Human pancreatic carcinoma cell lines HPAF II, BxPC3, PANC1; human breast cancer cell lines MCF-7, T-47D, MDA-MB-453; and human colon adenocarcinoma cell lines Caco2 and LS174T were obtained from American Type Culture Collection. HPAF II, MCF-7, LS174T, and Caco2 cells were cultured in Eagle's MEM (Sigma-Aldrich), PANC1 cells were cultured in DMEM (Sigma-Aldrich), BxPC3 and T-47D cells were cultured in RPMI 1640 (Sigma-Aldrich), and MDA-MB-453 cells were cultured in Leibovitz's L-15 medium (Invitrogen, Carlsbad, CA, USA). All media were supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin (Sigma-Aldrich) and 100 μg/ml streptomycin (Sigma-Aldrich).
Isolation of human colonic crypts
Fresh normal and tumor tissue samples were obtained from colorectal carcinoma specimens that were obtained surgically at Iwate Medical College Hospital. Normal colonic mucosa was taken from an area distant from the tumor. The tumor samples were obtained from the tumor-rich area of carcinomas. Crypt isolation from the normal and neoplastic mucosa was performed as previously reported [20
]. Briefly, fresh normal mucosae and tumors were minced with a razor into minute pieces and then incubated at 37°C for 30 minutes in calcium- and magnesium-free Hanks' balanced salt solution (CMF) containing 30 mmol/L ethylene-diaminetetraacetic acid (EDTA). Following this procedure, the tissue was stirred in CMF for 30-40 minutes. Normal and neoplastic glands were separated from the lamina propria mucosa or fibrous stroma. The isolated crypts were immediately fixed in 70% ethanol and stored at 4°C until used for DNA and RNA extraction, and were also embedded in paraffin for immunohistochemical staining. All studies using human materials in this article were approved by the ethical committees of Kagoshima University hospital and other hospitals.
After completion of endoscopic retrograde pancreatography, pancreatic juice was collected using endoscopic nasopancreatic drainage (ENPD) or pancreatic stenting in 2 patients with PDAC and 5 patients with IPMN at Osaka Medical College Hospital [23
]. Collection of the samples was approved by the ethical committee of the hospital and informed consent was obtained from each patient. All studies using human materials in this article were approved by the ethical committees of Kagoshima University hospital and other hospitals.
Extraction and quantification of mRNA
Extraction of RNA
Total RNA was extracted from the cell lines, human colonic crypts and pancreatic juices using a RNeasy Mini kit (QIAGEN, Chuo-ku, Tokyo, Japan). Total RNA of 1 μg was then reverse transcribed with a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA).
Real time PCR assay
For quantification of MUC1 mRNA in the ethanol-fixed isolated crypts from human colonic normal mucosa and carcinoma lesions, the real time PCR assay was performed as described previously [7
]. The primers and probes were designed and synthesized by Applied Biosystems. The product number of the Target Assay Mix used for MUC1 was Hs00410317. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; product number 4310884E) was used to calibrate the original concentration of mRNA; i.e., the concentration of mRNA in the cell was defined as the ratio of target mRNA copies versus GAPDH mRNA copies. In this analysis, data from three separate experiments were averaged.
For confirmation of gene expression level in cell lines, semiquantitative RT-PCR were performed using a Fast Cycling PCR kit (QIAGEN, Japan). The RT-PCR products were subjected to electrophoresis on 1% agarose gel. Differentially expressed genes were detected using the primer pairs (shown in Additional file 1
: Table S1).
Extraction of DNA and bisulfite modification
DNA from cell lines, ethanol-fixed human crypt sections, and pancreatic juice samples was extracted using a DNeasy Tissue System (QIAGEN, Chuo-ku, Tokyo, Japan). Bisulfite modification of the genomic DNA was carried out using an Epitect Bisulfite Kit (QIAGEN, Chuo-ku, Tokyo, Japan).
