Direct Regulation of microRNA Biogenesis and Expression by
Estrogen Receptor Beta in Hormone-responsive Breast Cancer
O. Paris et al.
Supplementary Information
Supplementary Materials and methods
Microarray analyses; MicroRNA expression profiling data analysis; Immunohistochemistry.
Supplementary References
7 references.
Supplementary Tables S1-S4
Supplementary Figures S1-S6 with legends
Supplementary Materials and methods
Microarray analyses. For miRNA expression profiling, both biological (multiple experiments) and
technical (multiple arrays) replicates were performed. In the case of cells lines analyzed in standard
growth conditions (complete medium), 800 ng of purified total RNA were fluorescently labelled,
amplified and hybridized in duplicate on Illumina v.2 MicroRNA Expression Bead Chips (Illumina
Inc.). Also, 100 ng of the same from control cells (MCF-7 wt) and from TAP-ERβ expressing cells
were fluorescently labelled, amplified and hybridized in duplicate on Agilent Human microRNA
Microarray 18x15K v.3 (Agilent Technologies, Inc.). In each case analyses were performed
according to protocols provided by the array manufacturer. For cells arrested at G0-G1 and then
stimulated with 17-estradiol (+E2) and for FFPE tumor tissue samples analysis, RNA was
fluorescently labelled and amplified in triplicate, pooled and hybridized to Illumina v.2 MicroRNA
Expression BeadChips (Illumina Inc.), according to the protocols provided by the array
manufacturer.
For mRNA expression profiling, 500 ng total RNA from C-TAP-ER, C-TAP-ER and N-TAPER were reverse transcribed, as described previously (Cicatiello et al., 2010; Ravo et al., 2008)
and used for synthesis of cDNA and biotinylated cRNA according to the Illumina TotalPrep RNA
amplification kit (Ambion; code IL1791) sample preparation kit protocol. For each sample, 700 ng
of cRNA were hybridized for 18 hours at 55°C on Illumina HumanWG-6 version 2.0 BeadChips
containing 48701 probes (Illumina Inc.), according to the manufacturer’s protocol. The BeadChips
was scanned using Illumina BeadArray Reader 500 according to the manufacturer’s standard
methods.
The microarray data were deposited in the Array Express repository database with the following
accession numbers: E-TAMB-1053 for miRNA expression profiling performed in breast cancer cell
lines in standard growth conditions, E-TAMB-1055 for miRNA expression profiling performed in
starved and hormone-stimulated breast cancer cell liness; E-TAMB-1054 for miRNA expression
profiling in FFPE tumor samples; E-TAMB-1052 for mRNA expression profiling in breast cancer
cell lines.
MicroRNA expression profiling data analysis. For analysis of miRNA expression profiling data
generated with Illumina microarrays, the fluorescence intensity files were loaded into the Illumina
GenomeStudio v2009.1 software for quality control and expression analysis. First, the quantile
normalization algorithm was applied on the raw datasets to correct systematic errors. This
normalization equalizes distribution, median and mean of probe intensities among all sample, as the
normalization distribution is chosen by averaging each quantile across samples. For differential
expression analysis, technical replicates were grouped together and miRNAs with a detection pvalue below 0.01, corresponding to a false positive rate of 1%, were considered as expressed.
Statistical significance was calculated with the Illumina DiffScore, a proprietary algorithm that uses
the bead standard deviation to build an error model. Only miRNAs with a DiffScore ≤-20 and ≥20,
corresponding to a p-value of 0.01, were considered as statistical significant.
