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SUPPLEMENTAL INFORMATION
Materials and Methods
Cell culture and reagents
PANC-1 cells were maintained in DMEM medium (10% FBS, 50 units/ml penicillin,
50µg/ml streptomycin sulfate). PANC10.05 were cultured in RPMI-1640 medium with
10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate
(10% FBS, 0,01mg/ml bovine insulin (Sigma-Aldrich, Saint-Louis, MO, USA), 50
units/ml penicillin, 50µg/ml streptomycin sulfate). BxPC-3 cells were cultured in
RPMI-1640 medium with 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose,
1.5 g/L sodium bicarbonate (10% FBS, 50 units/ml penicillin, 50µg/ml streptomycin
sulfate) and HEK293FT cells were maintained in DMEM medium (10% FBS, 1%
nonessential amino acids, 1% geniticin, 50 units/ml penicillin, 50µg/ml streptomycin
sulfate). Human umbilical vein endothelial cells (HUVEC) were used for in vitro
angiogenesis assays. Cells were grown in EGM-2 medium (PromoCell, Heidelberg,
Germany) with 10% FBS, meanwhile EBM-2 medium (PromoCell, Heidelberg,
Germany) with 4% FBS were used during experiments. Passages lower than 10
were used through the experiments. All cell culture media and reagents were from
Gibco BRL (Gran Island, NY, USA) unless mentioned differently. All cell lines were
maintained at 37oC with 5% CO2.
MG-132 and BAY-117082 chemical inhibitors were purchased from Calbiochem
(Millipore, Billerica, MA, USA) and human recombinant TNFα was from Sigma
(Sigma-Aldrich, Saint-Louis, MO, USA). Human recombinant TNF-α was used at a
final concentration 20ng/ml.
Transfections
All transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA) according to manufacturer’s instructions. siRNAs targeting ANXA2, KRAS
and RELA, as well as the non-targeting control siRNA were purchased from Ambion
(Ambion, Austin, TX, USA). The second siRNA against ANXA2 gene (siANXA2#2)
was purchased from Dharmacon (Lafayette, CO, USA). For each gene, 2 individual
siRNAs were used (Supplementary Table S6). miRIDIAN miRNA mimics and the
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negative control were obtained from Dharmacon (Lafayette, CO, USA). siRNAs and
miRNA mimics were used at a final concentration of 30nM.
Stable cell line construction using retroviral vectors
Human miR-206 gene was subcloned from pCMV-miR206 (Origene, MD, USA) into
the SgfI and MluI sites of pCR2.1 vector (Invitrogen, Carlsbad, CA, USA). MiR-206
gene was further shuttled into pBABE-puro retroviral vector using BamHI and XhoI
restriction enzymes. For the virus production, 2x106 HEK293FT cells were
transfected in 10cm plates with 3.5 μg of pBABE-empty or pBABE-miR206 vector
along with 3.5 μg of the gag-pol plasmid pHIT60 and 800ng of the envelope plasmid
pMD2.G using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). Cells were
allowed to produce viruses for 48 hours before PANC10.05 cells were infected.
Transduced cells were selected in 2 μg/ml puromycin (Invitrogen, Carlsbad, CA,
USA).
Luciferase reporter assays
For the NF-κB luciferase reporter assay, HEK293FT (0.8x104 cells/well), PANC-1
and PANC10.05 (1x104 cells/well) cells were transfected in 96-well plates with the
miRNA mimics, together with 100ng of the NF-κB reporter 3xKBL (kindly provided by
Prof. George Mosialos) and 20ng (in case of PANC-1 and PANC10.05) and 2.5ng (in
case of HEK293FT) of pMIR-REPORT β-gal vector (Ambion, Austin, TX, USA). 48h
after transfection cells were washed in PBS (Gibco, Gran Island, NY, USA) and lysed
in 100μl lysis buffer (25mM TrisP04 pH=7.8, 1mM CDTA pH=7.8, 10% Glycerol, 1%
Tritin-X100, 10 ul of fresh 0.2M DTT/ml). Luciferase activity was determined using a
firefly luciferase assay reagent (40 mM Tricine, 2 mM ATP, 10 mM MgSO4, 0.5 mM
EDTA , 10 mM DTT, 1 mM Coenzyme A, 1 mM D-Luciferin) and β-galactosidase
activity which was used for normalization, was measured by beta-glo® Luminescent
Assay Kit (Promega, Madison, WI, USA). 50μl of the lysate were mixed with 50 µl of
firefly luciferase assay reagent and after an incubation time of 1 min, luminescence
intensity of each well was measured using the TECAN infinite200 microplate reader
(Tecan, Linz, Austria). 5 µl of lysate were subsequently used for normalization
measurement of β-galactosidase activity. 20 µl of β-galactosidase substrate β-Glo
(Promega, Madison, WI, USA) were added to each well by the injector; the reaction
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was incubated for 10 min and luminescence intensity of each well was measured
using a plate reader. To validate direct targeting of miRNAs, psiCHECK2 vectors
(Promega, Madison, WI, USA), containing the respective 3’UTRs, were cotransfected with mimic miRNAs in PANC-1 cells. 48h after transfection Renilla and
firefly luciferase activities were determined using the TECAN infinite200 microplate
reader (Tecan, Linz, Austria). Renilla luciferase activity was determined using a
Renilla luciferase reagent (10 mM DTT, 8 mM EDTA, 0.05 mM Coelenterazine).
