E-Cadherin Is Transcriptionally Activated via Suppressionof ZEB1 Transcriptional Repressor by Small RNAMediated Gene Silencing
Minami Mazda, Kenji Nishi, Yuki Naito, Kumiko Ui-Tei*
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan

Abstract
RNA activation has been reported to be induced by small interfering RNAs (siRNAs) that act on the promoters of several
genes containing E-cadherin. In this study, we present an alternative mechanism of E-cadherin activation in human PC-3
cells by siRNAs previously reported to possess perfect-complementary sequences to E-cadherin promoter. We found that
activation of E-cadherin can be also induced via suppression of ZEB1, which is a transcriptional repressor of E-cadherin, by
seed-dependent silencing mechanism of these siRNAs. The functional seed-complementary sites of the siRNAs were foundin the coding region in addition to the 39 untranslated region of ZEB1 mRNA. Promoter analyses indicated that E-boxes,
which are ZEB1-binding sites, in the upstream promoter region are indispensable for E-cadherin transcription by the siRNAs.
Thus, the results caution against ignoring siRNA seed-dependent silencing effects in genome-wide transcriptional
regulation. In addition, members of miR-302/372/373/520 family, which have the same seed sequences with one of thesiRNAs containing perfect-complementarity to E-cadherin promoter, are also found to activate E-cadherin transcription.
Thus, E-cadherin could be upregulated by the suppression of ZEB1 transcriptional repressor by miRNAs in vivo.
Citation: Mazda M, Nishi K, Naito Y, Ui-Tei K (2011) E-Cadherin Is Transcriptionally Activated via Suppression of ZEB1 Transcriptional Repressor by Small RNAMediated Gene Silencing. PLoS ONE 6(12): e28688. doi:10.1371/journal.pone.0028688
Editor: Akio Kanai, Keio University, Japan
Received October 14, 2011; Accepted November 13, 2011; Published December 21, 2011
Copyright: ß 2011 Mazda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by grants from MEXT, the Genome Network Project, and Cell Innovation Project (MEXT) to KUT. No additional
external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ktei@bi.s.u-tokyo.ac.jp

Ago in a quasi-helical form to serve as the entry or nucleation site
for small RNAs in the RISCs [14]–[16].
Small RNAs are also known to induce transcriptional genesilencing (TGS). Originally, TGS was reported in plants andyeast [17], [18], in which small RNAs induce silencing of specificgenes containing homologous sequences at the transcriptional
level. Recently, TGS was shown to be induced in human cells byexogenous siRNAs that have sequence complementarity to the
genomic DNA in the promoter regions of several genes
containing Elongation factor-1 (EF1A) in 293FT cells [19]–[21], E-cadherin (CDH1) in HCT116 cells [22], immunodeficiency virus-1 (HIV-1) coreceptor (CCR5) in HEK293T cells[23], progesterone receptor (PR) in T47D cells [24], [25],ubiquitin C (UbC) in 293Gt cells [26], and vascular endothelial
growth factor (VEGF) in HeLa and ARPE-19 cells [27]. Recent
studies demonstrated that TGS is associated with biochemical
modification of critical residues in the histone tails and silent-statechromatin marks, not only at the target promoter region but also
at the downstream region, which in turn is associated withrepressive chromatin structures [20], [23], [28], [29]. The protein
components responsible for TGS were partially identified andfound to contain Ago proteins, DNA methyltransferase 3a(DNMT3a), DNMT1, and histone deacetylase (HDAC1) [20],[21], [23], [25], [26], [29]. Although DNA methylation insiRNA-directed TGS has not been observed in mammalian cells[22], the process is considered to operate through epigenetic
modifications similar to those in plants and yeast.

