Cell lines and cultures
I) Five cell lines of human ESCC cells (OE21, TE5, TE8, TE10, and TE11), a non-malignant human esophageal squamous cell line immortalized by SV40 infection, Het-1A, 2 human Barrett's adenocarcinoma cell lines (Bic-1 and Seg-1), 3 human gastric adenocarcinoma cell lines (AGS, AZ521 and KATOIII), 2 colorectal adenocarcinoma cell lines (Caco-2 and DLD1), a human cervix epithelioid carcinoma cell line (HeLa), a human lung adenocarcinoma cell line (A549), and human hematological malignant cell lines (acute promyelotic leukemia, HL60; human T cell lymphoblast-like cell line, Jurkat; and histiocytic lymphoma, U937) were cultured. The AZ521, KATOIII, DLD-1, HeLa, A549, HL60, and U937 cells were purchased from the Japanese Collection of Research Bioresources Foundation (Sennan, Japan). The OE21, Het-1A, AGS, and Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). The TE5, TE8, TE10, and TE11cells were purchased from Riken Bioresource Center Cell Bank (Tsukuba, Japan). Bic-1 and Seg-1were kindly provided by Dr. D.G. Beer (Department of Surgery, Section of General Thoracic Surgery, Michigan Medical School, Ann Arbor, MI). The OE21, TE5, TE8, TE10, TE11, Het-1A, U937, HL-60, DLD-1, Jurkat, and KATOIII cells were grown in RPMI 1640 medium, while the HeLa, A549, and Caco-2 cells were maintained in Dulbcco's modified Eagle medium. Both media were supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine, and all cell lines were cultured in a humidified incubator with 5% CO2 at 37°C.
Patients and Clinical samples
ESCC patients who underwent esophagoscopy between June 2007 and December 2010 were recruited. After obtaining informed consent, 3 biopsy samples each were taken from the ESCC tumor and the matched normal-appearing surrounding esophageal mucosa under endoscopic observation. Two of these samples were placed immediately into 1 mL of RNAlater (Applied Biosystems, Foster City, CA) for RNA isolation later. The other specimen was fixed in 10% formalin and embedded in paraffin for histopathology. The paraffin-embedded biopsy specimens were cut into 5-μm-thick sections and stained with hematoxylin and eosin, and the three pathologists (T.N., M.N., and T. H.) classified the ESCC differentiation.
Total RNA including miR from the tissue samples and cultured cells was extracted using a commercial kit (mirVana RNA™ Isolation kit, Applied Biosystems) according to the supplier's instructions. Quality of total RNA was determined on a Bioanalyzer (Bioanalyzer RNA Nano kit, Agilent, Santa Clara, CA), and the RNA was quantified using a Nanodrop-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Extracted RNA samples were stored at -80°C until used.
MiR array hybridization and analysis
To find specific miR(s) for ESCC cells, total RNA was extracted from OE21 and TE10 cells, representative well and moderately differentiated human ESCC cell lines, respectively, and the non-malignant human esophageal squamous cell line, Het-1A. The isolated RNA samples were subjected to comprehensive analysis of miRNA expression patterns with the microarray-based technology, an Agilent Human miRNA array chip version 1 (Agilent), containing 15,000 probes corresponding to 470 unique human miRs and 64 human viral miRs cataloged in the Sanger database version 9.1. One hundred ng of each total RNA aliquot were treated with calf intestine phosphatase (GE Healthcare, Chalfont St Giles, UK), denatured using DMSO (Sigma, St Louis, MO), and directly labeled with Cy3 using T4 RNA ligase (GE Healthcare). Labeled samples were hybridized to the miR array 8 × 15 k (G4470A) platforms in SureHyb chambers (Agilent), washed with the buffer supplied (Agilent), according to the manufacturer's instructions, and scanned using an Agilent Scanner (G2505B). Data were extracted using Feature Extraction Software 9.3 and GeneSpring software (Agilent). To identify miRs that were differentially expressed between the ESCC cell lines and Het1A cells, supervised analysis was performed using significance analysis of microarrays (SAM, Stanford University, Stanford, CA). The differences in miR expressions were considered significant if the fold change of expression values was >2.0 and the p value was < 0.05 using the t-test.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for miRs