Mutations within each of the predicted target sites of ANXA2 and KRAS 3’-UTRs
were generated by site-directed mutagenesis (Supplementary table S7).
Antibodies and immunoblotting
For Western Blotting, cells were lysed in ice cold M-PER lysis buffer (Pierce,
Rockford, IL, USA) containing protease inhibitor Complete Mini (Roche, Basel,
Switzerland) and anti-phosphatase PhosSTOP (Roche, Basel, Switzerland). Protein
concentrations were determined by BCA Protein Assay Reagent Kit (Pierce,
Rockford, IL, USA) and proteins were denatured with 4× Roti Load (Roth, Karlsruhe,
Germany) at 95°C for 5 minutes. Then, proteins (20µg) were separated by 12% SDSPAGE, blotted onto a PVDF membrane Immobilon-P (Millipore, Billerica, MA, USA)
and exposed to primary antibodies after blocking in 5% milk in TBS-T for 1h at room
temperature. The following antibodies were used in 5% milk/TBS-T: mouse
monoclonal anti-ANXA2 (610069) (BD Biosciences, Bedford, MA, USA), mouse
monoclonal anti-KRAS (ab55391) (Abcam, Cambridge, MA, USA), rabbit
polyclonal NF-κB p65 (A)(SC-372) (Santa Cruz Biotechnologies, California, CA,
USA) and rabbit monoclonal GAPDH (14C10) (CST#2118) (Cell Signaling
Technology, Danvers, MA, USA). Blots were probed with IRDye®680 or IRDye®800
conjugated antibodies (H+L) (LI-COR, Lincoln, NE, USA) and bands were visualized
using an Odyssey scanner (LI-COR, Lincoln, NE, USA). Primary antibodies were
used at a 1:1000 dilution (except for KRAS antibody which was used at a 1:200
dilution) and secondary antibodies at a 1:10.000 dilution.
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Electrophoretic Shift Mobility Assay (EMSA)
For the EMSA experiments the LightShift Chemiluminescent EMSA kit (#20148,
Pierce, Rockford, IL, USA) was used, according to the manufacturer's instructions.
Briefly, nuclear extracts from transfected cells were prepared using the NE-PER kit
(Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Protease
and phosphatase inhibitors (Roche, Basel, Switzerland) were added to the buffers
contained in the kit. Protein concentration was determined using the BCA kit assay
(Pierce, Rockford, IL, USA) as described above. The oligonucleotide sequences that
contain the wild type or mutated binding site of NF-κB transcription factor in the
LMP1 promoter were previously described [1]. The sequences to construct the
double-stranded oligos were the following:
WT:
5’ aggcccggGGGGATTTGCggggtct 3’
MUT: 5’ aggcccggGGGGAcccGCggggtct 3'
the capital letters correspond to the NF-κB binding site, and the red letters to
mutated nucleotides. The oligos were ordered from Sigma and were modified with
biotin at the 5’ end and were HPLC purified.
Annealed oligos (50 μM) were diluted using a 1:2000 dilution. 2 μl of the diluted
annealed oligos were incubated with 500ng of nuclear extract in binding buffer (1x
binding buffer provided with the kit, 0.05 μg/μl Poly (dldC), 5% glycerol, 0.1% NP-40,
5 mM MgCl2) for 20min at room temperature. DNA-protein complexes were resolved
by electrophoresis in a 5% polyacrylamide gel in 0.5xTBE at 100V. The transfer to a
positively charged nylon membrane (Amersham Biosciences, New Jersey, NJ, USA)
was performed at 380mA (~100V) for 1h at 4 oC in 0.5xTBE. When the transfer was
complete, the membrane was immediately crosslinked at 1200 μjoules with UV
crosslinker (Stratagene, Cambridge, MA, USA). The membrane was probed with
stabilized streptavidin-horseradish peroxidase-conjugate and the bands were
visualized by chemoluminescence (Amersham Biosciences, New Jersey, NJ, USA).
