Supplemental materials
Pancreatic Cell Lines and Tissues
The following 15 pancreatic cancer cell lines were used: AsPC-1, BxPC-3, KP-1N,
KP-2, KP-3, PANC-1 and SUIT-2 (gifts from Dr. H. Iguchi, National Shikoku Cancer
Center, Matsuyama, Japan); MIA PaCa-2 (Japanese Cancer Resource Bank, Tokyo,
Japan); CAPAN-1, CAPAN-2, CFPAC-1, H48N, HS766T and SW1990 (American Type
Culture Collection, Manassas, VA); NOR-P1, which was established from a metastatic
subcutaneous tumor of a patient with pancreatic tumor in our laboratory [23]. An
immortalized human pancreatic ductal epithelial cell line (HPDE6-E6E7 clone 6),
which was established from normal pancreatic ductal epithelium (24), was kindly
provided by Dr. Ming-Sound Tsao (University of Toronto, Toronto, Canada). All cells
were maintained as previously described [24,25]. Tissue samples were obtained from
the primary tumor of each resected pancreas or peripheral tissues at a distance from the
tumor at the time of surgery at Kyushu University Hospital (Fukuoka, Japan) as
described previously [26]. Experienced pathologists performed histologic examinations
of all tissues adjacent to the specimens. The study was approved by the Ethics
Committee of Kyushu University and conducted according to the Ethical Guidelines for
Human Genome/Gene Research enacted by the Japanese Government.
Laser-microdissection
Laser-microdissection was performed as described previously [27,28]. Briefly, frozen
tissue samples were cut into 8-µm-thick sections. One section was stained with
hematoxylin and eosin (H&E) for histologic examination. Invasive ductal carcinoma
(IDC) cells, pancreatic intraepithelial neoplasia (PanIN) cells and normal ductal
epithelial cells were selectively isolated from frozen sections (IDC cells from 20
pancreatic tumor sections; PanIN cells from 11 sections and normal ductal epithelial
cells from 10 sections of normal pancreas) using a laser-microdissection and pressure
catapulting system (LMPC; P.A.L.M. Microlaser Technologies, Bernried, Germany)
according to the manufacturer’s instructions.
miRNA Microarray Expression Analysis
To isolate the miRNA fractions from the total RNA samples for miRNA microarray
analysis, 20 µg of total RNA was fractionated and cleaned up using a flash PAGE™
fractionator system and reagents (Ambion) according to the manufacturer’s instructions.
Microarray analyses were carried out using a FilgenArray miRNA 384 (Filgen, Nagoya,
Japan) containing mirVana™ miRNA Probe Set ver. #1 (Ambion) as shown in
Supplemental materials. Probes were resuspended and spotted on a Nexterion Slide E
(Schott AG, Mainz, Germany) using a GeneMachines OmniGrid (Genomic Solutions,
Ann Arbor, MI). Chemically synthesized oligoribonucleotides (Ambion) or purified
miRNAs from the indicated cells were labeled with a mirVana™ miRNA Labeling Kit
(Ambion) and amine-reactive dyes according to the manufacturer’s recommendations.
The amine-modified miRNAs were cleaned up and coupled to an NHS-ester-modified
Cy5 dye (GE Healthcare, Piscataway, NJ). The labeled miRNAs were hybridized with
the slides. The hybridized slides were washed and dried prior to a high-resolution scan
on a GenePix 4000B (Axon Instruments, Foster City, CA). The raw data were
normalized and analyzed with Array-Pro Analyzer Version 4.5 (Media Cybernetics,
Silver Spring, MD).
Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) for
Analysis of miRNA Expression
RNA samples containing miRNA fractions were prepared from all cultured cells and
microdissected cells using a mirVana™ miRNA Isolation Kit (Ambion, Austin, TX)
according to the manufacturer’s protocol. Cultured cells were analyzed by qRT-PCR
with SuperTaq Polymerase (Ambion) and a mirVana™ qRT-PCR miRNA Detection Kit
(Ambion) according to the manufacturer’s instructions. For analyses of microdissected
cells, qRT-PCR was performed with a TaqMan® MicroRNA Reverse Transcription Kit
(Applied Biosystems, Foster City, CA), TaqMan® 2× Universal PCR Master Mix
(Applied Biosystems), a miR-10a-specific TaqMan® probe and miR-10a-specific
primers according to the manufacturer’s instructions. For each set of primers, negative
controls lacking a template or reverse transcriptase were performed. The miRNA
expression levels in each sample were normalized by the expression level of U6 snRNA.
qRT-PCR was carried out in a Chromo4 Real-Time PCR Detection System (Bio-Rad
Laboratories, Hercules, CA). All reactions were performed in triplicate.
