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SUPPLEMENTAL DATA
Supplemental Experimental Procedures:
siRNA, Plasmids and Lentiviral constructs:
Putative miR-301b binding sites on 3’UTR of NR3C2 were synthesized and inserted into the EcoRI/XbaI
sites pMIR-REPORT™ (Ambion) to generate NR3C2-3’UTR 1367-1388 and NR3C2-3’UTR 1736-1758.
Mutant construct, NR3C2-3’UTR 1736-1758 MUT, was generated by a substitution of twelve nucleotides
within the two core binding sites of NR3C2 3’UTR. Lentiviral MIF overexpression constructs (pLOCMIF) and MIF knockdown constructs (pGIPZ-shRNA1 and shRNA2) were purchased from Open
Biosystems (Rockford, IL). NR3C2 construct (EX-T8653-Lv203) was purchased from Genecopoeia
(Rockville, MD). Precursor miRNA and inhibitor of miR-301b, and NR3C2 siRNA were purchased from
Ambion (Grand Island, NY). Stable cell lines were established by infection with lentiviral particles
produced by transfecting 293T cells with package mix from Genecopoeia (Rockville, MD). Transfections
were carried out using lipofectamine 2000 reagent (Invitroge, Carlsbad, CA). Primers for plasmid
constructs were purchased from IDT (Integrated DNA Technologies). Detail information on primer
sequence and Taqman probes for qRT-PCR are provided in the Supplementary Table S8.
RNA Extraction, Quantitative RT-PCR, and Microarray Expression Profiling
Total RNA was extracted from frozen pancreas samples with Trizol (Invitrogen, Carlsbad, CA). RNA
from cultured cells was isolated using the Total RNA extraction kit (Norgenbiotek: Thorold, Canada).
RNA quality was determined by Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was reversetranscribed using Multi Scribe reverse transcriptase (Applied Biosystem, Foster City, CA). Geneexpression levels were measured by Quantitative RT-PCR (qRT-PCR) using probes set (Supplementary
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Table S8) from Applied Biosystems with an ABI GeneAmp PCR System 9600. microRNA (miRNA) and
mRNA expression profiling was performed at the microarray core facility of the National Cancer Institute,
Frederick, MD. miRNA profiling was performed by using nCounter miRNA Expression Assays
(Nanostring technologies,Seattle, WA) and Affymetrix GeneChip Human 1.0 ST arrays were used for
mRNA expression profiling according to the manufacturer’s protocol. Global miRNA array analysis was
performed by importing all data into nSolver Analysis Software v1.0 (Nanostring Technologies) and
normalized to the geometric mean of the 100 miRNAs with the highest expression values. Normalized
data was imported into Partek Genomic Suite V6.5 (St. Louis, MO) for further analysis. Prior to analysis,
data were filtered where any value less than 10 was omitted and any miRNA missing in 50% of samples
were excluded. Principal components analysis and Cox regression analysis was performed using Partek.
To analyze mRNA expression profile all arrays were first RMA normalized and gene expression
summaries were created for each gene by averaging all probes set for every gene. Further analysis was
performed on gene-summarized data. GSE 62452 also includes gene expression profiling data of 45
tumors and matching nontumor samples that we have earlier reported as GSE28735 in gene
expression omnibus. GSE62498. The batch effect between the two sets of mRNA expression
profiling data was removed using Partek genomic suite. The miRNA and mRNA microarray
expression data have been deposited to Gene Expression Omnibus with accession numbers GSE
62498 (URL access:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cfkvuuigrxadbkh&acc=GSE62498 and
GSE 62452 (URL access:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ybmrwcguvncxvol&acc=GSE62452).
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Cell proliferation, Migration and Invasion
Cells were seeded in quadruplicate at 5×103 cells/well in a 96-well culture plates. Cell counting kit-8
(WST-8) colorimetric assay (Dojindo, Kumamoto, Japan) was used to determine cell proliferation at 12hr intervals and the absorbance values were measured with a microplate spectrophotometer (Molecular
Devices, Tucson, AZ). Migration and invasion assays were performed with 24-well BD FALCON Cell
Culture Insert and BD BioCoat Matrigel Invasion Chamber (BD Biosciences: Bedford, MA) using
manufacturer’s protocol.