RNA isolation and Quantitative Real-Time PCR
Total RNA and microRNA were isolated from cells using miRNeasy Mini kit (Qiagen,
Hilden, Germany) according to the manufacturer’s instructions. For mRNA, cDNA
synthesis was carried out with the Revert Aid H Minus First Strand cDNA Synthesis
Kit (Fermentas, St Leon-Rot, Germany). The qRT-PCR reactions for target genes
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were performed using ABI Prism 7900HT Sequence Detection System (Applied
Biosystems, Weiterstadt, Germany), using probes from the Universal Probe Library
(Roche, Penzberg, Germany). The housekeeping genes HPRT1 and GAPDH were
used for normalization of mRNA analysis (Supplementray table S8). miRNA specific
primer sets for miR-206 and RNU48 (TaqMAN® MicroRNA Assays), TaqMAN® Fast
Universal PCR Master Mix (2x), and TaqMan® MicroRNA Reverse Transcription Kit
were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). RNU48 was used
for normalization of miRNA analysis. The relative gene expression level of one gene
compared to control was given by the following equation: fold expression level =
2−ΔΔCt.
ELISA
Transfected PANC-1 and PANC10.05 cells were cultured in full growth medium for
48h. Then, IL-8, CXCL1 and VEGF-C secretion was determined in the supernatant
using the human IL-8 QuantiGlo Chemiluminescent and the human CXCL1 and
VEGF-C Quantikine ELISA kits (R&D Systems, Inc, Wiesbaden-Nordenstadt,
Germany)
according to the manufacturers’ instructions.
Measurements for
absorbance or luminescence were performed using the TECAN infinite200 platereader (Tecan, Linz, Austria).
VEGFA was detected in supernatants of transfected PANC-1 and PANC10.05 cells
by fluorophore-linked immunosorbent assay (FLISA) using a modified version of the
ELISA kit provided by R&D Systems (Minneapolis, MN, USA). Cells were transfected
in 6-well plates as indicated above and incubated after transfection for 72h in full
growth medium. Supernatants were collected, cleared by centrifugation and stored at
-80°C prior to quantification of VEGFA. All incubations were performed at room
temperature and all washing steps were performed using 0.05% PBS-Twees20.
Standards of different concentrations ranging from 5 ng/ml to 39 pg/ml were prepared
in blocking buffer using recombinant human VEGF165 (293-VE, R&D Systems,
Minneapolis, MN, USA). Briefly, a high-binding 96-well micro-assay plate (Greiner
bio-one, Frickenhausen, Germany) was coated with 3µg/ml capture antibody (clone
26503, R&D Systems, Minneapolis, MN, USA) overnight, blocked using 1% BSA in
PBS for 2 hours and incubated with standards and samples for 2 hours. VEGFA was
detected using 200 ng/ml biotinylated detection antibody (BAF293, R&D Systems,
Minneapolis, MN, USA) for 2 hours followed by 0.4µg/ml Streptavidin, Alexa Fluor
680 conjugate (Molecular Probes, Invitrogen, Carlsbad, CA, USA). The plate was
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scanned using as Odyssey Near-Infrared scanner (LI-COR Biosciences, Lincoln, NE,
USA). Intensities were retrieved using the GenePix software (Molecular Devices,
Biberach, Germany) and analyzed using MS Excel.
Cancer cell invasion and migration assays
PANC-1 and BxPC-3 cells were transfected with 30nM siRNAs or miRNAs as
described above. 24h later, cells were starved in 0.5% FBS medium for 24h. 1.5x105
cells were seeded in 0.5% FBS medium in the invasion 24-well plates (BD Biocoat,
Matrigel Invasion chamber, 8.0µm, BD Biosciences, Bedford, MA, USA). Full growth
medium was used as chemoattractant in the lower chamber. Cells were allowed to
invade for 24h in case of PANC-1 and 48h in case of BxPC-3 and the number of
invaded cells was determined by flow cytometry (FACSCalibur, BD Biosciences,
Bedford, MA, USA). For trans-well migration assays 1x105 cells were seeded in 0.5%
FBS medium in the 24-well boyden chambers (Corning, 8.0μm, Thermo Scientific,
Schwerte, Germany). PANC-1 and BxPC-3 cells were allowed to migrate for 24h.
The number of migrated cells was determined by flow cytometry (FACSCalibur, BD
Biosciences, Bedford, MA, USA).
For RTCA migration experiments, transfections were performed as described above.
Cells were then starved in 0.5% FBS medium for 24 h and seeded in RTCA Cim-16
plates (RTCA; xCELLigence Roche, Penzberg, Germany) in serum-free medium.
Cells were seed in 0.5% FBS medium. Full growth medium was used as a
chemoattractant in the lower chamber. Measurements were performed in a timeresolved manner using the RTCA device (RTCA, xCELLigence Roche, Penzberg,
Germany).
Plasmin production assay
PANC-1 cells were transfected in 6-well plates as described above. 24 h after
transfection, transfected cells were trypsinized and re-seeded on a 24-well plate
(100,000 cells/well) in full growth medium. The next day, the plasmin production was
measured. Cells were washed with 1xPBS and detached from the plate using 200 μl
per well of enzyme-free dissociation solution (Sigma-Aldrich, Saint-Louis, MO, USA).
