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SUPPLEMENTAL FILE
Inhibition of S-Adenosylmethionine-Dependent Methyltransferase Attenuates TGF-β1induced EMT and Metastasis in Pancreatic Cancer: Putative Roles of miR-663a and miR4787-5p
Hardik R. Mody2, 3, Sau Wai Hung3, Mohammad Al-Saggar3, Jazmine Griffin3, and
Rajgopal Govindarajan1, 2, 3
SUPPLEMENTAL MATERIALS & METHODS
Materials and Reagents
DZNep was synthesized in the Chu Laboratory (University of Georgia, GA) as described
previously and also purchased from APExBIO (Houston, TX). Gemcitabine was purchased
from ChemieTek (Indianapolis, IN). Recombinant human TGF-β1 protein was purchased from
R&D systems (Minneapolis, MN). Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5dipheny ltetrazoliumbromide (MTT), and crystal violet were purchased from Sigma-Aldrich (St.
Louis, MO). Dimethylsulfoxide (DMSO) was obtained from Macron Chemicals (Center Valley,
PA). The bicinchoninic acid (BCA) protein assay reagent and West Pico Chemiluminiscent
substrate were obtained from Pierce Chemical (Rockford, IL). All cell culture media were
purchased from Cellgro Corning (Manassas, VA) except for the human keratinocyte basal
media that was purchased from Gibco ThermoFisher Scientific (Waltham, MA). Plasticwares
for cell culture were procured from Nunc ThermoScientific (Waltham, MA). Matrigel-coated
transwell inserts (8 µm pore size) were obtained from BD Biosciences (#3544480). The
Quantikine TGF-β1 ELISA kit (#DB100B) was purchased from R&D Systems.
Antibodies
The rabbit polyclonal TGF-β antibody (ab66043) was purchased from Abcam (Cambridge,
MA), and the mouse monoclonal Smad3 (sc-101154) and rabbit polyclonal p-Smad3 (sc130218) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
rabbit polyclonal Caspase-3 (#9662), rabbit polyclonal p-Smad2 (#3101), rabbit monoclonal
Smad2 (#3122) & vimentin (#5741), and mouse monoclonal cytokeratin 8/18 (#4546)
antibodies were purchased from Cell Signaling Technology (Danvers, MA), and the mouse
monoclonal N-cadherin antibody was purchased from BD Biosciences (San Jose, CA). The
mouse monoclonal β-actin (A1978) antibody was purchased from Sigma-Aldrich (St. Louis,
MO). The mouse E-cadherin antibody was kindly received from Dr. Parmender Mehta
(University of Nebraska Medical Center, Nebraska). The mouse monoclonal EZH2 antibody
(05-1319) was purchased from Millipore (Massachusetts).
Primers and Constructs
The Taqman primer probes for TGF-β1 (Hs00998133_m1), EZH2 (Hs01016789_m1), ACTB
(Hs99999903_m1), GAPDH (Hs99999905_m1), GUSB Hs00939627_m1, pri-miR-663a
(Hs03304850_pri), miR-4787-5p (464332_mat), and RNU6B (001093) were purchased from
Applied Biosystems (Foster City, CA). MiR-663a and miR-4787-5p (custom synthesized)
lentiviral constructs for overexpressing miRNAs were purchased from System Biosciences
(Mountain View, CA). The GIPZ EZH2 lentiviral shRNAs (RHS4531-EG2146) were purchased
from Dharmacon GE (Lafayette, CO).
Scratch Wound Assay
Cells cultured in 6 cm dishes were grown to 90% confluence. A sharp scratch was created
across the cell monolayer with a sterile scalpel blade. The cells were washed with PBS to
remove cellular debris and replenished with growth media with or without various treatments.
Cells were allowed to grow for various time intervals and wound closure images were
recorded. Percent wound closure was determined using Image J software by measuring the
average differences in wound areas.
Transwell Invasion Assay
Matrigel-coated transwell inserts were rehydrated with cell media containing 0.1% BSA and
growth media containing 10% FBS was added to the lower chamber of the plate. 0.5x105 cells
in 200 µL suspensions were added to the upper chamber and the transwell inserts were
incubated for 48 h in 5% CO2. At the end of 48 h, the non-invading cells were removed
carefully by gently scrubbing the upper surface of the transwell with a cotton swab. The cells
invaded on the lower surface of the membrane were fixed with cold methanol, stained with
0.1% crystal violet, and counted for quantification.
