoter regions were amplified using primers designed against 14-3-3 sigma (5’- GGTATTGTGAAAGTGGATTTGA -3’ and 5’- ACTATCCAACAAACCCAACAC -3’), 14-3-3 eta (5’- AGTAGGTGAYGTTATTTTGAAA -3’ and 5’- ACCCAACCTCAAAAAATAAC -3’), and 14-3-3 zeta (5’- GGAAATTTTTTTTTTGGTTTGT -3’ and 5’- AATTTTCCTACCCAAATAAAACTTT -3’). When used to amplify bisulfite-treated, genomic DNA, these primer pairs generate products of 702, 651, and 655 base pairs long, respectively. PCR was conducted using Platinum® Taq DNA Polymerase (Invitrogen, Grand Island, NY) according to the manufacturer’s guidelines. Reactions were run for 35 cycles using an annealing temperature of 55 °C and 1 μL of template bisulfite-treated DNA. Resultant PCR products were analyzed on a 1 % TAE agarose gel to verify sample quality. DNA bands of the desired mass were excised and subsequently purified using QIAquick® Gel Extraction Kit (Qiagen), according to manufacturer guidelines. Samples were eluted into 30 μL buffer TE for immediate use.
Single-copy isolation of 14-3-3 promoter regions by subcloning
Purified fragments of 14-3-3 promoter regions were ligated into bacterial cloning vectors using pGEM®-T Easy Vector System I (Promega, Madison, WI) according to the manufacturer’s guidelines. Ligated constructs were immediately used to transform DH5α competent cells and grown on IPTG/β-Gal/Ampicillin positive agar plates for 24 h. Twelve white colonies were randomly selected from each plate and grown in 2 mL liquid cultures overnight. Plasmid DNA was then extracted and purified using AxyPrep Easy-96 Plasmid DNA Kit (Axygen, Union City, CA) according to the manufacturer’s guidelines.
Sequencing and analysis of bisulfite-treated 14-3-3 promoter DNA
Sequencing of 14-3-3 promoter constructs was conducted by The University of Arizona Genetics Core Facility using an Applied Biosystems 3730 DNA Analyzer. Cloned sequences for each 14-3-3 isoform were aligned against established gene sequences using a ClustalW alignment algorithm and BioEdit© Software (Ibis Biosciences, Carlsbad, CA).
Plasmids
The T24-C3 vector (activated H-ras inserted into pBR322 plasmid) was obtained from Dr. Radhakrishnan, University of Arizona, AZ, USA. FNpCDNA3 vector was obtained via Addgene (plasmid 45346). The Flag-14-3-3 eta plasmid was created by PCR amplification of 14-3-3 eta cDNA (16) followed by sub-cloning into the FNpCDNA3 vector using BamHI/EcoRI restriction enzymes (NEB, MA).
Cell culture and transfection conditions
NIH3T3 cell line was obtained from ATCC (American Type Culture Collection). Cells were grown in Dulbecco’s modified minimal essential medium (Cellgro, VA) supplemented with 100 U of penicillin, 100 mg of streptomycin, and 5 % fetal bovine serum (Sigma) and maintained in a humidified atmosphere of 5 % CO2.
Transformation assay
Early passages of NIH3T3 cells were plated in 30 mm, 6-well plates at a density of 300,000 cells/well. 1 μg of each plasmid mixed in FuGene 6 transfection reagent (Promega) was slowly added to culture plates under gentle agitation. After 24 h, the transfected cells were trypsinized and counted. About 5,000 cells were plated into 100 mm dishes, each. Cells were grown in DMEM with 5 % fetal calf serum, changed every 84 h. After 21 days, cells were stained with 0.05 % crystal violet solution (Thermo Scientific #88101) and foci were counted using a ColCount colony counter (Oxford Optronix, UK).
Western blotting
To verify exogenous protein expression, cell lysates were collected 96 h after transformation. Total protein was extracted and analyzed by SDS-PAGE. Two rabbit-primary antibodies were used to probe for total Ras (Cell Signaling antibody #3339) and Flag peptide (Cell Signaling antibody #2368), incubated overnight in 5 % BSA at a dilution of 1:800. Uniform protein loading was verified using mouse anti-β-actin primary antibody (Sigma #A3853), also incubated overnight in 5 % BSA at a dilution of 1:800. Anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch antibody #111-035-003) and anti-mouse HRP-conjugated secondary antibody were both used at a 1:10,000 dilution in 5 % blotting-grade blocker (Bio-Rad #170-6404).
Statistical analysis
Statistics were calculated using R© version 2.13.2 software (R Foundation for Statistical Computing, Vienna, Austria). A two-tailed Student’s t-test and Mann–Whitney U-test were used to compare differences in 14-3-3 mRNA expression between tumor and non-tumor sample groups. P-values less than 0.01 were considered significant.