The reaction was stopped using growth medium. The cells were centrifuged for 4 min
at 2,500 rpm and the cell pellet was washed with 1xPBS. To trigger plasmin (Pm)
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production from plasminogen (Pg), tPA (Sigma-Aldrich, Saint-Louis, MO, USA) was
added to the cells diluted in 50 μl of PBS and to a final concentration of 1 μg/ml. The
cells were incubated on ice for 30 min. Then, the cells were washed with 1xPBS and
0.4 μM human glu-plasminogen (American Diagnostica, Pfungstadt, Germany) was
added in 100 μl of PBS together with 10 μl of Spectrozyme®PL (American
Diagnostica, Pfungstadt, Germany). The plasmin production was measured after
incubation for 40 min at 37 oC, by absorbance at 405nm, using a TECAN infinite200
plate-reader.
Cancer cell proliferation and cell cycle assays
Cell proliferation and viability was assessed by WST-1 assay (Roche, Penzberg,
Germany) according to the manufacturer’s instructions. 5000 cells/well were seeded
in 100 μl of growth medium into transparent 96-well plates and transfected as
described above. 10µl of WST-1 reagent was added to each well and absorbance at
450nm was measured using a TECAN infinite200 plate-reader (Tecan, Linz, Austria).
For 7-AAD assays, transfected cells were analyzed after growing either
asynchronously in full growth medium or synchronously. In order to succeed cell
cycle synchronization, transfected cells were grown for 48h until reaching 90%
confluency, Then, the cells were washed with 1xPBS and were subsequently
incubated in serum deprivation conditions (medium supplemented with 0.5%FBS) for
48h to cause G0 arrest. Then cells were re-seeded in 6-well plates and cultured in
full growth medium (supplemented with 10% FBS) to trigger cell cycle initiation for
24h. Asynchronous cells were analyzed 72h after transfection, in full growth medium
culturing conditions. Transfected cells were washed with 1xPBS and were trypsinized
at 37 oC and 5% CO2.The cells were fixed and permeabilized using 1 ml of ice cold
90% methanol while vortexing. The cells were incubated on ice for 30 min,
centrifuged at 1500rpm for 5min and resuspended in 500 μl of 7-AAD (Actinomycin
D, 7 Amino) staining solution (Sigma-Aldrich, Saint-Louis, MO, USA) diluted 1:200 in
PBS. Samples were incubated at 4 oC for 90 min and were then subjected to flow
cytometric analysis (FACSCalibur, BD Biosciences, Bedford, MA, USA).
In vitro angiogenesis assays
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PANC-1 conditioned medium (0.5% FBS) was collected 48h after transfection with
the respective siRNAs and miRNAs.
Cell proliferation of HUVECs was measured with MTT (Sigma-Aldrich, Saint-Louis,
MO, USA) at 72 h after seeding 1x103 cells into 96 well-plates treated with mixture of
EBM-2 and 25% conditioned medium (n=6/group). Absorbance at 490 nm was
detected by spectrometer.
For capillary-like tube formation assay, 1x105 cells were incubated on Matrigel (BD
Biosciences, Bedford, MA, USA) in EBM-2 medium with mixture of EBM-2 and 25%
conditioned medium in 24 well-plates for 6 h (n=6 fields/group). Images were
photographed by light microscopy (Nikon, Danderyd, Sweden). Tube numbers were
analyzed by Adobe Photoshop CS5.
The motility of HUVEC cells responses to the tumor cell secreted factors were
assayed by using the modified Boyden chamber technique. Approximately 6 × 104
cells were added into each of the upper wells of the Boyden chamber (n=6/group),
which contained the mixture of EBM-2 and 25% conditioned medium in the lower
chamber. The cells were incubated over night at 37°C, after which the Boyden
chamber was disassembled and cells attached to the filter were fixed in methanol
and stained with a Giemsa solution (Sigma-Aldrich, Saint-Louis, MO, USA). Cells that
had migrated through the filter were counted using a light microscope (Nikon,
Danderyd, Sweden) and plotted as numbers of migrating cells per optic field.
All assays were performed at least three times and observed similar results.
Mouse Tumor Models
4 week-old SCID mice were used for tumor studies. Approximately 5 × 106 of PANC1 tumor cells or PANC10.05 cells were subcutaneously injected into the back along
the mid-dorsal line. Tumor volumes were measured two to three times per week and
were calculated according to the standard formula (volume = length × width × width ×
0.52). PANC-1 formed tumors were transfected in vivo weekly with a mixture of 10 μg
of mirVana miR-206 mimics or miRNA Negative control (Ambion, Austin, TX, USA)
and in vivo-jet PEI (Polyplus, NY, USA) in 0.015% collagenase II (Sigma-Aldrich,
Saint-Louis, MO, USA) solution intratumorally when tumor size reached at 0.2 cm3.
Mice were sacrificed 7 days after the last intratumoral injection of miRNA mimics.