Preparation of the samples for MSE method
The target sequence containing CpG sites important for expression in respective mucin promoter regions were determined using a massARRAY analysis that has been published previously [2
]. All the PCR primers were designed to avoid CpG site. The primers are bisulfite genomic sequencing type (Table ). In addition, the target PCR primers were designed to be optimal sample fragment sizes (100 bp to 700 bp) for DGGE (Table B).
Synthetic oligonucleotides used in MSE and conventional bisulfite-DGGE
Preparation of the samples
In the MSE method, a nested-PCR approach was used. In the first round of PCR, bisulfite treated DNA was amplified using the nested primer sets (Table A). The cycling conditions consisted of an initial denaturation step at 95°C for 5 min, then 40 cycles of 96°C for 5 s, Tm°C for 5 s, and 68°C for 3 s (the value of Tm shown in Table1). The information of each PCR product size and primer location at this initial PCR reaction is shown in Table A. For the second round of PCR, this product was diluted 1:50 in water, and 2 μl of the dilution were amplified using the target primer sets (Table B). The PCR parameters were as above, except that the annealing temperatures for the MUC1, MUC2, MUC3A, MUC4, MUC5AC and MUC17 reactions were 53, 51, 52, 57, 62 and 53°C respectively. The information of each PCR product size and primer location at the second PCR reaction is shown in Table B. All steps of PCR were performed using a Fast Cycling PCR kit (QIAGEN, Japan).
Information of PCR product size and primer location
Denaturing gradient gel electrophoresis (DGGE)
After the PCR, DGGE was performed as described by Schäfer and Muyzer [24
] using the D-Code system (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was performed with 1-mm thick 10% polyacrylamide gels (ratio of acrylamide to bisacrylamide, 40:1) submerged in 1Χ TAE buffer (40 mM Tris, 40 mM acetic acid, 1 mM EDTA, pH 7.5) at a constant temperature of 60°C. PCR products in amounts ranging from 5 μl were applied to the individual lanes on the gel. The electrophoresis conditions for the target gene fragment were 14 h at 70 V in a linear denaturant gradient (MUC1, 30%-40%; MUC2, 25%-35%; MUC3A, 25%-35%; MUC4, 25%-45%; MUC5AC, 30%-40%; MUC17, 35%-45%). After electrophoresis, the gels were incubated for 30 min in Milli-Q water containing GelRed (dilution 1:10000) (Biotium, Hayward, CA, USA) and photographed using AE-6905 N (ATTO). The emission intensity of the band was measured using Image J (NIH).
Conventional bisulfite-DGGE for comparison
In the conventional bisulfite-DGGE method, the bisulfite treated DNA was amplified using the target primer sets (Table B). PCR parameters were as above, except that the annealing temperatures for the MUC1, MUC2, MUC3A, MUC4, MUC5AC and MUC17 reactions were 53, 51, 52, 57, 62 and 53°C respectively. Information on each PCR product size and each primer location is shown in Table B. All steps of PCR were performed using a Fast Cycling PCR kit (QIAGEN, Japan). After the PCR, these products were applied to DGGE.
MUC1 protein expression was assessed by immunohistochemistry using an anti-MUC1 monoclonal antibody (MAb) clone 014E (Mab MUC1-014E), which was developed by Yonezawa et al. [25
], in ethanol-fixed paraffin-embedded 4-μm sections of the isolated human normal and neoplastic crypts. Immunohistochemistry was performed by the immunoperoxidase method as follows. Antigen retrieval was performed using CC1 antigen retrieval buffer (Ventana Medical Systems, Tucson, AZ, USA) for all sections. Following incubation with MAb MUC1-014E (diluted 1:5) in phosphate-buffered saline (PBS, pH 7.4) with 0.1% bovine serum albumin, sections were stained on a Benchmark XT automated slide stainer using a diaminobenzidine detection kit (Ventana Medical Systems).