For miRNA expression profiling generated with Agilent microarrays, raw data were obtained with
the Agilent Feature Extraction (AFE) software v.10.7 (Agilent Technologies, Inc.). The AFE
algorithms estimate a single intensity measure for each miRNA, referred to as total gene signal
(TGS). The AFE-TGS is estimated by multiplying the total probe signal by the number of probes
per gene. After getting the raw data, we identified miRNAs differentially expressed in hormoneresponsive human breast cancer cells ERβ+ vs ERβ-, using GeneSpring gx v.11 software (Agilent
Technologies, Inc.). We performed data normalization to the 75th percentile; detected miRNAs were
then filtered based on their flag values P (Present) present in the data file. The GeneSpring software
considers the "gIsGeneDetected" as the flag column and marks microRNA marked with '0' as
Absent and '1' as Present. Only miRNAs 'present' in at least one sample were further considered and
are displayed. Finally miRNA differential expression between two given conditions was determined
calculating ERβ+/ ERβ- ration (fold-change) with a cut-off of ≥1.2.
RNA expression profiling data analysis was performed as described earlier (Cicatiello et al., 2010;
Ravo et al., 2008). Transcripts were considered detected when the corresponding detection p-value
was ≤0.01. Differentially expressed RNAs were selected when DiffScore was ≤-40 or ≥40, values
corresponding to a p-value of 0.0001. Raw data were normalized as described above using
GenomeStudio v. 2009.1 and analyzed with GeneSpring. Background was subtracted and spots
corresponding to absent or low-quality signals were removed from the analysis before global
median normalization. Expression data were median-centered by using a per-chip 50th percentile
method that normalizes each chip on its median, allowing comparison among chips. miRNAs with a
detection p-value below 0.05 were considered as expressed. Hierarchical clustering was performed
with the Manhattan algorithm. To identify differentially expressed miRNAs between ER+ and
ER- samples, a fold-change cut-off of ±1.2 was applied.
Immunohistochemistry. Formalin-fixed paraffin-embedded breast specimens were cut on
SuperFrost Plus slides (Menzel-Glaser). Antigen retrieval was performed by microwave at 430 W
(1mM citrate buffer, pH6.0) for two cycles of 10 minutes each and one of 5 minutes for anti ER
monoclonal antibodies (MoAbs) and by thermostatic bath at 96°C (10 mM/L citrate buffer, pH 6.0)
for 40 minutes for ER PgR, HER2 and Ki67. Sections were incubated with the anti-ER-1 MoAb
PPG5/10 (GeneTex, dilution 1:15; Skliris et al., 2002) overnight at 4°C, with the anti-ER MoAb
6F11 (Novocastra), the anti-PgR MoAb 1A6 (Novocastra), the anti-Ki67 MoAb MIB-1 (Dako) and
the anti-HER2 polyclonal antibody (A0485, Dako) for 30 min at room temperature. Positive and
negative controls were included for each antibody and in each batch of staining. The
immunoreactions were revealed by a streptavidin-biotin enhanced peroxidase system (Super
Sensitive Link-Label IHC Detection System, Biogenex) using 3-amino-9-ethylcarbazole (Dako) as
chromogenic substrate. Evaluation of the immunohistochemical results, was performed
independently and in blinded manner by two investigators (M.M. and F.N.) using a light microscope
(Nikon, Eclipse 55i) equipped with a software to capture images (Eureka Interface System).
ER
carcinoma cell nuclei, and positive when a moderate/strong staining reaction was observed in 20%
to 100% of neoplastic cell nuclei. This cut-off, which was in agreement to Shaaban et al. (2003) and
Gruvberger-Saal et al. (2007), was generated using the Classification and Regression Tree (C&RT)
analysis. We introduced into the model three variables (nodal status, ER expression, relapses) as
described earlier (Novelli et al., 2008). ER and PgR were considered positive when >10% of the
neoplastic cells showed a distinct nuclear immunoreactivity whereas Ki67, based on the median
value of a referral series of BC patients, was regarded as high if >15% of the cell nuclei were
immunostained. HER-2 immunostaining was performed following the manifacturer’s protocol and
protein overexpression was determined as defined in the HercepTest kit guide: score 0, 1+, 2+, 3+.
All IHC 2+ tumors were analyzed with Chromogenic in situ hybridization (CISH) as previously
described (Vocaturo et al., 2006), in order to determine the HER2 gene copy level. Cases with score
0, 1+ and 2+ lacking amplification were considered negative whereas amplified cases with score 2+
and cases with score 3+ were considered positive.