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Animals were sacrificed using a lethal dose of CO2. All mouse studies were approved
by the Northern Stockholm Experimental Animal Ethical Committee.
Whole-Mount Staining
Immunohistochemical staining of whole-mount tissue samples was performed. Tumor
tissues were fixed with 4% PFA overnight and cut into small pieces. Tissues were
digested at room temperature with 20 mM proteinase K in 10 mM Tris buffer (pH 7.5)
for 5 min, followed by incubation in 100% methanol for 30 min. Tissues were washed
three times with PBS and incubated overnight at 4°C in PBS containing 3% skim milk
in 0.3% Triton X-100, followed by incubation with the indicated combinations of a rat
monoclonal anti-mouse CD31 (1:200; BD-Pharmingen, Bedford, MA, USA) and a
rabbit polyclonal anti-LYVE-1 (1:200; AngioBio, Del Mar, CA, USA). Samples were
thoroughly rinsed, and blood vessel and lymph vessel were detected using
fluorescently labeled secondary antibodies of Alexa Fluor 555-labeled goat anti-rat
(1:200; Invitrogen, Carlsbad, CA, USA), and Cy5-labeled goat anti-rabbit (1:200;
Millipore, Billerica, MA, USA). After washing, tissues were mounted using
Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and were
analyzed by confocal microscopy (Nikon C1 Confocal microscope, Nikon
Corporation, Japan).
Hematoxylin and eosin staining (H&E)
Tumor tissues were fixed with 4% paraformaldehyde overnight, and paraffinembedded tissues were cut in sections 5 mm thick, followed by staining with
hematoxylin–eosin (H&E) (Sigma-Aldrich, Saint-Louis, MO, USA). Images were
photographed by light microscope (Nikon, Danderyd, Sweden) and pictures were
overlapped using Adobe Photoshop CS software.
Microarray data
Publically available microarray data on matched miRNA and mRNA profiling of 25
PDAC tumors and 7 non-malignant pancreatic tissues (accession number:
GSE32688), as well as the data on 45 matched pairs of PDAC tumor tissue and their
adjacent healthy counterparts (accession number: GSE28735) were downloaded
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from GEO database (http://www.ncbi.nlm.nih.gov/geo). Differential expression of
miRNAs between tumor and normal samples was assessed using bioconductor’s
limma R-package. miRNAs with a p-value<0.01 (Benjamini-Hochberg corrected)
were included into a heatmap visualization using euclidean distance with the ward
clustering method.
Statistical analysis
Data are presented as mean ± SD, unless indicated differently. Samples were
analyzed by unpaired two-tailed Student’s t-test, unless mentioned otherwise and pvalues <0.05 were considered as being statistically significant. p-values <0.05, <0.01
and <0.001 are indicated with one, two and three asterisks, respectively.
SUPPLEMENTARY TABLES
Supplementary Table S1. Average Ct and ΔCt values of miR-206 analyzed in
normal and PDAC cell lines by TaqMan qRT-PCR run at standard mode.
Cell line
Average Ct
Average ΔCt
Threshold
HPDE
35.60
11.09
0.2
HPNE
35.38
9.93
0.2
PANC10.05
35.95
12.52
0.2
PANC-1
36.42
12.03
0.2
CAPAN-1
35.81
11.61
0.2
MiaPaca-2
36.57
12.15
0.2
CFPAC-1
36.12
11.49
0.2
BXPC-3
35.71
10.76
0.2
Supplementary Table S2. Average Ct and ΔCt values of miR-206 analyzed in
PANC-1 and PANC10.05 cell lines before and after miR-206 overexpression by
TaqMan qRT-PCR run at standard mode.
Cell line
Treatment
Average Ct
Average ΔCt
Threshold
PANC-1
miR-control
34.85
10.36
0.2
10
(30nM)
PANC-1
miR-206
(5nM)
21.21
-3.4
0.2
PANC-1
miR-206
(30nM)
20.24
-4.58
0.2
PANC10.05
miR-control
(30nM)
34.95
10.47
0.2
PANC10.05
miR-206
(5nM)
21.41
-3.13
0.2
PANC10.05
miR-206
19.93
-4.43
0.2
(30nM)
PANC-1_xenograft
miR-control
30.48
0.07
0.2
PANC-1_xenograft
miR-206
23.92
1.20
0.2
PANC10.05_pBABEempty
-
35.15
10.6
0.2
PANC10.05_pBABEmiR-206
-
24.50
-1.49
0.2
Supplementary Table S3. Average Ct and ΔCt values of all genes analyzed before
miR-206 mimic overexpression by TaqMan qRT-PCR run at standard mode.
