supplementary materials for - science signaling · 6/10/2011 · supplementary materials for ......

www.sciencesignaling.org/cgi/content/full/4/177/ra41/DC1

Supplementary Materials for

TRPS1 Targeting by miR-221/222 Promotes the Epithelial-to-Mesenchymal Transition in Breast Cancer

Susanna Stinson, Mark R. Lackner, Alex T. Adai, Nancy Yu, Hyo-Jin Kim, Carol

O’Brien, Jill Spoerke, Suchit Jhunjhunwala, Zachary Boyd, Thomas Januario, Robert J. Newman, Peng Yue, Richard Bourgon, Zora Modrusan, Howard M. Stern, Søren Warming, Frederic J. de Sauvage, Lukas Amler, Ru-Fang Yeh, David Dornan*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 14 June 2011, Sci. Signal. 4, ra41 (2011)

DOI: 10.1126/scisignal.2001538 The PDF file includes:

Materials and Methods References Fig. S1. Plot of miR-221, miR-222, and miR-200c expression across luminal and basal-like cell lines. Fig. S2. Triple-negative breast cancers have more abundant miR-221/222 than ER/PR breast cancers. Fig. S3. Phase contrast and immunofluorescence images of MCF10A cells transfected with miR-221/222. Fig. S4. miR-221/222 abundance correlates with that of the mRNA encoding vimentin. Fig. S5. miR-221/222 abundance inversely correlates with that of the mRNA encoding E-cadherin. Fig. S6. miR-221/222 abundance inversely correlates with that of the mRNA encoding TRPS1. Table S1. Luminal-basal specific differential gene expression. Table S2. miR-221/222 gene targets down-regulated in basal-like subtype and in MCF10A cells overexpressing miR-221/222. Table S3. Filtered candidate target genes for miR-221/222 based on CRS.

Stinson et al. Supplemental Material

Page 1 of 16

Supplemental Materials and Methods

Luciferase Reporters

The FOS-like antigen 1 (FOSL1) binding site upstream of the miR-221/222 gene cluster,

as well as mutated binding sites, was cloned into the pGL3 Promoter Vector (Promega).

Sense and antisense oligonucleotides containing the binding site or mutated binding site

(underlined) and 24 nucleotides on each side were annealed and cloned between nheI and

xhoI (boxed). FOSL1 binding site sense: 5’-

ACGCGTGCTAGCTGTGTGGTGGCTGGGCACTGCAAAATGAGTCAGTTCCTGC

AGTTTGGTTACAAGTTCCTCGAGATCTGC-3’ antisense: 5’-

GCAGATCTCGAGGAACTTGTAACCAAACTGCAGGAACTGACTCATTTTGCAG

TGCCCAGCCACCACACAGCTAGCACGCGT-3’. FOSL1 mutated binding site sense:

5’-

ACGCGTGCTAGCTGTGTGGTGGCTGGGCACTGCAAAATGGGCCCGTTCCTGC

AGTTTGGTTACAAGTTCCTCGAGATCTGC-3’ antisense: 5’-

GCAGATCTCGAGGAACTTGTAACCAAACTGCAGGAACGGGCCCATTTTGCAG

TGCCCAGCCACCACACAGCTAGCACGCGT-3’. Cells were co-transfected with

luciferase reporters (200 ng/ml final concentration), phRL-CMV transfection control

plasmid (Promega, 50 ng/ml final concentration), and miRNA inhibitors or mimics. 18 to

72 hours after transfection, Dual Glo (Promega) was used to measure luciferase activity.

Retrieval and Construction of pMir TRPS1 3’UTR


Page 2 of 16

A human bacterial artificial chromosome (BAC) clone containing the 3’UTR of TRPS1,

CTD-2122H18, was obtained from Invitrogen. To introduce the recombineering

machinery into the bacteria containing the BAC, the cells were electroporated with

pSIM18(1). The retrieval vector for the 3’UTR was amplified using pBR322 as template.