Supplementary References
Cicatiello L, Mutarelli M, Grober OM, Paris O, Ferraro L, Ravo M, et al. (2010). Estrogen receptor
alpha controls a gene network in luminal-like breast cancer cells comprising multiple transcription
factors and microRNAs. Am J Pathol 176: 2113-2130.
Gruvberger-Saal SK, Bendahl PO, Saal LH, Laakso M, Hegardt C, Edén P, et al. (2007). Estrogen
receptor beta expression is associated with tamoxifen response in ERalpha-negative breast
carcinoma. Clin Cancer Res 13: 1987-1994.
Novelli F, Milella M, Melucci E, Di Benedetto A, Sperduti I, Perrone-Donnorso R, , et al. (2008).
A divergent role for estrogen receptor-beta in node-positive and node-negative breast cancer
classified according to molecular subtypes: an observational prospective study. Breast Cancer Res
10: R74.
Ravo M, Mutarelli M, Ferraro L, Grober OMV, Paris O, Tarallo R, et al. (2008). Quantitative
expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor
biopsies by oligonucleotide microarrays. Lab Invest 88: 430-440.
Shaaban AM, O'Neill PA, Davies MP, Sibson R, West CR, Smith PH, et al. (2003). Declining
estrogen receptor-beta expression defines malignant progression of human breast neoplasia. Am J
Surg Pathol 27: 1502-1512.
Skliris GP, Parkes AT, Limer JL, Burdall SE, Carder PJ, Speirs V (2002). Evaluation of seven
oestrogen receptor beta antibodies for immunohistochemistry, western blotting, and flow cytometry
in human breast tissue. J Pathol; 197:155-62.
Vocaturo A, Novelli F, Benevolo M, Piperno G, Marandino F, Cianciulli AM, , et al. (2006).
Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and
ThinPrep-processed breast cancer fine-needle aspirates: a sensitive and practical method in the
trastuzumab era. Oncologist 11: 878-886.
Supplementary Table S1. Relationship between ERβ expression and bio pathological variables
Variable
Histotype
Invasive ductal carcinoma
Invasive lobular carcinoma
pT
T1
T2
T 3-4
pN
Negative
Positive
G
1-2
3
ERα
Negative (≤10%)
Positive (>10%)
PgR
Negative (≤10%)
Positive (>10%)
HER2
Negative (0/1+)
Positive (2+/3+)
Ki-67
Low (≤15%)
High (>15%)
N° of cases (%) ERβ+ (%) ERβ- (%) p-value 1
37 (92.5)
3 (7.5)
20 (54)
2 (67)
17 ( 46)
1 (33)
0.673
28 (70)
9 (22.5)
3 (7.5)
18 (64)
3 (33)
1 (33)
10 (36)
6 (67)
2 (67)
0.197
27 (67.5)
13 (32.5)
21 (78)
1 (8)
6 (22)
12 (92)
<0.0001
29 (72.5)
11 (27.5)
19 (66)
3 (27)
10 (34)
8 (73)
0.03
3 (7.5)
37 (92.5)
2 (67)
20 (54)
1 (33)
17 (46)
0.67
9 (22.5)
31 (77.5)
6 (67)
16 (52)
3 (33)
15 (48)
0.43
28 (70)
12 (30)
15 (54)
7 (58)
13 (46)
5 (42)
0.78
24 (60)
15 (62.5)
16 (40)
7 (44)
9 (37.5)
9 (56)
0.24
pT: Tumor size; pN: Lymphnodes; G: Grading; ERα: Estrogen Receptor alfa; ERβ: Estrogen
Receptor beta; PgR: Progesterone Receptor.