Enzyme Linked Immunosorbent Assay
Cells were seeded at 3x103/well in 96-well plates and cultured in serum-containing medium.
After 24 h of incubation, the culture media was replaced with fresh media containing reduced
serum (2% FBS). The supernatant was collected at different time intervals (72-144 h) and
assayed for TGF-β1 concentration using the Quantikine TGF-β1 ELISA kit as per the
manufacturer’s instructions. The basal levels of TGF-β1 in FBS in the absence of cells were
also measured for normalization. Briefly, the samples were activated by acidification with 1N
HCl for 10 minutes and then neutralized by 1.2 N NaOH in 0.5 M HEPES buffer. Activated
samples were added to individual wells of a 96-well plate pre-coated with a TGF-β1-specific
monoclonal antibody. Titrated dilutions of purified recombinant human TGF-β1 were used to
plot a standard curve. After 2 h of incubation at room temperature, the unbound substrates in
the samples were removed by washing the wells four times. Subsequently, the sample wells
were incubated with an enzyme-linked anti-TGF-β1 polyclonal antibody for an additional 2 h.
The unbound antibody was removed by washing four times. Finally, the sample wells were
incubated with a mixture of tetramethylbenzidine and hydrogen peroxide for 30 minutes, and
OD measurements at 450 nm (background at 540 nm) were determined for quantification.
MicroRNA Microarray
2.5x104 cells were seeded in 6 cm dishes and replenished with fresh growth media with or
without various treatments at 24 h. At the end of treatment periods, cells were lysed and total
RNA was extracted with the miRVana isolation kit (Ambion, Inc., Austin, TX). In addition, RNA
samples from normal pancreatic and PDAC tissues were also used in the study. The samples
were evaluated for RNA integrity, enriched for small RNA enrichment, and loaded on
microarray chips that consisted of probe sets detecting 1900 miRNA transcripts referenced in
the Sanger miRBase Release 18.0. (LC Sciences, Houston, TX). In addition, the microarray
chip also included perfect matched and single-basepair mismatched control probes for a 20mer RNA oligo that served as a positive control and was spiked into the RNA samples before
labeling with Cy3 or Cy5. Each sample was repeated at least twice. Data were analyzed by
subtracting the background from measured signals and then normalized using the Locallyweighted Scatterplot Smoothing (LOWESS) Regression method. MiRNAs were considered
detectable if the signal intensity was higher than three times that of the background and the
spot coefficient of variation was <0.5 (spot CV = standard deviation/signal intensity). The ratio
of signal intensities between two samples was transformed into log2 scale and differentially
expressed miRNAs (detectable) were represented by a 2-color system: green indicating lowest
expression and red indicating highest expression. The microarray data was deposited in GEO
repository (Accession GSE86383).
Luciferase In Vitro Reporter Assay
The luciferase binding assays were carried out as mentioned previously with a few
modifications. Briefly, 5x103 cells were seeded per well in a 96-well plate. After 24 h, each well
was transfected with 0.1 µg of control or TGF-β1 3’UTR target vector (ID: HmiT018044;
GeneCopoeia), 10 µl of Opti-MEM (Invitrogen), and 0.3 µl of Xtreme Gene HP DNA
Transfection Reagent (Roche) as per the manufacturer’s protocol. 24 h post transfection, cells
were lysed with a passive lysis buffer, and the lysates were subjected to the Dual-Luciferase
assay (Promega) as per the manufacturer’s instructions.
Knockdown of MiRNAs
The LNA-based anti-miR-663a and anti-miR-4787-5p (miRCURY LNA-based miRNA
inhibitors) were purchased from Exiqon (Vedbaek, Denmark). Cells were seeded in 6 cm
dishes and grown overnight. Within 24 h of seeding, cells were transfected with either LNA663a or LNA-4787-5p at a concentration of 5-50 nM with the help of a transfection agent,
Xtreme Gene HP DNA Transfection Reagent (Roche). After 36-48 h of transfection, cells were
lysed and subjected to various applications.