Long oligos were used, with ~80 bp serving as homology arms for the retrieval of the 3’

UTR. The primer sequences are (italics corresponds to pBR322, added SpeI sites are

underlined):

TRPS1 pBR fwd: 5’-

GCAAACAATCTTCAGGCAGCAAAGATGTCTGTTACATCTAAACTTGAATAAT

AAAGTTTTACCACCAGTTACACAACTAGTGATACGCGAGCGAACGTGA-3’;

TRPS1 pBR rev: 5’-

CATAAGACATTACAAGCTATTGAATTCCCATCAAGAAAACCTATTTCTATTTA

ATTGTGCTAAGTGCTAAGGTTACTAGTTTAGACGTCAGGTGGCAC-3’

PCR conditions were 95°C for 30 s, 58°C for 30 s and 72°C for 3 min, for 25 cycles.

Primers were from Integrated DNA Technologies (IDT), and the Expand High-Fidelity

PCR System, Roche Applied Science, was used. After PCR, 1 µl of DpnI was added to

the 50uL reaction for 1 hr at 37°C to remove all plasmid template. The reaction mix was

run on a 1% agarose gel, and the PCR product was purified (GFX kit, GE healthcare) and

eluted in 30ul ddH2O.

Bacteria containing pSIM18 as well as CTD-2122H18 BAC were heat-shocked and made

electrocompetent. 150 ng of the purified retrieval PCR product was electroporated into

the cells using standard settings. After electroporation, the bacteria were recovered in 1

ml LB for 1 hr in a 32°C shaking water bath, then plated on carbenicillin 50 µg/ml LB


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agar and incubated at 37°C overnight. Correct retrieval clones were confirmed by

restriction digest of minprep DNA.

The SpeI fragment containing the TRPS1 3’UTR was then subcloned into the pMir

luciferase vector and checked for correct orientation.

Insertion of seedbox mutation into 3’UTR

An em7-neomycin cassette was amplified with oligos containing 46 bp homology arms

corresponding to the sequence flanking the wildtype seedbox in the 3’UTR. The primer

sequences are (italics corresponds to either em7 or neomycin, added AscI or SwaI sites

are underlined):

TRPS1 em7 AscI fwd: 5’-

GCATTAGAGTCAGTTCTGGCTCTGCCTAGCTGTTTACATTTGCAAAGGCGCGC

CGTTGACAATTAATCATCGGCATAG -3’;

TRPS1 neo SwaI rev: 5’-

ATCTACTGCAATAGAAATAGTTGCTTCATTACCTTGTTTGCTATCAATTTAAA

TCAGAAGAACTCGTCAAGAAGGCG -3’

PCR conditions were 95°C for 30 s, 58°C for 30 s and 72°C for 1 min, for 25 cycles.

After completion of the PCR, 1 ml of DpnI was added to the 50 ml reaction for 1hr at

37°C to remove all plasmid template. The reaction mix was run on a 1% agarose gel, and

the PCR product was purified and eluted in 30 ul of ddH20. pMir luciferase TRPS1

3’UTR plasmid and em7-neo PCR product were then co-transformed into heat-shocked

and electrocompetent SW102 cells (2) using electroporation. The bacteria were recovered

in 1 ml LB for 1 h in a 32°C shaking water bath, and then plated on kanamycin 50 µg/ml


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LB agar and incubated at 32°C overnight. Kan-resistant clones were analyzed by

restriction digest, and the em7-neo selection marker was cut out of a targeted clone by

AscI/SwaI restriction digest. The digest was run out on a 1% gel and the vector was gel

purified. The mutated seedbox with 144bp (5’) and 124bp (3’) flanking homologies to

the TRPS1 3’UTR was synthesized by Blue Heron Biotechnology. Sequence is as

follows (mutation underlined):

CTAGTAAATATTAATGTATTACATTTCAAATAATGGTGCCTGACATATTGAAT

AATTATTTTCTACAGTGTACGTATGCAACAAAGATATTCCATCATGCATTAGA

GTCAGTTCTGGCTCTGCCTAGCTGTTTACATTTGCAAAACTAGCAAACAAGGT

AATGAAGCAACTATTTCTATTGCAGTAGATATCCTTTTGTGTGTGTGTGTGTG

CATTAAAGTTGTAAACGGTAACATGAAACAAATGAAAGTTCTTGCTATAATG

GTATGG

The linearized pMir 3’UTR vector and mutant seedbox synthesis fragment were co-

transformed into heat-shocked and electrocompetent SW102 cells. After electroporation,

the bacteria were recovered in 1 ml LB for 1 h in a 32°C shaking water bath, then plated

on carbenicillin 50 µg/ml LB agar and incubated at 32°C overnight. Correctly modified

clones were identified by restriction digest and confirmed by sequencing.