Cell line
Gene
Average Ct
Average ΔCt
Threshold
PANC-1
ANXA2
19.97
-1.66
0.1
PANC-1
KRAS
32.95
11.24
0.1
PANC-1
CCL2
22.24
0.60
0.1
PANC-1
IL8
28.51
7.54
0.1
PANC-1
CXCL1
26.08
4.43
0.1
PANC-1
CSF2
33.84
12.20
0.1
11
PANC-1
VEGFA
32.32
11.38
0.1
PANC-1
VEGFC
24.87
3.22
0.1
PANC-1
MMP9
33.88
11.07
0.1
PANC10.05
ANXA2
18.56
-2.30
0.1
PANC10.05
KRAS
29.53
8.11
0.1
PANC10.05
CCL2
28.70
7.83
0.1
PANC10.05
IL8
29.16
8.30
0.1
PANC10.05
CXCL1
27.68
6.81
0.1
PANC10.05
CSF2
26.55
5.68
0.1
PANC10.05
VEGFA
33.05
12.19
0.1
PANC10.05
VEGFC
26.40
5.53
0.1
PANC10.05
MMP9
34.85
14.28
0.1
Supplementary Table S4. Average Ct and ΔCt values of all genes analyzed after
miR-206 mimic overexpression by TaqMan qRT-PCR run at standard mode.
Cell line
Gene
Average Ct
Average ΔCt
Threshold
PANC-1
ANXA2
22.21
0.26
0.1
PANC-1
KRAS
33.83
12.01
0.1
PANC-1
CCL2
24.54
2.59
0.1
PANC-1
IL8
29.38
8.56
0.1
PANC-1
CXCL1
28.46
6.52
0.1
PANC-1
CSF2
36.59
14.64
0.1
PANC-1
VEGFA
33.12
11.81
0.1
PANC-1
VEGFC
26.22
4.28
0.1
PANC-1
MMP9
35.49
14.83
0.1
PANC10.05
ANXA2
19.84
-1.25
0.1
PANC10.05
KRAS
31.02
8.90
0.1
PANC10.05
CCL2
29.56
9.72
0.1
12
PANC10.05
IL8
29.92
8.33
0.1
PANC10.05
CXCL1
29.18
7.99
0.1
PANC10.05
CSF2
29.13
7.08
0.1
PANC10.05
VEGFA
34.04
12.57
0.1
PANC10.05
VEGFC
27.86
6.16
0.1
PANC10.05
MMP9
36.27
14.79
0.1
Supplementary Table S5. Average Ct and ΔCt values of KRAS and ANXA2 genes
analyzed in PANC-1 and PANC10.05 cell lines by TaqMan qRT-PCR run at standard
mode, upon gene knockdowns.
Cell line
Treatment
Gene
Average Ct
Average ΔCt
Threshold
PANC-1
siControl
KRAS
31.76
10.58
0.1
PANC-1
siKRAS#1
KRAS
35.70
13.72
0.1
PANC-1
siKRAS#2
KRAS
35.89
13.89
0.1
PANC-1
siControl
ANXA2
18.95
-1.62
0.1
PANC-1
siANXA#1
ANXA2
24.86
3.61
0.1
PANC-1
siANXA#2
ANXA2
24.09
2.65
0.1
PANC10.05
siControl
KRAS
30.43
9.36
0.1
PANC10.05
siKRAS#1
KRAS
32.74
11.32
0.1
PANC10.05
siKRAS#2
KRAS
32.79
11.28
0.1
PANC10.05
siControl
ANXA2
19.43
-1.89
0.1
PANC10.05
siANXA#1
ANXA2
22.31
1.03
0.1
PANC10.05
siANXA#2
ANXA2
22.35
1.08
0.1
BxPC-3
siControl
ANXA2
19.87
-1.09
0.1
BxPC-3
siANXA#1
ANXA2
22.52
1.32
0.1
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Supplementary Table S6. Sequences of siRNAs used.
Gene
Company
siRNA no.
Sequence
ANXA2#1
Ambion
s1385
GAACUUGCAUCAGCACUGATT
ANXA2#2
Dharmacon
Custom designed
AGACCAAAGGUGUGGAUGAUU
KRAS#1
Ambion
s7939
CUAUGGUCCUAGUAGGAAATT
KRAS#2
Ambion
s7940
GCCUUGACGAUACAGCUAATT
RELA
Ambion
s11914
CCCUUUACGUCAUCCCUGATT
Supplementary Table S7. Sequences of PCR primers used for cloning and
mutagenesis of ANXA2 and KRAS 3’-UTR.
Name
Sequence (5'->3')
ANXA2 FW_XhoI
CAGTTCTCGAGGAAAGCGCTGCTGTACCTGTGTGG
ANXA2 RV_NotI
GTCAAGCGGCCGCGCGACACTTGGATAGGGGCAAC
ANXA2 FW_mut
CAAAGAAATGAAGTCCCCAAGGAGTTGGA
ANXA2 RV_mut
GACTTCACTTCCAACTCCTTGGTTACTTCATTTC
KRAS FW_XhoI
GCGCGCCTCGAGCAATCCATTAGCGACAGTAGG
KRAS RV_NotI
GCGCGCGCGGCCGCGCAAACAGTTCAAATTTCATGA
KRAS FW_mut
CTCCTGGTAACAGTAATACTAAGCATTGTTTTAGTAACCAG
KRAS RV_mut
CTGGTTACTAAAACAATGCTTAGTATTACTGTTACCAGGAG
Supplementary Table S8. Sequences and Universal Probe Library (UPL) probe
numbers for genes quantified by TaqMan qRT-PCR.