RT2 Profiler PCR Array
Cells were cultured in 6 cm dishes at a density of 2.5x104 in growth media with or without
various treatments for 24 h. At the end of treatment periods, cells were lysed, and total RNA
was extracted with the E.Z.N.A. Total RNA kit (Omega bio-tek, Norcross, GA). Total RNA was
transcribed into cDNA using the RT2 First Strand Kit (#330401, Qiagen Sciences, Hilden,
Germany) as per the manufacturer’s instructions. Subsequently, cDNA was loaded into the
RT2 Profiler PCR Array (TGF-β Signaling Pathway Array, #PAHS-035Y, Qiagen Sciences).
384-well plates containing 4 primer assay sets of 96 different genes were amplified and
quantified using the RT2 SYBR Green ROX qPCR Mastermix (#330521, Qiagen Sciences) as
per the manufacturer’s instructions. The 96 genes, listed in Table S2, consists of 84 pathwayrelated genes, 5 housekeeping genes, 7 quality control genes including 1 genomic DNA
control gene, 3 reverse-transcription controls, and 3 PCR controls. The Ct values were
generated from real-time PCR and normalized with the 5 housekeeping genes. The Ct values
were then analyzed with the help of a web-based PCR Array Data Analysis Software
(SABiosciences).
Orthotopic Pancreatic Tumor Xenograft Model
Animal experiments were performed as per our protocol (AUP# A2014 04-021-Y1-A0) that was
approved by the Institutional Animal Care and Use Committee (IACUC) at the University of
Georgia. We generated pancreatic cancer xenograft models by adapting to published
procedures. Eight week old female athymic nude mice (homozygous; 002019, Jackson
Laboratories) were used for this study. 1x105 cells were seeded in 10 cm dishes and were
allowed to grow up to 90% confluence. Cell suspensions were prepared at a concentration of
1x105 cells/100 µl in sterile HBSS containing 1% v/v serum-free Matrigel and implanted
orthotopically in the tail region of the pancreas. The cell suspension was allowed to solidify in
order to avoid leakages outside the pancreas. Mice were monitored for changes in their weight
twice a week. Seven days after the cancer cells were injected, mice were injected IP with
either DZNep (3 mg/kg) or saline as a control twice a week. Ten weeks after cell injection,
tumors were excised and assessed for weight and size for end-point assays. Tumor volume
was measured with a digital caliper and calculated by formula, tumor volume = π/6 (L x S x S)
where L = longest diameter and S = shortest diameter of the tumors. In addition, the spleen,
liver, kidneys, and lungs were monitored for metastatic tumor lesions and tissue sections at
normal-tumor interface were collected for histological examination of metastatic foci. To study
the effects of DZNep on tumor regression, 2x106 cells/100 µl in sterile HBSS containing 1% v/v
serum-free Matrigel was used as above. After 28 days of tumor implantation, tumor-bearing
mice were treated with DZNep (5 mg/kg) every alternate day, for five doses and untreated and
DZNep-treated mice (n=3 for each group) were sacrificed after 40 days of tumor implantation,
tumors were harvested, and tumor volumes were measured.
Statistical analysis
Data analysis was conducted using GraphPad Prism 6 (Graphpad software Inc.). The
statistical significance of the data was determined using Student’s t-test between two groups,
or using one-way (one variable) or two-way (two independent variables) analysis of variance
(ANOVA) with Tukey’s multiple comparisons test for multiple experimental groups. All P-values
<0.05 were considered significant unless otherwise stated. P-values <0.05, <0.01, and <0.005
are indicated with *, **, and *** respectively unless otherwise stated.
SUPPLEMENTAL TABLES
Table S1. Clinical and histological characterization of human pancreatic tumor tissue samples
isolated from patients.
See Excel file Table S1
Table S2. Genes profiled for RT2 Profiler PCR Array of TGF-β Signaling Pathway.