Cumulative Regulatory Score

The Cumulative Regulatory Score, CRS, is a heuristic to rank the potential cumulative

regulatory effect several miRNAs have on one target gene. CRS was designed to

establish context-dependent miRNA target identification. For example, a candidate list

can be established based on biologically-relevant criteria, such as luminal or basal-

specific gene expression. The score is defined as


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CRSg ! ng,m * tm"# $%m&F

' * tgF

' = CRSg,FF

'

where tm is the t-statistic of the miRNA m; tg is the t-statistic of the target gene g; ng,m is

the number of target sites for m in the 3’UTR of g; the inner sum is over all miRNAs in

the same family F (miRNAs with the same seed sequences); the outer sum is over all

miRNA families that target g. The t-statistic is calculated using the limma package in R

(3). The CRS can be determined for a single family of miRNAs by excluding the outer

sum or for a single miRNA by excluding both sums. For our analysis, we calculated the

CRS for every gene in the context of luminal-specific expression. The number of target

sites, ng,m, draws from TargetScan 3.0 predictions(4) and is equal to 0 if a gene is not

predicted to be a target. Some comforting implications arise from CRS. CRS scores are

better (numerically lower) if g and m are strongly anti-correlated (opposing t-statistics),

and g has more target sites for m. The ng,m scale factor is appropriate because the

additivity of target sites has previously been demonstrated(5). Note that CRS assumes the

seed sequences are independent between miRNAs and families, and also assumes the

miRNA microarray platform can distinguish between family members. As a result, the

most useful conclusions derived from CRS are between families, and not within families

because family members tend to have positively correlated expression.

The CRS estimate produces positive and negative scores because some miRNAs are

positively correlated to genes that are predicted targets whereas others are anti-correlated.

The positive correlation of miRNAs and potential targets could be due to the false


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positive prediction of targets or weak t-Statistics. In either case, we consider positive

CRS scores as a sort of null distribution, whereas negative (and biologically meaningful)

CRS scores represent the mRNA::miRNA combinations of interest. To generate a high

quality list for specific mir-221/222 targets, we performed the following enrichment

strategy. Assuming negative scores were “True” and positive scores were “False”, we

sorted the absolute value of the CRS estimates in descending order, and used precision-

recall analysis to estimate a good candidate cutoff. Precision is the same as positive

predictive value or TP/(TP+FP), whereas recall is the same as sensitivity or true positive

rate, and it is equal to TP/TotalTrues. We used a precision ~70% at ~13.5% recall when

sorting the scores by absolute values. Note that ‘True’ targets (negative CRS scores)

beyond the ~13.5% recall start to become increasingly diluted with ‘False’ targets

(positive CRS scores). This gave 9 top ranked genes that are likely targeted by mir-

221/222: TRPS1, IRX5, ESR1, SHANK2, VASH1, ZNF385, TP53INP1, CROP,

CDKN1B. These 9 genes had negative CRS scores that tended to be much higher than the

distribution of positive CRS scores, and represent a good set of hypotheses to investigate

for future research. Note that ESR1 and CDKN1B have already been confirmed as targets

of miR-221/222.

References:

1. W. Chan, N. Costantino, R. Li, S. C. Lee, Q. Su, D. Melvin, D. L. Court, P. Liu, A recombineering based approach for high-throughput conditional knockout targeting vector construction. Nucleic acids research 35, e64 (2007).

2. S. Warming, N. Costantino, D. L. Court, N. A. Jenkins, N. G. Copeland, Simple and highly efficient BAC recombineering using galK selection. Nucleic acids research 33, e36 (2005).


Page 7 of 16

3. G. K. Smyth, Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 3, Article3 (2004).

4. B. P. Lewis, I. H. Shih, M. W. Jones-Rhoades, D. P. Bartel, C. B. Burge, Prediction of mammalian microRNA targets. Cell 115, 787 (2003).

5. J. G. Doench, C. P. Petersen, P. A. Sharp, siRNAs can function as miRNAs. Genes & development 17, 438 (2003).


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Fig. S1. Plot of miR-221, miR-222, and miR-200c expression luminal and basal-like

cell lines. Blue and red represents expression values from microarrays in luminal or

basal-like cell lines, respectively.