1 2 test
Supplementary Table S2. Sixty-seven miRNAs differentially expressed in ER positive vs in
ER negative breast tumors
miRNA
Foldchange
p-value
miRNA
1,55
-1,23
-1,36
-1,76
1,61
-1,50
1,30
1,31
1,51
-2,47
0,03300
0,01330
0,04961
0,00478
0,00491
0,00436
0,01662
0,01692
0,00260
0,01408
HS_76
hsa-miR-106a:9.1
hsa-miR-1248
hsa-miR-1273
1,37
-1,45
1,60
-1,21
3,65
0,01582
0,02278
0,03216
0,04955
0,01661
hsa-miR-1285
hsa-miR-129*
hsa-miR-1300
hsa-miR-1301
hsa-miR-1304
hsa-miR-1321
hsa-miR-138-1*
hsa-miR-146b-5p
hsa-miR-155
hsa-miR-181b
hsa-miR-19b
hsa-miR-200b*
hsa-miR-202
hsa-miR-21*
hsa-miR-216a
hsa-miR-300
hsa-miR-30a
hsa-miR-30a*
hsa-miR-323-3p
2,20
-1,45
1,63
-1,34
1,90
-1,30
1,89
-1,24
-1,29
-1,22
1,24
1,23
1,23
-1,26
1,51
1,30
1,25
1,47
-1,75
0,04450
0,01608
0,01434
0,01527
0,01300
0,02943
0,02667
0,01200
0,04829
0,02224
0,03712
0,01470
0,03878
0,04976
0,04616
0,00264
0,04286
0,03410
0,01749
HS_108.1
HS_1341
HS_170 1
HS_240 1
HS_251.1 1
HS_266.1
HS_276.1
HS_286_a (hsa-miR-758)
HS_40
HS_44.1
HS_69 a
a
1
Foldchange
p-value
hsa-miR-329
hsa-miR-337-5p
hsa-miR-342-5p
hsa-miR-34b
hsa-miR-367
hsa-miR-409-3p
hsa-miR-424*
hsa-miR-431
hsa-miR-450b-3p
hsa-miR-454*
-1,57
-1,48
1,33
-1,64
1,20
-1,45
-1,56
-2,16
-1,36
-2,09
0,04835
0,03259
0,00626
0,02189
0,01750
0,03871
0,03287
0,00322
0,04701
0,01347
hsa-miR-487b
hsa-miR-491-5p
hsa-miR-499-3p
hsa-miR-505
hsa-miR-518e
hsa-miR-518f/d-5p/520c5p/526a
hsa-miR-520d-3p
hsa-miR-548d-3p
hsa-miR-616
hsa-miR-616*
hsa-miR-625*
hsa-miR-662
hsa-miR-663b
hsa-miR-7
hsa-miR-708*
hsa-miR-801:9.1
hsa-miR-873
hsa-miR-886-3p
hsa-miR-922
hsa-miR-92b*
hsa-miR-941
solexa-2526-361
solexa-7764-108
-1,46
1,32
1,32
-1,43
-1,28
0,03464
0,04791
0,03770
0,01903
0,04002
1,36
1,61
-1,28
-1,42
-1,44
1,43
1,41
-1,57
-1,51
-1,50
1,45
1,49
-1,65
-1,22
-1,60
-1,46
1,52
2,33
0,01679
0,01742
0,03891
0,01638
0,02911
0,02250
0,04948
0,04488
0,01951
0,01346
0,04721
0,02599
0,03621
0,01549
0,02824
0,02612
0,02596
0,00998
The miRNA probe in the array matches multiple loci in the genome
Bold: miRNAs differentially expressed also in ER–positive vs ER–negative hormone-responsive
human breast cancer cells in culture
Supplementary Tables S3. Estrogen receptor binding sites within 10kb from loci encoding ER-responsive miRNAs
S3-A
miRNA
differentially
expressed in
ER+ vs ERcells
Closest ER
binding site
upstream1
Closest ER
binding site
downstream1
Closest ER
binding site
upstream1
Closest
ERbinding site
downstream1
Closest ER
binding site
upstream1
ER+ cells
Closest ER
binding site
downstream1
ER- cells
0 (overlapping)
HS_166.1
–
–
–
–
–
HS_266.1
0 (overlapping)
0 (overlapping)
0 (overalapping)
0 (overlapping)
