Step 1
Treatment of normal and tumoral prostate
epithelial cells with AZA:
PrEC, PC3, DU145, LNCaP
Step 2
Profiling of 650 miRNAs by RT-qPCR in
treated and untreated cells
Step 3
MiRNAs upregulated by AZA in any tumor
cell line and with an upstream CpG island
Step 4
73 miRNAs
MiRNAs unresponsive to treatment (or
minimally compared to tumor cells) in PrEC
Step 5
62 miRNAs
MiRNAs with downregulated expression as
compared to PrEC
38 miRNAs
Step 6
MSP: miRNAs methylated in tumor cell line
but not in PrEC
7 miRNAs
Step 7
MSP in human FFPE specimens
Step 8
1 miRNA
MiRNAs with cancer-specific methylation:
gain-of-function experiments in vitro
Figure S1: Study Workflow
a
Determination of the optimal number of
reference targets
1.20
0.20
0.18
1.00
0.16
0.14
0.12
0.10
0.08
0.06
0.80
geNorm V
geNorm M
Average expression stability of remaining
reference targets
0.60
0.40
0.04
0.02
0.00
0.20
0.00
V2/3
1.2
5
1.0
4
0.6
PC3
Control
3
PrEC
PC3
PrEC
Determination of the optimal number of
reference targets
0.85
0.16
0.80
0.14
0.75
0.12
0.55
geNorm V
geNorm M
Average expression stability of remaining
reference targets
0.60
V7/8
0
0.0
0.65
V6/7
1
0.2
0.70
V5/6
2
0.4
c
V4/5
AZA
0.8
R.U.
R.U.
b
V3/4
0.10
0.08
0.06
0.50
0.04
0.45
0.02
0.40
0.00
V2/3
V3/4
V4/5
V5/6
V6/7
V7/8
Figure S2: Candidate reference gene search in vitro and in vivo. (a) Genorm analysis of candidate reference genes across
PrEC, PC3, DU145, LNCaP cells untreated and treated with AZA. Candidates included: RNU6B, U18, miR-210, miR-30e-5p,
miR-191, miR-197, miR-331 and miR-103. SnoRNAs were employed since they find ubiquitous use as reference genes.
MiRNAs 210, 30e-5p, 197 and 331 were chosen according to Mestdagh et al1. MiR-191 and miR-103 were included as per
Peltier and Latham2. TaqMan miRNA assays (Applied Biosystems) were employed to measure expression levels by a multiplex
RT reaction. The 20uL RT reaction contained the following reagents available in the Taqman miRNA reverse transcription kit
(Applied Biosystems): 0.4uL dNTP, 4uL reverse transcriptase, 2uL 10X RT buffer, 0.25uL RNase inhibitor, 20ng RNA and 4uL 5X
primer pool. The primer pool consisted of the Taqman RT primers each diluted 1:4 from 250nM to 62.5nM. The 16uL qPCR
reaction contained 2X Taqman qPCR master mix, 0.4uL of Taqman probe and 3uL of 5-fold diluted cDNA. Standard curves
were done to ensure an amplification efficiency from 90 to 110%. qPCR was carried out on the Stratagene Mx3000P. Data
was imported into qBasePLUS (Biogazelle) for geNorm analysis3 and relative quantification. (b) miR-132 expression in cell lines
averaged over 3 biological replicates and normalized to the geometric mean of miR-331 and miR-103. R.U. = relative units.
(c) GeNorm analysis of candidate reference genes in 20 FFPE prostate specimens consisting of 20 normal and 20 cancerous
samples indicates the geometric mean of miR-191 and miR-103 as the optimal normalization factor (same protocol as above).
a
b
Figure S3: miR-132 expression correlated with tumor stage and Gleason score. Samples (GSE21032)4 were clustered by
(a) average Gleason score and (b) pathologically determined stage . Red asterisks indicate average miR-132 expression
for each group and dashed line indicates decreasing trend. Pearson correlation coefficients and associated p-values are
indicated at the bottom left. Overall, the mean miR-132 expression of low Gleason score (≤7) versus high Gleason score
(≥8) had a decreasing trend (6.81 versus 6.56; p=0.19 Student’s t-test). High Stage was significantly associated with
lower miR-132 expression (p=0.004, Student’s t-test for T2a-T2b versus T3a-4).