0 2 4 6 8 10 12 14

log2(Expression)

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across


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Fig. S2. Triple negative breast cancers have more abundant miR-221/222 than

ER/PR breast cancers. RNA was extracted from primary breast tumors and abundance

of miR-221/222 was determined by qRT-PCR. Statistical significance was determined

using a Wilcoxon test.

-


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Fig. S3. Phase contrast and immunofluorescence images of MCF10A cells

transfected with miR-221/222. Cells were viewed four days post-transfection with 100

nM of either scrambled control mimic or miR-221/222 mimic in triplicate. Upper panel:

Phase contrast images. Lower left panel: Cells stained with Hoescht nuclear stain (blue)

and Phalloidin-AlexFluor546 (red). Lower right panel: Cells stained with Hoescht

nuclear stain (blue) and vimentin (red).


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Fig. S4. miR-221/222 abundance correlates with that of the mRNA encoding

vimentin. RNA was extracted from primary breast tumors (n = 27, a subset from Figure

S2) and vimentin mRNA abundance was determined. Statistical significance for

correlation was determined by a Spearman rank correlation.


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Fig. S5. miR-221/222 abundance inversely correlates with that of the mRNA

encoding E-cadherin. RNA was extracted from primary breast tumors (n = 27, a subset

from Figure S2) and E-cadherin mRNA abundance was determined. Statistical

significance for correlation was determined by a Spearman rank correlation.


Page 13 of 16

Fig. S6. miR-221/222 abundance inversely correlates with that of the mRNA encoding TRPS1. RNA was extracted from primary breast tumors (n = 27, a subset from Figure S2) and TRPS1

mRNA abundance was determined. Statistical significance for correlation was

determined by a Spearman rank correlation.


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Name logFC t P.Value adj.P.Val B

hsa-miR-222 -6.1389 -7.5218 0.0000 0.0000 9.0457

hsa-miR-221 -6.3296 -7.1339 0.0000 0.0000 8.0806

hsa-miR-146a -4.1826 -5.6077 0.0000 0.0006 4.1256

hsa-miR-203 4.0934 5.1355 0.0000 0.0017 2.8710

hsa-miR-138 -3.4977 -4.9999 0.0000 0.0018 2.5103

hsa-miR-26b 1.7994 4.9926 0.0000 0.0018 2.4909

hsa-miR-34a 2.1894 4.9079 0.0000 0.0019 2.2658

hsa-miR-29a -2.5257 -4.8836 0.0000 0.0019 2.2012

hsa-miR-30a-5p -1.2388 -4.7280 0.0001 0.0025 1.7886

hsa-miR-136 -2.0683 -4.6973 0.0001 0.0025 1.7071

hsa-miR-425 1.5985 4.5497 0.0001 0.0031 1.3170

hsa-miR-100 -3.9965 -4.5229 0.0001 0.0031 1.2462

hsa-miR-30a-3p -2.3252 -4.4706 0.0001 0.0034 1.1087

hsa-miR-155 -3.3616 -4.4239 0.0001 0.0036 0.9860

hsa-miR-375 3.9213 4.2985 0.0002 0.0048 0.6571

hsa-miR-18a-AS -1.2451 -4.1884 0.0002 0.0062 0.3701

hsa-miR-30e-5p -1.1351 -4.0618 0.0003 0.0082 0.0422

Table S1. Luminal-basal specific differential gene expression.

A list of significantly differentially expressed miRNAs according to moderated t-statistic

calculations. Luminal-basal specific is defined in a two category t-test of luminal vs.

basal cell lines.


Page 15 of 16

Table S2. miR-221/222 gene targets down-regulated in basal-like subtype and in

MCF10A cells overexpressing miR-221/222.