(chr17: 5232117-52323792)
(chr17: 5232117-52323792)
(chr17:523287-52324146)
(chr17:523287-52324146)
–
–
–
–
–
–
-5986
–
–
–
–
–
–
-5498
–
-5459
–
–
–
–
–
–
hsa-let-7a (hsa-let7a-3 gene)
hsa-miR-1257
hsa-miR-1285
(miR-1285-2 gene)
hsa-miR-181c*
hsa-miR-23a*
hsa-miR-27a*
hsa-miR-24-2*
hsa-miR-23b*
hsa-miR-27b*
hsa-miR-24-1* /
hsa-miR-189:9.1
hsa-miR-30a*
(chr2:70339139-70339830)
-3032
(chr19:13843046-13843481)
–
(chr2:70339100-70339829)
-2998
(chr19:13842946-13843515)
(chr22:44880878-44881307)
(chr11:64368360-64368992)
2970
(chr20:59958529-59959027)
-7039
401
-7082
302
-6791
191
(chr19:13815512-13815956)
(chr19:13807463-13808000)
(chr19:13815555-13816119)
(chr19:13807490-13808099)
(chr19:13815264-13816060)
(chr19:13807532-13808210)
-7181
254
-7224
155
-6933
44
(chr19:13815512-13815956)
(chr19:13807463-13808000)
(chr19:13815555-13816119)
(chr19:13807490-13808099)
(chr9: 13815264-13816060)
(chr19:13807532-13808210)
-7939
101
-7382
2
0 (overlapping)
0 (overlapping)
(chr19:13815512-13815956)
(chr19:13807463-13808000)
(chr19:13815555-13816119)
(chr19:13807490-13808099)
(chr19:13807532-13808210)
(chr19:13807532-13808210)
-5704
4878
-5667
4698
-5716
4437
(chr9:96881097-96881607)
(chr9:96892285-96893483)
(chr9:96881256-96881644)
(chr9:96892105-96892943)
(chr9:96880936-96881595)
(chr9:96891841-96892253)
-5941
4641
-5904
4461
-5953
4197
(chr9:96881097-96881607)
(chr9:96892285-96893483)
(chr9:96881256-96881644)
(chr9:96892105-96892943)
(chr9:96880936-96881595)
(chr9:96891841-96892253)
-6517
4094
-6480
3914
-6529
3650
(chr9:96881097-96881607)
(chr9:96892285-96893483)
(chr9:96881256-96881644)
(chr9:96892105-96892943)
(chr9:96880936-96881595)
(chr9:96891841-96892253)
0 (overlapping)
0 (overlapping)
(chr6:72169848-72170426)
(chr6:72169848-72170426)
–
–
–
–
hsa-miR-32*
hsa-miR-330-5p
hsa-miR-338-3p
hsa-miR-365
–
-2139
(chr19:50836324-50836783)
-9728
(chr17:76724072-76724572)
-946
(miR-365-2 gene)
(chr17: 26925148-26925597)
hsa-miR-548d3p (miR-548d-2
–
gene)
hsa-miR-616*
hsa-miR-663b
hsa-miR-935
–
9615
–
(chr9:110838378-110838715)
9586
(chr9:110838112-110838744)
–
–
–
–
–
–
–
–
–
–
–
–
-743
–
-936
–
–
(chr17:26924937-26925800)
3158
–
(chr17:62894141-62894909)
–
-2852
(chr12:56202161-56202718)
3280
(chr17:62894161-62894787)
–
(chr17:26925077-26925607)
–
-2791
(chr12:56202100-56202671)
3304
(chr17:62894122-62894763)
–
-531
823
-2027
603
-931
811
(chr2:132731654-132732045)
(chr2:132729595-132730186)
(chr2:132733150-132733597)
(chr2:132729599-132730406)
(chr2:132732054-132732613)
(chr2: 132729555-132730198)
–
–
–
252
–
Bold: miRNAs differentially expressed in ER+ vs ER- breast tumors
1
Distance in bps