Table S3. List of miRNAs downregulated (fold difference ≥1.5) in MIA PaCa-2 when compared
with HPDE
See Excel file Table S3
Table S4. List of thirty-five miRNAs commonly downregulated (fold difference ≥1.5) in MIA
PaCa-2 and PDAC tissues
Common downregulated miRNAs
hsa-miR-141-3p
hsa-miR-200c-3p
hsa-miR-376c
hsa-miR-4484
hsa-miR-625-5p
hsa-miR-139-5p
hsa-miR-1207-5p
hsa-miR-30c-1-3p
hsa-miR-4508
hsa-miR-4492
hsa-miR-1915-3p
hsa-miR-3196
hsa-miR-5001-5p
hsa-miR-4532
hsa-miR-3676-5p
hsa-miR-663a
hsa-miR-638
hsa-miR-4800-3p
hsa-miR-762
hsa-miR-4516
hsa-miR-2861
hsa-miR-3665
hsa-miR-4687-3p
hsa-miR-4466
hsa-miR-3960
hsa-miR-192-5p
hsa-miR-3940-5p
hsa-miR-3656
hsa-miR-27b-3p
hsa-miR-148a-3p
hsa-miR-4443
hsa-miR-3607-5p
hsa-miR-1587
hsa-miR-7-1-3p
hsa-miR-130a-3p
MIA PaCa-2 vs HPDE
PDAC tissues
Fold Difference of Downregulation Fold Difference of Downregulation
672.1199115
9.77379508
288.4859078
1.717909927
265.9947843
13.18054908
52.32463449
2.305993175
16.98572076
1.988085433
10.97996802
2.064592832
10.85292421
7.924640582
7.853344687
2.017539583
7.62191826
1.530602625
7.304689364
2.879563246
5.822332039
1.752757762
5.15526543
1.966384186
4.718478402
4.202919898
4.654390816
1.909715373
4.492434785
3.079881789
4.369336395
1.773408738
3.981428989
2.093784266
3.880171259
3.19769181
3.823977939
2.621684831
3.725870031
2.006320563
3.343542932
4.986606502
3.282366966
1.955893066
3.243226359
2.37024532
3.098171207
2.667956503
2.96405239
1.500310288
2.803425649
1.921633839
2.175500636
1.701546302
2.075061517
1.464649221
1.981355193
2.768060498
1.87938843
10.12862266
1.858338429
1.599765439
1.737741946
1.972748446
1.644532807
2.413178251
1.622476508
3.171943009
1.59694242
1.875172455
Table S5. List of miRNAs upregulated (fold difference ≥1.5) in MIA PaCa-2 after DZNep (8 h
and 72 h) treatment
See Excel file Table S5
Table S6. DZNep partially reversed the downregulation of miRNAs seen in PDAC
See Excel file Table S6
Table S7. GC content of miRNAs induced by DZNep (seed sequence; red)
Table S8. Bioinformatic predictions of DZNep-induced miRNAs to target players in the TGF-β
signaling pathway (TGF-β1 or –β2), receptors, (TGFBR1, TGFBR2, or TGFBR3) or Smads
(Smad2, Smad3, or Smad4)
See Excel file Table S8
SUPPLEMENTAL FIGURES
Figure S1. Growth inhibitory responses to TGF-β1 are lost in pancreatic cancer cells. A
and B. Pancreatic cancer cell lines and HPDE treated with TGF-β1 at various concentrations
(A) or at 10 ng/mL (B) for 72 h and cellular proliferation measured with an MTT assay.
Significant growth inhibitions were noticed with HPDE and HPAF II but not with other cell lines.
Points, mean of triplicate; bars, SD. n=3
Figure S2. DZNep retains growth inhibitory properties in the presence of TGF-β1 in
pancreatic cancer cells. A and B. Pancreatic cancer cell lines treated with TGF-β1 (10
ng/mL), DZNep (50 µM), or both for 72 h. Cells were subjected to fixation, staining with crystal
violet, and images acquired (A) or MTT cellular proliferation assay (B). bars, SD. n=3
Figure S3. DZNep increases epithelial markers and decreases mesenchymal markers in
pancreatic cancer cells. Whole cell lysates (50 µg) from cells treated with DZNep were
subjected to Western blotting analysis for various EMT markers. β-actin, the internal loading
control, is shown with a representative blot.
Fig. S4. DZNep resists TGF-β1-induced E-cadherin mRNA loss in MIA PaCa-2. Relative
transcript levels of E-cadherin measured in TGF-β1 (10 ng/mL; 48 h) and/or DZNep (2 or 20
µM; 48 h)-treated cells with qRT-PCR using respective taqman assays. GUSB used as an
internal control. Bars, SD. UD = undetected.
Fig. S5. DZNep did not significantly reduce TGF-β1 transcript levels in pancreatic
cancer cells. Relative transcript levels of TGF-β1 measured in DZNep (20 µM; 72 h)-treated
cells with qRT-PCR using a taqman assay. GUSB used as an internal control. Bars, SD.