Transfections
Cells were transfected by electroporation with a Nucleofector System (Amaxa
Biosystems, Koln, Germany) as described previously [26]. Cells (1-2 × 106) were
collected and resuspended in 98 µL of Nucleofector Solution (Amaxa Biosystems),
followed by the addition of 2 µL of 50 µM anti-miRNA or control nonsense inhibitors
(anti-miR miRNA inhibitors from Ambion; miRCURY LNA knockdown probe from
Exiqon, Vedbaek, Denmark; final concentration, 1µM), or pre-miR-10a or control
precursors (Ambion). After the first electroporation, the transfected cells were
resuspended in regular culture medium for 48 hours, and then subjected to a second
electroporation with the same protocol used for the first electroporation.
Cell Proliferation Assay
Cells were transfected with 100 pmol of the indicated miRNA inhibitors or control
nonsense inhibitors (Ambion) and seeded in 24-well plates at a density of 3-5  104
cells/well. Cell proliferation was analyzed at various time points by measuring
propidium iodide (PI) incorporation as described previously [29].
Invasion Assay
The invasiveness of cancer cells was evaluated by counting the number of cells
invading a Matrigel-coated transwell as reported previously [26]. Briefly, transwell
inserts with 8-µm pores were coated with Matrigel (20µg/well for SUIT-2 cells, 40
µg/well for PANC-1 cells). Cancer cells were transfected with the various miRNA
inhibitors and seeded in the Matrigel-coated transwell inserts. After incubation (24
hours for PANC-1 cells, 36 or 72 hours for SUIT-2 cells), the cells invading the lower
surface of the Matrigel-coated membrane were fixed with 70% ethanol, stained with
H&E and counted in 5 randomly selected fields under a light microscope. The invasion
assays were independently performed three times and similar results were obtained in
all experiments. Each experiment was performed in triplicate.
Quantitative Analysis of HOXA1 Levels by One-step qRT-PCR
One-step qRT-PCR was performed using a QuantiTect SYBR Green RT-PCR Kit
(Qiagen, Tokyo, Japan) with a Chromo4 Real-Time PCR Detection System (Bio-Rad
Laboratories) as described previously [28]. We designed and used specific primers for
HOXA1 (forward, 5’-GGGTGTCCTACTCCCACTCA-3’; reverse,
5’-GGACCATGGGAGATGAGAGA-3’), and used previously described primers for
18S rRNA [28]. The expression level of HOXA1 mRNA was normalized by the
corresponding expression level of 18S rRNA.
Inhibition of HOXA1 Expression by RNA Interference
Inhibition of HOXA1 expression was achieved by RNA interference with small
interfering RNAs (siRNAs) against HOXA1 (siRNA-1: sense, ggaugaaagucaaaagaaaTT,
antisense, uuucuuuugacuuucauccTT; siRNA-2: sense, ccgaaugaagcaaaagaaaTT,
antisense, uuucuuuugcuucauucggTT; B-Bridge, Mountain View, CA) as described
previously [26]. To verify the specificity of the knockdown effects, we used a control
siRNA (B-Bridge). Cells were transfected with 100 pmol of the appropriate siRNA
using Nucleofector (Amaxa Biosystems) according to the manufacturer’s instructions.
Western Blot analysis
Protein expression was analyzed by western blotting as described previously [21] with
the following primary antibodies: anti-HOXA1 (Aviva Systems Biology, San Diego,
CA); anti-HOXA2 and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).
Appropriate secondary antibodies (Santa Cruz Biotechnology) were used. For detection,
ECL Plus Western Blotting Detection Reagents were used (Amersham, Little Chalfont,
UK).
Statistical Analyses
For microarray data analysis, the mean of the triplicate values for each miRNA was
normalized and analyzed with Microarray Data Analysis Tool Ver. 1.2. Differences in
the expression levels were analyzed by Student’s t-test. In the analyses of
microdissection samples, each sample was analyzed in duplicate. Any sample showing a
deviation of more than 10% in the values was tested a third time. The data were
analyzed by the Mann-Whitney U test and Spearman rank correlation test when normal
distributions were not obtained. For in vitro experiments, values were expressed as the
mean ± SD. All experiments were repeated more than three times independently.
One-way analysis of variance (ANOVA) was used to analyze differences among three
groups, while Student’s t-test was used to evaluate differences between two groups.