Relative miR-132 expression
a
60,000
***
50,000
40,000
***
30,000
20,000
10,000
0
Ctrl cells PremiR-132 PremiR-132
Attached Detached
cells
cells
b
Control, 72hrs
miR-132, 72hrs
350
350
G1 52%
S 29%
G2/M 19%
280
210
Counts
Counts
210
G1 52%
S 25%
G2/M 23%
280
140
140
70
0
70
0
1
10
100 1000 10000
FL2-H
1
10
100 1000 10000
FL2-H
PI
c
Ctrl, 72hrs
10000
1000
miR-132, 72hrs
10000
Necrotic: 1.8%
Apoptotic: 2%
4.5%
1000
Live:95%
100
10
10
92%
PI
100
3%
1
1
1
100
10000
1
100
10000
Fluorescein DA
Figure S4: Flow cytometric cell cycle and vitality analysis of PC3 cells transfected with a scrambled
sequence or premiR-132 at 72 hours. (a) Analysis of PremiR-132 transfection efficiency by RT-qPCR of
mature miR-132 at 72 hours. See Figure S1b for procedure.***p-value<0.001, Student’s t-test. (b) Cell
cycle analysis at 72 hours post-transfection shows no differences between control and miR-132expressing cells. Similar results were obtained at 48 hours (data not shown). See Viticchiè et al.5 for
experimental procedure. (c) Quantitative analysis of cell viability and death by flow cytometric
measurement of cellular fluorescence after staining with fluorescein diacetate and propidium iodide on
the FacsCalibur flow cytometer (Becton Dickinson)6. The percentages indicate necrotic (top left),
apoptotic (bottom left) and live cells (bottom right). Cells were harvested at 72 hours post-transfection
and stained for 5’ with propidium iodide and fluorescein DA to a final concentration of 4ug/mL and
100mM, respectively.
Z-scores (tumor versus normals)
4
3
2
1
0
-1
-2
10
Z-scores (tumor versus normals)
HB-EGF mRNA expression
Tumor samples (n=110)
TALIN2 mRNA expression
8
6
4
2
0
-2
Tumor samples (n=110)
-4
Figure S5: HB-EGF and TALIN2 mRNA expression for 110 prostate cancer tumors4 for which
miRNA data was also available. Black bars indicate significant expression modulations at a zscore cutoff of ±2 (dashed line). Data were downloaded from the cBio cancer genomics portal
(http://www.cbioportal.org). Significant changes occurred in 7.3% of cases for HB-EGF where
100% of these demonstrated an upregulated expression. For TALIN2, 20.8% of cases had
deregulated expression, where 95.6% of these demonstrated an upregulated expression.
Supplementary references:
1.
Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F, Vandesompele J . A
novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 2009; 10: R64.
2.
Peltier HJ, Latham GJ. Normalization of microRNA expression levels in quantitative RT-PCR assays:
identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA
2008; 14: 844-52.
3.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of
real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome
Biol 2002; 3:RESEARCH0034.
4.
Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS et al. Integrative genomic profiling of
human prostate cancer. Cancer Cell 2010; 18: 11-22.
5.
Viticchiè G, Lena AM, Latina A, Formosa A, Gregersen LH, Lund AH et al. MiR-203 controls proliferation,
migration and invasive potential of prostate cancer cell lines. Cell Cycle 2011; 10: 1121-31.
6.
Bartkowiak D, Högner S, Baust H, Nothdurft W, Röttinger EM. Comparative analysis of apoptosis in HL60
detected by annexin-V and fluorescein-diacetate. Cytometry 1999; 37: 191-6.