Fig. S6. Heat map showing differential miRNA expression in MIA PaCa-2 (PanCan) (n=2)
compared against HPDE (n=2). Clustering of miRNAs differentially expressed in MIA PaCa-2
versus HPDE. Heat maps with statistically significant (p<0.1) changes in miRNA expression
shown by a 2-color system (light green, lowest expression; dark red, highest expression). Each
row represents expression levels of a single miRNA while each column represents a single
sample. n=2.
Fig. S7. DZNep exclusively induced miR-663a and miR-4787-5p expression and not
other epigenetic drugs or nucleoside analogs. Expression levels of miR-663a and miR4787-5p were measured in drugs-treated (1µM, 8 h) MIA PaCa-2 with qRT-PCR using
respective taqman probes. GUSB and U6B were used as an internal control for assaying miR663 and miR-4787-5p. Bars, SD. n=2.
Fig. S8. Knockdown of EZH2 by short-hairpin RNA induces miR-663a/miR-4787-5p
expression and decreases TGF-β1 protein levels in pancreatic cancer. A and B. Stable
knockdown of EZH2 in pancreatic cancer cells, MIA PaCa-2 using short-hairpin RNAs #1-5.
Protein (A) and transcript (B) levels of EZH2 in MIA PaCa-2 transduced with shControl or
shEZH2#1-5 analyzed by Western blotting and qRT-PCR, respectively. β-actin was used as a
loading control for Western blotting (A) while qRT-PCR was performed with EZH2 taqman
probe using GUSB as an internal control (B). shEZH2#3 showed maximum knockdown of
EZH2 as compared with shControl and was utilized for further experiments. C. Knockdown of
EZH2 increased expression of miR-663a and miR-4787-5p in MIA PaCa-2 as analyzed by
qRT-PCR using respective taqman probes. GUSB and U6B were internal controls for miR663a and miR-4787-5p respectively. D. Knockdown of EZH2 decreased TGF-β1 protein levels
in MIA PaCa-2 as determined by Western blotting. Bars, SD. n=2 or 3.
Fig. S9. Stable overexpression of miR-663a and miR-4787-5p in pancreatic cancer cells.
Expression levels of miRNAs in MIA PaCa-2 and PANC-1 stably overexpressing miR-663a or
miR-4787-5p analyzed by qRT-PCR with respective taqman probes. GUSB and RNU6B were
used as internal controls. Bars, SD. n=3.
Fig. S10. LNAs antagonized miR-663/4787-induced decreases in cell migration of PANC1. PANC-1 stably overexpressing miR-663a or miR-4787-5p were treated with LNA-663a or
LNA-4787-5p (50 nM), respectively, and monolayers were subjected to a scratch wound
assay. Wound closure images were acquired after 24 h and 48 h at a magnification X4.
Fig. S11. DZNep treatment induces tumor regression in an orthotopic pancreatic cancer
xenograft model. A. Schematic presentation of dosing scheme for DZNep in tumor-bearing
mice. After 28 days of tumor inoculation, untreated mice (n=3) sacrificed, tumors harvested,
and measured for tumor volumes. Tumor-bearing mice treated with DZNep (5 mg/kg) every
alternate day for five doses and untreated/DZNep-treated tumors (n=3 for each group)
harvested after 40 days of tumor implantation and tumor volumes measured. B. Excised tumor
specimens under indicated conditions.
Fig. S12. TGF-β1-induced auto-stimulatory effects in PANC-1 and not in MIA PaCa-2:
Inhibition of autostimulatory effects by DZNep. A and B. Cells treated with TGF-β1 (10
ng/mL; 48 h) and/or DZNep (2 or 20 µM; 48 h) were subjected to qRT-PCR (A) and Western
blotting analysis for relative quantification of TGF-β1 transcript and protein levels as well as pSmad3 protein levels with β-actin as a loading control. Bars, SD.
Fig. S13. DZNep or miR-663a/miR-4787-5p induced no effects on apoptosis in vivo.
Whole tumor lysates from indicated groups were subjected to Western blotting analysis for
relative quantification of cleaved caspase-3 as a marker for apoptosis. Representative blot with
β-actin as a loading control is shown.