A list of genes that are decreased in basal vs. luminal subtype breast cancer cell lines and

in MCF10A cells expressing miR-221/222 mimic. ANOVA was run on 54 basal and

luminal breast cell lines and MCF10A cells treated with miR-221/222 mimic or control

and the list was filtered on miR-221/222 predicted targets based on TargetScan 5.0.

probe ID gene description

gene

symbol

pvalue

(basal vs

luminal)

ratio

(basal vs

luminal)

fold

change

(basal vs

luminal)

pvalue

(MCF10A

mimic vs

control)

ratio

(MCF10A

mimic vs

control)

fold

change

(MCF10A

mimic vs

control)

210239_at iroquois homeobox 5 IRX5 2.32E-08 0.076344 -13.0987 2.21E-05 0.468204 -2.13582

222651_s_at trichorhinophalangeal syndrome I TRPS1 2.13E-09 0.077465 -12.909 8.69E-06 0.146571 -6.82265

243386_at castor zinc finger 1 CASZ1 2.33E-07 0.13 -7.69234 0.007521 0.198786 -5.03053

214855_s_at GTPase activating Rap/RanGAP domain-like 1 GARNL1 1.28E-15 0.201494 -4.96292 0.000341 0.320624 -3.11892

213508_at chromosome 14 open reading frame 147 C14orf147 4.68E-08 0.20276 -4.93193 3.99E-05 0.060915 -16.4163

226045_at fibroblast growth factor receptor substrate 2 FRS2 0.000671 0.251274 -3.97972 0.000212 0.40672 -2.45869

212310_at melanoma inhibitory activity family, member 3 MIA3 0.000295 0.282735 -3.53688 7.51E-05 0.312399 -3.20103

1553227_s_at bromodomain and WD repeat domain containing 1 BRWD1 1.62E-08 0.294848 -3.39157 0.000596 0.326321 -3.06447

209586_s_at prune homolog (Drosophila) PRUNE 3.31E-08 0.38278 -2.61247 8.92E-05 0.246807 -4.05176

225954_s_at midnolin MIDN 0.000987 0.485532 -2.0596 0.001668 0.39944 -2.50351

221744_at WD repeat domain 68 WDR68 2.82E-05 0.521975 -1.9158 7.50E-06 0.437055 -2.28804

227112_at transmembrane and coiled-coil domain family 1 TMCC1 0.005756 0.533137 -1.87569 6.75E-06 0.424676 -2.35474

226119_at protein-L-isoaspartate (D-aspartate) O-methyltransferase domain containing 1 PCMTD1 0.014609 0.580794 -1.72178 0.003747 0.417613 -2.39456

218128_at nuclear transcription factor Y, beta NFYB 0.005503 0.589614 -1.69603 6.48E-05 0.341201 -2.93083

229418_at chromosome 17 open reading frame 63 C17orf63 0.002616 0.591174 -1.69155 0.000325 0.1264 -7.91138

213229_at dicer 1, ribonuclease type III DICER1 0.012694 0.611474 -1.63539 0.004241 0.486179 -2.05686

202550_s_at VAMP (vesicle-associated membrane protein)-associated protein B and C VAPB 0.001983 0.617837 -1.61855 5.52E-05 0.275772 -3.62619

225888_at chromosome 12 open reading frame 30 C12orf30 0.003405 0.649243 -1.54025 2.60E-05 0.38186 -2.61876

212652_s_at sorting nexin 4 SNX4 0.001954 0.653071 -1.53123 0.000141 0.199977 -5.00057

208942_s_at SEC62 homolog (S. cerevisiae) SEC62 0.000505 0.699807 -1.42897 2.06E-05 0.113318 -8.82474

223130_s_at myosin regulatory light chain interacting protein MYLIP 0.047672 0.976139 -1.02444 1.87E-05 0.230185 -4.34433


Page 16 of 16

Gene Symbol logFC t P.Value

TRPS1 -1.2551 -13.892 7.10E-44

IRX5 -0.73186 -8.1007 5.46E-16

CDKN1B -0.65233 -7.2204 5.18E-13

TP53INP1 0.39419 4.3632 1.28E-05

VASH1 -0.18017 -1.9943 0.0461

CROP -0.16178 -1.7907 0.0733

SHANK2 0.16109 1.7830 0.0746

ESR1 -0.15521 -1.7179 0.0858

ZNF385 -0.011407 -0.12626 0.90

Table S3. Filtered candidate target genes for miR-221/222 based on CRS.

A filtered list of significantly differentially expressed miRNAs according to basal

vs. luminal differential expression and downregulation by miR-221/222 in

MCF10A cells. A p-value of <0.05 was required for significance.

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