Colony formation and Drug Cytotoxicity Assays
0.5–1× 103 cells were seeded in 6-well plate in the absence or presence of gemcitabine (Company Name,
Ellisville, MO) for 48 hr. Cells were allowed to form colonies for 2 weeks. Plates were fixed and stained
with crystal violet and colonies >2 mm in diameter were scored. For drug sensitivity assay, 5×10 3 cells
were seeded in 96-well plates and treated with 10nM gemcitabine for 4 days. The cytotoxicity was
assessed by Cell Counting kit-8. All assays were performed in triplicate and repeated at least three times.
Immunoblotting, Immunohistochemistry, Immunoflorescence and Luciferase reporter assay:
5 µm thick paraffin sections were incubated with primary antibodies (see Supplementary Table S9).
Signals were amplified using biotinylated IgG, followed by horseradish peroxidase-conjugated avidinbiotin complex (Vectastain ABC Kit, Vector Lab, Burlingame, CA) and diaminobenzene (DAB) as the
chromogen (Dako Envision System, Dako, Carpinter, CA). Immunostaining was scored blindly by Boardcertified pathologists assigning the intensity and prevalence score. Briefly, the intensity was assigned a
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score of 0-3, representing negative, weak, moderate or strong expression, whereas, prevalence was
assigned a score of 0-4 representing <10%, 10–30%, >30–50%, >50–80% and >80% cells showing
NR3C2 expression. The overall quantitation was then achieved by multiplying the intensity and
prevalence score as described earlier.
Immunoblotting
Cells were lysed with RIPA buffer (Invitrogen, CA). Proteins were electrophoresed under reducing
conditions on 4–12% acrylamide gels (Invitrogen, Carlsbad, CA), and then transferred onto a PVDF
membrane (Invitrogen, Carlsbad, CA). The membrane was incubated for 60 min with 0.1% Tween 20
(TBS) containing 5% nonfat milk at room temperature. Incubation with primary antibody
(see Supplementary Table S9) was carried out overnight at 4°C, followed by secondary ECL anti-Rabbit
or anti-Mouse IgG HRP-linked antibody (GE Healthcare, Pittsburgh, PA) incubation for 1hr at room
temperature. Protein was visualized using a Super-Signal West Dura Extended Duration Substrate
detection Kit (Thermo Scientific, MA, US) using manufacturer’s instructions.
Luciferase Reporter Assay
Cells were cultured in 96-well plates and co-transfected with 50nM of Pri-miR-301b, or Anti-miR-301b
(or 50nM negative control), 100ng of luciferase reporter pMIR-REPORT NR3C2 1367-1388 or NR3C23’UTR 1736-1758, and 1ng of pRL-CMV Renilla luciferase reporter. Cells were incubated for 48 hr then
washed twice in PBS and harvested for firefly/Renilla luciferase assay using the Dual-Luciferase Reporter
Assay System (Promega, City,State).
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Immunofluorescence
Approximately, 1.5×104 Panc-1, Capan-2 stable sh-NR3C2 and control cells were seeded on 8-well LabTek II chamber slides. After 36 hrs, cells were washed with PBS and fixed in 4% paraformaldehyde in
PBS buffer at room temperature for 20 min, rinsed twice and permeabilized with 0.2% Triton X-100 in
PBS for 10 minutes, rinsed again with PBS then blocked in PBS containing 5% goat serum for 60 minutes
at room temperature before overnight incubation with anti-E-cadherin and anti-vimentin antibodies
(Supplementary Table S9). Cells were then washed three times with PBS for 15 min and incubated with
anti-rabbit or anti-mouse Alexa Fluor secondary antibodies (Life Technology, Carlsbad, CA) for 1 hr.
Cells were then washed for 15 min, and mounted using the Slowfade Gold Antifade reagent with DAPI
(s36939) (Life technology, Carlsbad, CA). All samples were subjected to confocal microscopy and
photographed at identical exposure times.