Introduction
Small RNA molecules, including small interfering RNAs(siRNAs) and microRNAs (miRNAs), are crucial regulators of
posttranscriptional gene silencing (PTGS) referred to as RNA
silencing or RNA interference (RNAi). RNAi is an evolutionarily
conserved pathway induced by siRNAs, 21–23-nucleotide (nt)double-stranded RNAs (dsRNAs) with 2-nt 39 overhangs. The
siRNAs incorporated into cells are transferred to an RNAi effector
complex called the RNA-induced silencing complex (RISC) [1],[2]. The RISC assembles on one of the two strands of the siRNA
duplex and is activated upon the removal of the passenger strand[3]–[5]. The activated RISC is a ribonucleoprotein complex
minimally consisting of the core protein Argonaute (Ago) andsingle-stranded siRNA, which acts as a guide to recognize mRNAs
with sequence complementarity [6]–[8]. Usually, a guide strand ofsiRNA forms base-pairing with perfectly complementary mRNA
sequences in siRNA-mediated RNAi. However, it can also giverise to the silencing of other genes with incompletely complementary sequences. This phenomenon is referred to as the seeddependent off-target effect. A growing body of evidence fromgenome-wide experiments indicates that the target recognition
mechanism is similar to that of miRNA-mediated gene silencing.
The transcripts with sequences complementary to the seed region,
nucleotide positions 2–8 from the 59 end of the RISC-loaded
siRNA strand or miRNA, are mainly reduced [9]–[14]. This isprobably because the seed nucleotides are present on the surface of
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E-Cadherin Activation via Small RNAs

repressors were measured by quantitative RT-PCR (qRT-PCR).
ZEB1 mRNA has at least two seed-complementary sequences ofeach dsEcad (Table S1). Accordantly, the expression level of ZEB1mRNA was significantly reduced to 35–48% by the transfection ofdsEcad215, dsEcad302, and dsEcad640 (Figure 2B). The expression level of SLUG mRNA was also significantly reduced by thetransfection of dsEcad640, but the seed-complementary site todsEcad640 was not found (Table S1), suggesting that the reductionof SLUG expression is not induced by the direct effect ofdsEcad640 but the secondary effect probably caused by the
inhibition of the other upstream regulator(s). In contrast, althoughseed-complementary site of either dsEcad215 or dsEcad302 wasfound in SLUG mRNA, dsEcad215 and dsEcad302 showed weak
but not significant silencing activities. No significant changes in theexpression levels of SNAIL or E12/E47 were observed by any
dsEcads, although both of them have a seed-complementary site todsEcad640 sense strand. These results suggested that all ofdsEcad215, dsEcad302, and dsEcad640 could downregulate theexpression level of a transcriptional repressor, ZEB1, by seeddependent silencing effects accidentally, but could not suppress theother repressors at least in PC-3 cells. The changes of ZEB1
protein levels were also examined by Western blot (Figure 2C). At
24 hour after transfection, no or little downregulation of ZEB1
protein was observed by dsEcads and siRNA targeting to ZEB1
coding region (siZEB1_CDS). The noticeable level of decrease wasobserved by siZEB1_CDS at 48 hour, and the slight decrease was
detected by dsEcad215 or dsEcad640. At 72 hour, ZEB1 protein
was severely repressed by siZEB1_CDS, and the unambiguous
repression by dsEcad215, dsEcad302, or dsEcad640 was also
observed. To investigate the involvement of each repressor in Ecadherin activation, knockdown experiments using siRNAs
targeting each of the transcriptional repressors were performed.
The expression level of E-cadherin was remarkably increased toabout 8-fold when siZEB1_CDS was transfected into PC-3 cells(Figure 2D). However, no or little E-cadherin activation wasobserved by siRNAs against the other repressors, although theexpression levels of these repressors were reduced to 24–55%(Figure 2E). These results suggest that the ZEB1 is one of thepotential candidates to be repressed by the seed-dependentsilencing effects of dsEcad215, dsEcad302, and dsEcad640, andlead to E-cadherin activation in PC-3 cells.

