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 selectivelyisolated 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 tothe manufacturer’s instructions.

miRNA Microarray Expression Analysis

To isolate the miRNA fractions from the total RNAsamples for miRNA microarray analysis, 20 µg of total RNA was fractionated and cleaned up using a flashPAGE™ fractionator system and reagents (Ambion) according to the manufacturer’sinstructions.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 purifiedmiRNAs from the indicated cells were labeled witha mirVana™ miRNA Labeling Kit (Ambion) and amine-reactivedyes according to the manufacturer’s recommendations. The amine-modified miRNAs were cleaned upand coupled to an NHS-ester-modified Cy5dye (GE Healthcare, Piscataway, NJ).The labeled miRNAs were hybridized with the slides. The hybridized slideswere washed and dried prior to a high-resolution scan on aGenePix 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 amirVana™ 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),amiR-10a-specific TaqMan® probe and miR-10a-specific primersaccording 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 theexpression 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 controlnonsense 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 controlnonsense 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, 40µg/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 ofHOXA1 mRNA was normalized by the corresponding expression level of 18S rRNA.

Inhibition of HOXA1 Expression by RNAInterference

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 thetriplicate 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. Inthe analyses of microdissection samples, each sample was analyzedin 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.