Name
Sequence (5'->3')
UPL Probe
ANXA2_L
CCTGCTCAGTATGACGCTTCT
#23
ANXA2_R
TCTGGAGCAGATGATCTCAATG
#23
CCL2_L
CCL2_R
COX2_L
COX2_R
CSF2_L
CSF2_R
AGTCTCTGCCGCCCTTCT
GTGACTGGGGCATTGATTG
CGCTTCAAGGTCAATGTGAA
GGCTCTGCTTGGTCAGGA
TCTCAGAAATGTTTGACCTCCA
GCCCTTGAGCTTGGTGAG
#40
#40
#72
#72
#1
#1
CXCL1_L
TCCTGCATCCCCCATAGTTA
#52
CXCL1_R
CTTCAGGAACAGCCACCAGT
#52
GAPDH_L
AGCCACATCGCTCAGACAC
#60
GAPDH_R
GCCCAATACGACCAAATCC
#60
HPRT-1_L
TGACCTTGATTTATTTTGCATACC
#73
HPRT-1_R
CGAGCAAGACGTTCAGTCCT
#73
IL8_L
AGACAGCAGAGCACACAAGC
#72
IL8_R
ATGGTTCCTTCCGGTGGT
#72
KRAS_L
TGGACGAATATGATCCAACAAT
#88
KRAS_R
TCCCTCATTGCACTGTACTCC
#88
MMP9_L
GAACCAATCTCACCGACAGG
#53
14
MMP9_R
GCCACCCGAGTGTAACCATA
#53
VEGFA_L
CGCAAGAAATCCCGGTATAA
VEGFA_R
AAATGCTTTCTCCGCTCTGA
#1
#1
VEGFC_L
TGCCAGCAACACTACCACAG
#27
VEGFC_R
GTGATTATTCCACATGTAATTGGTG
#27
Supplementary Figure Legends
Supplementary figure S1. a) Cell viability was assessed in PDAC cell lines 72 hours
after transfection with miR-206 or mimic control, using a WST-assay. Experiments
were performed with 6 replicates and data are shown as mean±SD (**p<0.01,
***p<0.001; Unpaired two-tailed t-test). b) Cell-cycle distribution of synchronized
PANC-1 or PANC10.05 cells, previously transfected with miR-206 mimic or miRcontrol, was measured by 7-AAD staining and quantified by flow cytometry.
Experiments were performed with 3 replicates and data are shown as mean±SD
(**p<0.01, ***p<0.001; Unpaired two-tailed t-test). c) Basal NF-κB activity was
determined in HEK293FT cells transfected with miR-206 mimic or negative control.
30nM of mimics, 100 ng of 3xKBL vector and 20 ng of pMIR-β-gal were cotransfected in 96-well plate format. 48h later, NF-κB luciferase activity was
determined and normalized to the β-galactosidase activity. Experiments were
performed in 6 replicates and data are shown as mean ± SD (**p<0.01, ***p<0.001;
Unpaired two-tailed t-test).
Supplementary figure S2. a) RelA/p65 total protein levels in PANC-1 cells 48 hours
after transfection with siRNAs were determined by Western blot, using total cell
lysates. GAPDH was used as loading control. b) Cell viability of PANC-1 cells was
determined 72 hours after transfection with siRNAs or treatment with 10 μΜ of NF-κB
chemical inhibitors (MG-132, BAY-117082) or vehicle (DMSO), using WST-assay.
Experiments were performed in 6 replicates and data are shown as mean±SD. c)
Cell cycle analysis of PANC-1 cells previously transfected with siRELA or negative
control, was performed using a 7-AAD assay. Cell phase proportions were quantified
by flow cytometry. Experiments were performed in 3 replicates and data are shown
as mean±SD. d) RTCA migration assay of PANC-1 cells treated with 10 μΜ of MG132, BAY-117082 or vehicle (DMSO) and of transfected PANC-1 cells with 30nM
siRELA or control. Experiments were performed with 4 replicates and data are shown
as mean±SD. Data were analyzed by unpaired two-tailed t-test; ***p<0.001.