Confusingly, transcriptional activation was shown to be directedby siRNAs with promoter complementarity in a process referred toas RNA activation (RNAa), which is reported to be induced bysiRNAs with perfect complementarity to the promoter sequencesof genes containing E-cadherin or p21WAF/CIP1 in PC-3 andMCF-7 cells, VEGF in HeLa cells [30], and PR in T47D andMCF7 cells [31], [32]. Although the precise mechanism of RNAa
remains uncertain, the process requires the Ago protein and isassociated with the 59 region of the siRNA guide strand
overlapping seed region [31], [32]. These are analogous to thealready known mechanism of RNA silencing. Thus, a possible
alternative pathway of RNAa is considered that siRNA seeddependent silencing on a transcriptional repressor causes theactivation of downstream genes. Recently, siRNA targeted to theHIV-1 LTR promoter was demonstrated to induce gene activation
via an indiscriminate off-target effect by suppressing the C10orf76
transcriptional regulator candidate [33].
In this study, we investigated whether the seed-dependent offtarget effects of siRNA also induce transcriptional activation in ahuman endogenous gene. E-cadherin is a central component ofcell–cell adhesion junctions required for the formation of epithelia,
and shown to be linked to the control of embryonic stem cell
(ESC)-like pluripotency [34], [35]. Since the transcriptional
regulation of E-cadherin has been well studied, we examined theseed-dependent off-target silencing effects of the RNAa-inducible
and E-cadherin promoter-directed siRNAs, dsEcad215, dsEcad320, and dsEcad640 [30], and miR-373 [36], which has thesame seed sequence with dsEcad640, reported previously [30],[36]. Our results showed that one of the alternative mechanisms
for activating E-cadherin expression involves the seed-dependent
off-target effects of these siRNAs on the Zinc finger E-box binding
homeobox 1 (ZEB1) transcriptional repressor of E-cadherin.
When ZEB1 was downregulated by seed-dependent siRNAsilencing, E-cadherin transcription was upregulated. Furthermore,
the members of miR-302a/372/373/520 family miRNAs, whichcontain the same seed sequence with dsEcad640, showed paralleleffects on ZEB1 expression, advocating the possibility that such amechanism is also functional in vivo.

Results
Seed-dependent off-target effects of siRNAs with perfectcomplementarity to the E-cadherin promoter

Identification of functional seed-complementary sites ofdsEcad215, dsEcad302, and dsEcad640 in ZEB1 mRNAIn human PC-3 cells, the siRNAs named as dsEcad215,
dsEcad302, and dsEcad640 (Figure 1A), which contain sequenceswith perfect complementarity to the promoter region of the Ecadherin gene, were reported to induce transcriptional activationof E-cadherin [30]. Upregulation of E-cadherin expression was not
significant at 24 and 48 hour after transfection, but it wassignificantly upregulated certainly confirmed by dsEcad215,
dsEcad302, and dsEcad640 at 72 hour after transfection(Figure 2A). However, the alternative possibility was consideredthat seed-dependent off-target effect of siRNA could activate Ecadherin transcription via suppression of its negative transcription
factor(s). The seed-dependent off-target effect is shown to be
induced by complementarity in the 7-nt seed region mainly
positioned 2–8 from the 59 end of the siRNA guide strand [14],[37], [38]. Among the known negative regulators of E-cadherin,
ZEB1 [39], ZEB2 [40], SNAIL [41], SLUG [42], and E12/E47[43] were found to have one or more complementary sequence(s)of seed regions positioned 2–8 or 1–7 of sense or antisense strandof dsEcad215, dsEcad302, or dsEcad640 in their mRNAs (Table
S1). At 72 hour after transfection of each of the chemicallysynthesized dsEcads into PC-3 cells, mRNA levels of these
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In ZEB1 mRNA, perfect complementary sequences to dsEcad215, dsEcad302, and dsEcad640 were not found. As shown in
Table S1 and Figure 3A, 2 seed-complementary sites (215a-1 and215a-2) of dsEcad215 antisense strand, each one seed-complementary site of dsEcad302 sense strand (302s-1) and its antisense
strand (302a-1), 3 sites of dsEcad640 sense strand (640s-1 and640s-2), and 2 sites of dsEcad640 antisense strand (640a-1 and640a-2) were found in the ZEB1 mRNA. Among them, 215a-1
and 215a-2, 640s-1 and 640s-2, and 640a-1 and 640a-2 weresituated in CDS, and 302s-1, 302a-1, and 640s-3 were in the
ZEB1 39 untranslated region (UTR).
The silencing activities of dsEcad215, dsEcad302, and dsEcad640 against each of seed-complementary sequences wereanalyzed using psiCHECK-SM reporter system. Twenty-threeoligonucleotides containing seed-complementary regions in ZEB1mRNA were inserted into the 39UTR of the Renilla luciferase genein psiCHECK-1 (Figure 3B). Each of these constructs and pGL3Control, which expresses firefly luciferase used as an internal
control, were transfected into HEK293 cells along with each ofdsEcads. One day after transfection, the relative luciferase2