Expression levels of miRs that showed significant differences based on the microarray results were analyzed by quantitative RT-PCR using various human malignant cell lines including ESCC and non-malignant Het-1A. cDNA was prepared from total RNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Predesigned TaqMan MicroRNA Assays including the primer set and TaqMan probe were purchased from Applied Biosystems. The reverse transcription reactions were performed in aliquots containing 50 ng total RNA,1.5 μl 1 × RT Primer, 1 μl 10 × RT Buffer, 0.15 μl 100 mM dNTP,1 μl reverse transcriptase, and nuclease-free water added up to 15 μl at 16°C for 30 min, followed by 42°C for 30 min and 85°C for 5 min. All PCR reactions were performed in 20-μl aliquots containing 1.33 μl miR RT products with 18.67 μl PCR master mixture (10 μl 2 × Universal PCR master mix, 1 μl each primer, 1 μl Taqman Probe, and 6.67 μl nuclease-free water), and run in triplicate on the 7500 Real-Time PCR system (Applied Biosystems). Thermal cycling was initiated with a first denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The cycle passing threshold (Ct) was recorded for each candidate miR, and a small RNA, U6B, was used as the endogenous control for data normalization. Relative expression was calculated using the formula 2-
DCt = 2-
Ct,Specific) as described in the ABI PRISM 7700 SDS relative quantification of gene expression protocol by PE Applied Biosystems. Similarly, total RNAs extracted from the neoplastic and non-neoplastic samples (esophagoscopic biopsies) were subjected to real-time quantitative RT-PCR for quantitation of miR-205 expression levels.
Northern blot analysis
Ten micrograms of total RNA were separated on 15% denaturing polyacrylamide gel and electrotransferred onto Nylon Membrane Positively Charged (Roche Diagnostics, Basel, Switzerland). Oligonucleotides complementary to mature miR-205 were labeled with digoxigenin by terminal transferase-mediated 3' end-labeling and used as probes. The sequence of oligonucleotides was 5'-cagactccggtggaaatgaagga-3'. The membrane was then hybridized with hybridization mixture (0.25 M Na2HPO4 [pH 7.2], 1 mM ethylenediamine tetraacetic acid (EDTA), 1% bovine serum albumin, 7% sodium dodecyl sulfate (SDS), 15% formamide, and the labeled probe) overnight at 43°C. After hybridization, the membrane was washed with wash mixture (20 mM Na2HPO4 [pH 7.2], 1 mM EDTA, 1% SDS) followed by the washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween-20). After blocking with 1% Blocking Reagent (Roche Diagnostics), the hybridized membrane was incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics). The membrane was then washed with the washing buffer. After equilibration with the detection buffer (0.1 M Tris-HCl [pH 9.5], 0.1 M NaCl), the membrane was incubated with the chemiluminescent substrate CDP Star (Roche Diagnostics). Detection was performed using a LAS3000 imaging system (Fujifilm, Tokyo, Japan).
Cultured cells were directly lysed for 30 minutes on ice with lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 mmol/L Na3VO4, and 1 mmol/L NaF]. After centrifugation at 13,000 g for 15 minutes, protein concentrations were measured using Bradford's reagent (Bio-Rad laboratories, Hercules, CA), and protein was denatured by boiling for 10 minutes. Protein (25 μg) was loaded onto sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and then transferred onto nitrocellulose membranes. After blocking with 5% milk in TBST (137 mmol/L NaCl, 25 mmol/L Tris, and 1 mmol/L disodium ethylenediaminotetraacetate containing 0.1% Tween-20), the membranes were incubated with mouse monoclonal anti-E-cadherin (1:1000, BD Biosciences, Franklin Lakes, NJ) and anti-N-cadherin (1:1000, BD Biosciences), and rabbit anti- ZEB1 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ZEB2 (1:200, Santa Cruz Biotechnology), anti-phospho (Ser473)-Akt (1:500, Cell Signaling Technology, Tokyo, Japan) and anti-β-actin (1: 1000, Santa Cruz Biotechnology) at 4°C overnight. After washing with TBST 3 times (10 minutes each), the membranes were incubated with their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. After washing with TBST 3 times (10 minutes each), bound antibodies were visualized using enhanced chemiluminescent substrates (Amersham, Arlington Heights, IL).