Supplemental Figure Legends:
Figure S1. A robust correlation existed between miRNA expression level determined by
microRNA array analysis and qRT-PCR in the test cohort. (A) A higher expression of miR-301b,
miR-15b, miR-10b, miR-93 and miR-590-5p, as determined by affymetrix gene expression array
analysis, were found in MIF-high tumors as compared with MIF-low tumors. Dot plots represent
the normalized log2 transformed miRNA expression values with the bars indicating median
values. P values were obtained using Student’s t-test. (B) Kaplan-Meier analysis generated by
using miRNA array expression with values above and below median grouped as high and low,
respectively. (C) miRNAs expression as determined by qRT-PCR strongly correlated with the
microRNA array expression data in test cohort, as assessed by Pearson correlation . Each data
point represents an individual tumor from a single patient.
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Figure S2. MIF regulates miR-301b expression in human pancreatic cancer cells. (A) mRNA
expression analysis by qRT-PCR in a panel of human pancreatic cancer cell lines showed a
positive correlation between MIF and miR-301b expression (Pearson Correlation, r=0.67, p=
0.01). (B and C) Stable knockdown of MIF in MiaPaCa-2 and Capan-2 cell-lines resulted in a
decrease in miR-301b expression as determined by qRT-PCR. (D and E) Stable MIFoverexpression in human pancreatic cancer cell lines Panc-1 and Capan-2, as confirmed by both
qRT-PCR and western blot analysis, led to an increase in the expression of miR-301b. qRT-PCR
data are represented as fold change with error bars representing standard deviation.
Figure S3. NR3C2 is a potential target of miR-301b. (A) Putative target genes of miR-301b, that
also associated with patients’ survival (Kaplan Meier analysis), were subjected to Gene Ontology
analyses. Twenty-six of these potential target genes were classified as cancer-related. (B) Five
putative targets of miR-301b were validated in miR-301b overexpressing Panc1 and Capan-2
pancreatic cancer cell lines. qRT-PCR analysis in control and pri-miR-301b transfected cells
were performed to determine target genes expressions. Overexpression of miR-301b
significantly decreased NR3C2 mRNA level in Panc-1 and Capan-2 cell lines. P values were
obtained by two-tailed Student's t- test. (C) Schematics of wild type and mutant NR3C2luciferase reporter constructs, generated to examine miR-301b mediated regulation of NR3C2.
The CMV promoter drives constitutive transcription of a chimeric mRNA containing the firefly
luciferase coding sequence fused to the NR3C2 3’UTR (pmiR-Luc-NR3C2 WT) or to the same
3’UTR mutated in all the miR-301b seed-pairing sequences (pmiR-Luc-NR3C2-3-MUT). (D)
Sequence validation of NR3C2 3’UTR luciferase constructs. The core-binding sites of wild type
and mutant reporter constructs are underlined in blue and red respectively. (E) Luciferase
reporter assay showed the suppressive effect of miR-301b on NR3C2 3’UTR luciferase reporter
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activity with binding sites 1736-1758. However, it did not affect the luciferase reporter activity
of the construct containing the other potential binding site spanning 1367-1388, in HEK-293
cells. Histogram represents the value of the Firefly luciferase activities normalized to Renilla
luciferase activities. Error bars represent standard deviation obtained from 3 independent
experiments.
Figure S4. Endogenous NR3C2 expression was negatively correlated with MIF and miR-301b in
a panel of human pancreatic cancer cell lines. (A and B) mRNA expression analysis by qRT-PCR
in a panel of human pancreatic cancer cell lines showed that Endogenous NR3C2 expression was
negatively correlated with MIF and miR-301b (Pearson Correlation).
Figure S5. NR3C2 enhances sensitivity to gemcitabine in pancreatic cancer cells. (A)
Endogenous expression of NR3C2 in a panel of pancreatic cancer cell lines as shown by
immunoblotting (left panel). Lenti-viral mediated generation of stable pancreatic cancer cell lines
overexpressing NR3C2 as shown by immunobloting (right panel). (B-C) NR3C2 expressing cells
showed a higher sensitivity to chemotherapeutic drug gemcitabine (10nm and 20nM) as
compared with control cells as determined by cell survival (B) and colony formation assay (C).