Supplementary figure S3. mRNA expression of known NF-κB target genes in
PANC-1 and PANC10.05 cells was determined after RNAi-induced knockdown of
RELA (a) or TNF-α stimulation of non-transfected PANC-1 cells (b) by qPCR. c)
mRNA expression of VEGFC in PANC-1 and PANC10.05 cells was assessed after
inhibition of non-canonical NF-κB signaling using simultaneously siRNAs against
RELB and NFKB2 genes. d) Knockdown efficiency of RELB and NFKB2 genes was
determined by qRT-PCR. e) mRNA expression levels of CXCL1, COX2, VEGFC, and
IL8 were determined in PANC-1 cells transfected with miR-206 or negative control
together with 500ng of pCMV-p65 or empty vector in 6-well plates, as analyzed by
qRT-PCR 48h after transfection. f) End-point trans-well migration assay of PANC-1
cells transfected with microRNA mimics or miR-206 along with 500ng of pCMV-p65
15
or empty vector. Transfected cells were seeded in 0.5% FCS medium and allowed to
migrate for 24h using full-growth medium as chemoattractant. The number of
migrated cells was quantified by flow cytometry. g) Overexpression of p65 in PANC-1
cells, transfected with 500ng of pCMV-p65 in 6-well plates, was verified in total cell
lysates by Western Blot. All experiments were performed in triplicates and data are
shown as mean±SD (*p<0.05; **p<0.01; ***p<0.001; Unpaired two-tailed t-test). .
Supplementary figure S4. mRNA expression of ANXA2 (a) and KRAS (b) was
analyzed in 45 matched normal-tumor pairs of PDAC patients (GEO accesion no.
GSE28735). ***p<0.001; Paired two-tailed t-test. c) The efficacy of miR-206 mimic
overexpression in PANC-1 and PANC10.05 was determined by qRT-PCR. Data are
shown as mean±SD (***p<0.001; Unpaired two-tailed t-test). d) Cell viability was
assessed in PDAC cell lines 72 hours after transfection with different concentrations
of miR-206 or mimic control, using a WST-assay. Experiments were performed with 6
replicates and data are shown as mean±SD (***p<0.001; Unpaired two-tailed t-test).
e) mRNA expression of known NF-κB target genes in PANC-1 and PANC10.05 cells
was determined 48h after transfection with 5nM of miR-206 mimic or negative
control. Experiments were performed with 3 replicates and data are shown as
mean±SD (**p<0.01, ***p<0.001; Unpaired two-tailed t-test).
Supplementary figure S5. Knockdown efficiency of two individual siRNAs against
either KRAS or ANXA2 genes was verified by analyzing total mRNA (a) and protein
(b) levels of the respective genes and proteins in PANC-1 cells transfected with
either negative control, siANXA2#1, siANXA#2, siKRAS#1 or siKRAS#2. Relative
mRNA expression was determined by qRT-PCR and changes at the protein level by
Western blot analysis. Experiments were performed with 3 replicates and data are
shown as mean±SD (***p<0.001; Unpaired two-tailed t-test). c) Basal NF-κB activity
was determined in PANC-1 cells transfected with 2 individual siRNAs against KRAS
or non-targeting control. NF-κB luciferase activity was normalized to the βgalactosidase activity. The experiment was performed with 6 replicates and data are
shown as mean±SD (***p<0.001; Unpaired two-tailed t-test).
Supplementary figure S6. a) Gross pathology images of excised PANC-1 tumors
from SCID mice injected intratumorally with miR-206 mimics showing necrotic and
hemorrhaging phenotype compared to the control tumors. b) Histological images of
PANC-1 tumors by H&E staining to visualize the tumor necrotic and hemorrhagic
area (red-braun). Representative images are shown from an analysis of 2 sections
per animal from a minimum of 4 mice per group. Scale bar, 250 μm. c) mRNA
expression levels of ANXA2 and KRAS in dissected fresh frozen tumors from PANC1 xenografts following in vivo transfection of miR-206 mimcs (n=2) or negative control
(n=3), as determined by qRT-PCR. Data are shown as mean±SD (*p<0.05,
***p<0.001; Unpaired two-tailed t-test).
Supplementary figure S7. Alterations in the secreted amounts of VEGFA after miR206 overexpression were determined in PANC-1 and PANC10.05 cell supernatants
by FLISA, 48 hours after transfection. Experiments were performed with three
replicates and data are shown as mean±SD (**p<0.01, ***p<0.001; Unpaired twotailed t-test).
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Supplementary figure S8. a) Total cell lysates of PANC-1 or PANC10.05 were
analyzed for KRAS protein levels 48 hours after transfection with miR-206 mimic or
control by Western blot. b) mRNA expression levels of KRAS, HRAS and NRAS in
PANC-1 and PANC10.05 cells 48h following transfection with miR-206 mimics or
negative control. c) Endogenous mRNA expression levels of KRAS, HRAS and
NRAS in PANC-1 and PANC10.05 cells are shown as ΔCt values normalized to the
average Ct value of HPRT1 and GAPDH. Experiments were performed with three
replicates and data are shown as mean±SD (*p<0.05, ***p<0.001; Unpaired twotailed t-test).
Supplemental references:
1. Demetriades C, Mosialos G (2009) The LMP1 promoter can be transactivated directly by
NF-kappaB. Journal of virology 83: 5269-5277.
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