(chr19:59177715-59178175)
361
(chr19:59177824-59178301)
S3-B
miRNA
differentially
expressed in
ER+ vs ERbreast tumors
HS_266.1
hsa-miR-1285
(hsa-miR-1285-2
gene)
hsa-miR-129*
(hsa-miR-129-1 gene)
hsa-miR-181b
hsa-miR-21*
hsa-miR-30a
Closest ER
binding site
upstream1
0 (overlapping)
Closest ER
binding site
downstream1
ER+ cells
0 (overlapping)
0 (overlapping)
(chr17:5232117-52323792)
(chr17:5232117-52323792)
-5498
–
(chr2:70339139-70339830)
Closest ER
binding site
upstream1
–
–
–
–
Closest
ERbinding site
downstream1
0 (overlapping)
–
–
–
–
–
–
–
–
-320
–
–
(chr17:523287-52324146)
(chr17:523287-52324146)
-5459
(chr2:70339100-70339829)
(chr1:197095054-197095581)
3028
(chr7:127638260127638719)
–
0 (overlapping)
0 (overlapping)
0 (overlapping)
0 (overlapping)
0 (overlapping)
0 (overlapping)
(chr17:55273240-55273891)
(chr17:55273240-55273891)
(chr17:55272893-55273703)
(chr17:55272893-55273703)
(chr17:55273348-55273732)
(chr17:55273348-55273732)
0 (overlapping)
0 (overlapping)
(chr6:72169848-72170426)
(chr6:72169848-72170426)
–
–
–
–
–
–
–
–
-839
–
0 (overlapping)
0 (overlapping)
(chr6:72169848-72170426)
hsa-miR-342-5p
–
–
–
–
hsa-miR-409-3p
–
–
–
–
hsa-miR-487b
–
–
–
–
hsa-miR-520d-3p
–
–
–
–
3158
–
hsa-miR-548d-3p
Closest ER
binding site
downstream1
ER- cells
(chr6:72169848-72170426)
hsa-miR-30a*
Closest ER binding
site upstream1
(hsa-miR-548d-2
gene)
–
hsa-miR-616
–
chr17:62894141-62894909)
–
-2855
(chr12:56202161-56202718)
3280
(chr17:62894161-62894787)
–
(chr14:99644461-99644906)
-9836
(chr14:100591066-100591554)
–
-2535
(chr19:58912177-58912627)
–
-2791
(chr12:56202100-56202671)
–
8438
(chr14:100591066-100591554)
–
3304
(chr17:62894122-62894763)
–
-2855
–
–
hsa-miR-625*
–
–
–
hsa-miR-663b
-531
823
-2027
(chr2:132731654-132732045)
(chr2:132729595-132730186)
(chr2:132733150-132733597)
(chr2:132729599132730406)
hsa-miR-922
–
–
–
–
hsa-miR-92b*
–
–
–
–
–
-6039
–
-5975
–
–
–
–
–
–
–
–
–
hsa-miR-941-1
hsa-miR-941-2
hsa-miR-941-3
(chr20:62014658-62015199)
-6343
(chr20:62014658-62015199)
-6458
(chr20:62014658-62015199)
–
–
–
-2791
hsa-miR-616*
(chr12:56202161-56202718)
–
603
(chr20:62014664-62015263)
-6282
(chr20:62014664-62015263)
-6394
(chr20:62014664-62015263)
Bold: miRNAs regulated by ER in hormone-responsive human breast cancer cells in culture
1
Distance in bps
(chr12:56202100-56202671)
-118
(chr14:65007006-65007455)
–
–
-931
811
(chr2:132732054-132732613)
(chr2:132729555-132730198)
-8011
–
(chr3:198893855-198894243)
7901
(chr1:153439588-153440193)
Supplementary Table S4. Primers used for qPCR analyses
ERβ binding Site
Chr Forward Primer (5’-3’)
Coordinates
Reverse primer (5’-3’)
Coordinates
ERG7838/39
6