Data are presented as means ± SD from 3 independent experiments. Statistical significance was
determined by using Student t-test.
Figure S6. NR3C2-knockdown increased proliferation, migration and invasion, and decreased
the sensitivity of pancreatic cancer cells to gemcitabine. (A) Decreased protein expression of
NR3C2 in stable NR3C2 shRNA lenti-virus infected pancreatic cancer cells as demonstrated by
western blot analysis. (B-D) NR3C2 knockdown resulted in increased proliferation, migration
and invasion and decreased the sensitivity of Capan-2 cells to gemcitabine.
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Figure S7. A lower endogenous NR3C2 expression was associated with EMT phenotype. (A)
Western blot analysis of NR3C2, E-cadherin and N-Cadherin in pancreatic cell lines. (B)
Representative phase-contrast microscope and immunofluorescence staining of E-cadherin in
Panc-1 and Capan-2 cells.
Figure S8. NR3C2 inhibits epithelial–to-mesenchymal transition. (A) Stable pancreatic cancer
cell lines were generated by infection with lenti-viral constructs, expressing either shRNAs
directed against the NR3C2 (sh-NR3C2) or shRNAs directed against the GFP gene (sh-Control)
and then selected with puromycin (2ug/ml) for 2 weeks. Expression of NR3C2 was confirmed by
qRT-PCR. (B) NR3C2 knockdown decreased E-cadherin in stable pancreatic cancer cell lines as
shown by immunofluorescence. DAPI was used for nuclear staining. (C) Quantification of
NR3C2 expression in sh-NR3C2 and sh-Control Panc 10.05 cells. (D) Representative phasecontrast micrograph of stable sh-NR3C2 showing mesenchymal phenotype as compared with shcontrol Panc 10.05 cells (×100). (E) Knocking down NR3C2 decreased E-cadherin expression as
visualized by immunofluorescence assay in Panc 10.05 cells.
Figure S9. MIF induced-miR-301b expression was blocked by transfection of anti-miR-301b.
miR-301b expression in control or MIF overexpressing cells transfected with miR-301b inhibitor
or control was quantified by qRT-PCR.
Figure S10. Inhibition of MEK did not affect the expression of miR-301b or NR3C2 in
pancreatic cancer cell lines. MIF-expressing stable Panc-1 and Capan-2 cells were treated with
MEK inhibitor (AZD6244) to examine the involvement of MEK/MAP Kinase pathway in MIFmediated regulation of miR-301b and NR3C2. The expression of miR-301b and NR3C2 were
determined 48 hours after treatment by qRT-PCR.
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Figure S11. NR3C2 expression is decreased in tumors as compared with adjacent nontumor
pancreas tissue in two independent cohorts. (A) NR3C2 was expressed at a lower level in
pancreatic tumors as compared to non-tumor tissue in the test (n=69) and (B) validation cohorts
(n=64). Dot plots represent the quantitative RT-PCR (n=3, 3 independent experiments) log 2transformed mRNA expression values with the bars indicating median values. P values were
obtained using Student’s t-test.
Figure S12. A lower expression of NR3C2 is associated with poor survival in multiple
independent cohorts. (A, B) A third validation was done using NR3C2 expression in the cohort
from “The Cancer Genome Atlas (TCGA). Primary tumors from metastatic PDAC cases showed
a lower expression of NR3C2 as compared with the primary tumors from nonmetastatic cases (A).
Kaplan-Meier analysis showing that a lower expression of NR3C2 is associated with poorer
survival in PDAC cases in TCGA cohort, which is consistent with the findings from test and first
validation cohorts (B). (C) Metastatic tumors expressed a lower level of NR3C2 as compared
with primary tumors in publicly available recent gene expression dataset (GSE71729). Dot plots
represent the normalized log 2 transformed NR3C2 expression values obtained by gene
expression microarray analysis. (D) Kaplan-Meier analysis showing that a lower expression of
NR3C2 is associated with poorer survival in PDAC cases (Log-Rank test).
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