MiR-205 precursor and anti-miR-205 inhibitor transfection
The OE21 cells were seeded (8 × 105 cells in 4 ml of RPMI1640 per dish) in 60-mm culture dishes and grown overnight. Transfection of miR-205 precursor, anti-miR-205 inhibitor, or each negative control (all purchased from Applied Biosystems) at indicated concentrations was introduced into the cell using 20 μl siPort NeoFX Transfection Agent (Applied Biosystems) in 500 μl Opti-MEM (GIBCO™, Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. The negative controls were scrambled oligonucleotides that were validated not to produce identifiable effects on known miR function (http://www.ambion.com/jp/catalog/ProdGrp.html?fkProdGrp=344, http://www.ambion.com/catalog/CatNum.php?17100). We confirmed successful transfections using real-time RT-PCR for miR-205.
Cell proliferation assay
Cellular proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI). OE21 cells were plated at a density of 3 × 103 cells/well on 96-well plates and grown overnight. For each well, anti-miR-205 inhibitor molecule, miR-205 precursor, or each scrambled negative control was introduced into each well at a concentration of 50 nM. Twenty-four hours later, the assay was initiated by adding 20 μL of MTS solution reagent to 100 μL of culture medium for each well. After incubation for 3 hours at 37°C, the plates were read in a microplate autoreader (Molecular Devices, Sunnyvale, CA) at wavelength of 490 nm. The results were expressed as the mean optical density for selected paradigms performed in duplicate.
Quantitation of apoptosis
OE21 cells were plated in 12-well plates at a density of 1 × 105cells per well and incubated overnight. Then, 50 nM anti-miR-205 inhibitor, miR-205 precursor, or each scrambled negative control was transfected. Twenty-four hours later, apoptosis was quantitated by assessing the characteristic nuclear changes of apoptosis (i.e., chromatin condensation and nuclear fragmentation) using fluorescence microscopy (Eclipse TE200; Nikon Instruments, Melville, NY) after DAPI (4',6'-diamidino-2-phenylindole dihydrochloride, Roche Diagnostics) staining at a concentration of 10 μg/mL for 15 minutes, as previously described .
Transwell invasion assay
OE21 cells were seeded at a density of 2.0 × 106/well on 60-mm Petri dishes, and 24 hours later, the cells were transfected with either 50 nM anti-miR-205 inhibitor or scrambled negative control. After 24 hours, the transfected cells were harvested by trypsinization, and washed twice in PBS, and 2.5 × 104 cells were transferred to the upper chamber, a BioCoat™ Matrigel™ Invasion Chamber (BD Biosciences) with inserts containing an 8-μm-pore-sized membrane with a thin layer of Matrigel in the 24-well Transwell plate filled with 500 μL serum-free RPMI1640 medium. In the lower chamber, 750 μL of the 10% FBS-containing medium were added. After incubation for 24 hours, the invaded cells were counted under microscopic observation using a Diff-Quick staining kit (Sysmex, Kobe, Japan ).
Wound healing assay
OE21cells were transfected with either 50 nM anti-miR-205 inhibitor or scrambled negative control. When cell confluence reached about 80% at 48-hours post transfection, wounds were created in confluent cells using a 200-μl pipette tip. The cells were then rinsed with medium to remove any free-floating cells and debris. Medium was then added, and culture plates were incubated at 37°C. Wound healing was observed at different time points within the scrape line, and representative scrape lines were photographed. Duplicate wells for each condition were examined, and each experiment was repeated three times.
ZEB1 and ZEB2 3'-UTR luciferase reporter assays
The 3'-UTRs for both ZEB1 and ZEB2 were PCR-amplified from genomic DNA as described previously . The Amplified 3'-UTRs were cloned downstream of the firefly luciferase coding region in the pMIR-REPORT™ (Applied Biosystems). OE21 cells were seeded in 24-well plates 24 hours prior to transfection. The following day, 200 ng of reporter plasmid along with 200 ng of control Renilla-luciferase plasmid were co-transfected using FuGENE® (Roche Diagnostics). Cells were collected 24 hours after transfection and assayed for luciferase activity using the Glomax 96 luminometer (Promega). To assess the effect of miR-205 on reporter activity, either 50 nM of miR-205 precursor (Applied Biosystems) or the negative control was co-transfected.
The differences between groups were analyzed using the unpaired, one-tailed, Student's t-test. Data were expressed as means ± standard error. Differences were considered statistically significant at p < 0.05. All examinations were conducted according to Good Clinical Practice and the Declaration of Helsinki, and they were approved by the Nagasaki University ethics committees.