TTGAAGTCCGAGGCAGTAGG
72169957-77
ATCCTCGACTGGAAGCTGTG
72170033-14
ERG9242
9
TGCCACTTCAGCACTTCAAA
96881191-210
CCCTCTGAGAACGGGTGTTA
96881278-59
ERG9243
9
CAGCCCCAGGACTTAAAGG
96894350-69
TGGGAGGCTCTGGTCTTTAC
96894411-392
pS2/TFF1
21
CTAGACGGAATGGGCTTCAT
42660162-43
GCTTGGCCGTGACAACAGTG
42660075-94
pri-miR
Chr Forward Primer (5’-3’)
Coordinates
Reverse primer (5’-3’)
Coordinates
TTCAGCTTTGTAAAAATGTATCAAAGAG
AT
TGCACCTGTTCTCCAATCTGC
72169875-904
Coordinates
Reverse primer (5’-3’)
Coordinates
pri-miR-30a
6
pri-miR-23b∼24-1
9
pre-miR
ATTGCTGTTTGAATGAGGCTTCAGTACTT 72170175-46
T
TCACATTGCCAGGGATTACCA
96887369-89
Chr Forward Primer (5’-3’)
96887459-39
pre-miR-30a
6
ATCCTCGACTGGAAGCTGTG
72170033-14
CTGCAAACATCCGACTGAAA
72169979-98
pre-miR-23b
9
AGGTGCTCTGGCTGCTTG
96887314-31
GTCGTGGTTGCGTGGTAA
96887401-384
pre-miR-24-1
9
CTCCGGTGCCTACTGAGC
96888124-41
TCCTGTTCCTGCTGAACTGA
96888190-71
pre-miR-27b
9
TGCAGAGCTTAGCTGATTGG
96887563-82
GTGAACAAAGCGGAAACCA
96887612-594
RPLP0
15 GGCGACCTGGAAGTCCAACT
119122952-33
CCATCAGCACCACAGCCTTC
119121560-79
Supplementary Figure Legends
Supplementary Figure S1. Heatmap summarizing the effects of the estrogen responsive
transcriptome of MCF-7 cell clones, showing changes in mRNA expression (fold-change) of cell
clones grown in standard culture condition.
Supplementary Figure S2. (A) Real-Time PCR validation of 10 miRNAs identified by
microarraya analysis in wt, N-TAP-ERβ and C-TAP-ERβ cells. (B) Correlation graph between
fluorescence intensity log2 value from microarray data and real-time rtPCR log2 data in ERβ+ and
ERβ- cells.
Supplementary Figure S3. (A) Heatmap and (B) Graphics showing miRNAs miR-30a* and miR23b*/-27b*/-24-1* fluorescence levels compared with those of their complementary strand in CTAP-ERα and TAP-ERβ cells upon stimulation with E2 for the indicated times. In panel (A)
fluorescence levels are reported as log2.
Supplementary Figure S4. Functional analysis according to Gene Ontology of predicted mRNA
targets of miRNAs differentially expressed between ERβ+ and ERβ- cells according to miRBase
(release 12).
Supplementary Figure S5. Immunohistochemistry of representative BC biopsies. (A) ERβ
negative, (B) and (C) ERβ positivity: 30-50%, (D) ERβ positivity: 80-90%.
Supplementary Figure S6, (A) Genome Browser view of ERβ and ERα binding sites within 10 kb
upstream or downstream from miR-23b/27b/24-1 cluster and ORF (c9orf3) in which the cluster is
present in Chromosome 9. (B) and (C) Validation of ERβ binding sites by ChIP and real time PCR
in wt, N-TAP-ERβ and C-TAP-ERβ cells stimulated for 45 min with 10-8M E2.
Figure S1
Figure S2
Figure S3
Figure S4
Figure S5
Figure S6