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Figure 1. Structures and sequences of siRNAs and miRNAs. Structures and sequences of siRNAs (A) and miRNAs (B) used in this study. The
same siRNAs shown in Li et al [30], [36], dsEcad215, dsEcad302, and dsEcad640, were chemically synthesized. Their guide strands are shown at thelower sides, and the passenger strands at the upper sides. The miRNAs were synthesized to form same structures shown in miRBase [51]. The miR302a duplex is composed of miR-302a and miR-302*; the miR-373 duplex, miR-373 and miR-373*; and the miR-520c duplex, miR-520c-5p and miR520c-3p. The opposite strand miRNAs of miR-372 and miR-520f are not annotated. The seed region is marked in gray. The Tm values of siRNA seed
duplexes are shown in (A). The solid lines indicate Watson–Crick base pairing; dotted lines indicate G:U wobble pairing.
doi:10.1371/journal.pone.0028688.g001

activities (Renilla luciferase activity/firefly luciferase activity) were
measured (Figure 3B). The significant silencing effects of theantisense strands of dsEcad215 and dsEcad640 were observed. In
both cases, the silencing activities against two different seedcomplementary sites (215a-1 and 215a-2, or 640a-1 and 640a-2)
were similar regardless of different sequences in non-seed regions.
The silencing activity of sense strand of dsEcad302 was alsosignificantly detected. The antisense strand of dsEcad302 showedvery weak and insignificant effect, and little or no activity was
detected by dsEcad640 sense strand. These results indicated thatfunctional RNA strands of dsEcads are dsEcad215 antisense
strand, dsEcad302 sense strand, and dsEcad640 antisense strand.
Their silencing efficiencies may simply reflect the thermodynamic
stabilities in siRNA seed duplexes that determine the efficiencies ofseed-dependent off-target effects of siRNAs [28], (see Figure 1 andDiscussion).
Usually, seed-dependent off-target effects are observed in
mRNAs which have seed-complementary sites in their 39UTRs.
However, the seed-complementary sites of the antisense strands of
dsEcad215 and the dsEcad640 were found in the ZEB1 CDS(Figure 4A). Therefore, we examined whether the repression of
ZEB1 by dsEcad215 and dsEcad640 was indeed the result oftargeting the CDS in the full-length context by luciferase reporter
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assays. The pLuc-CDS reporter, in which the CDS of ZEB1 was
expressed as a fusion protein with Renilla luciferase gene
(Figure 4A), and pLuc-39UTR reporter, in which ZEB1 39UTR
was inserted into the 39UTR of Renilla luciferase gene in
psiCHECK-1 vector, were constructed (Figure 4B). pLuc-CDS
or pLuc-39UTR, and pGL3-Control were transfected into
HEK293 cells with dsEcad215, dsEcad302, or dsEcad640, andthe luciferase activities were measured at 1 day after transfection.
Both dsEcad215 and dsEcad640 suppressed the expression ofRenilla luciferase activities in pLuc-CDS significantly to about
60%, but little effect was observed by dsEcad302, which has noseed-complementary site in the CDS (Figure 4A). In contrast,
dsEcad302, which has functional seed-complementary sites in the
39UTR, reduced luciferase activity of the pLuc-39UTR significantly to about 40%, whereas dsEcad215 and dsEcad640 showed
little or no silencing activities (Figure 4B). These results suggestthat dsEcad215 and dsEcad640 have the capabilities to reduce
ZEB1 expression by acting on the seed-matched sequences in the
CDS as targets, whereas dsEcad302 acts on ZEB1 39UTR.
To confirm that the target sites of dsEcad215 and dsEcad640
are their seed-complementary sites in ZEB1 CDS, the pLuc-CDS
with mutations in each of or all of the seed-complementary sites
were constructed (pLuc-CDS-m215 and pLuc-CDS-m640). The
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Figure 2. The effects of dsEcad215, dsEcad302, and dsEcad640 on E-cadherin and its transcriptional repressors in PC-3 cells. (A)Effects of dsEcad215, dsEcad302, and dsEcad640 (5 nM) on E-cadherin expression at 24, 48, and 72 hour after transfection. (B) Effects of dsEcads onthe expression of E-cadherin transcriptional repressors, ZEB1, SNAIL, SLUG, and E12/E47 at 72 hour after transfection. (C) Western blot analyses of the
ZEB1 protein expression at 24, 48, and 72 hour after transfection. (D) Effect of knockdown of each transcriptional repressor on E-cadherin activation at
72 hour after transfection. (E) Knockdown efficiency of each siRNA. P-values were determined by Student’s t-test (&; P,0.01, *; P,0.05).
doi:10.1371/journal.pone.0028688.g002

that of wild-type pLuc-CDS (53%). However, the silencing activityof dsEcad640 was completely inhibited when pLuc-CDS with
mutations in both seed-complementary sites of dsEcad640 (pLucCDS-m640-1+2) was used (Figure 5B). These results clearly
demonstrated that the seed-complementary sites of dsEcad215 anddsEcad640 in the ZEB1 CDS function as target sites of seeddependent silencing.

pLuc-CDS with mutations in each one of two seed-complementary sites of dsEcad215 antisense strand (pLuc-CDS-m215-1 andpLuc-CDS-m215-2) were transfected with dsEcad215 along with
control pGL3-Control, the relative luciferase activities weresignificantly reduced but slightly weak (64 and 79%, respectively)
compared to the result of wild-type pLuc-CDS (56%). However,the silencing activity was almost completely eliminated whenpLuc-CDS with mutations in both sites (pLuc-CDS-m215-1+2)
was used (Figure 5A). Similarly, the apparent silencing activities by
dsEcad640 were observed when each of two seed-complementarysites of dsEcad640 in the pLuc-CDS was mutated (pLuc-CDSm640-1 and pLuc-CDS-m640-2) at the low levels (relative
luciferase activities were 77 and 86%, respectively) compared to
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E-cadherin promoter assay
To examine the regulatory mechanisms of dsEcads on Ecadherin transcription, promoter analysis was carried out. We
generated two types of E-cadherin promoter-driven firefly
luciferase expression constructs: proE-cad178-Luc and proE4

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Figure 3. The seed-complementary sites of dsEcads in ZEB1 mRNA and silencing efficiencies of dsEcads against seedcomplementary regions. (A) The seed complementary sites in the ZEB1 mRNA. In the upper panel, the sites complementary to the seedsequences of dsEcad215 antisense strand (215a-1 and -2) are indicated by red inversed triangles, that of the dsEcad302 sense strand (302s-1) by light
blue, that of the dsEcad302 antisense strand (302a-1) by dark blue, those of the dsEcad640 sense strand (640s-1 to -3) by light green, and thedsEcad640a antisense strand (640a-1 and -2) by dark green. The numeral in each inversed triangle indicates the serial number of the seed
complementary site of each dsEcad counting from the 59 end of the ZEB1 mRNA. In the lower sequences, the black sequences indicate the seedcomplementary target sequences in ZEB1 mRNA. The left numbers in the inverted triangles correspond to those shown on ZEB1 mRNA in the upper
panel. The solid lines, Watson–Crick base pairing. Dotted lines, G:U wobble pairing. The numerals in parentheses indicate the corresponding positions
to 59 end of each siRNA in ZEB1 mRNA, counting from the 59end of the transcriptional start site (+1). (B) The silencing activity against each seedcomplementary site. The upper panel indicates the structure of psiCHECK-SM containing 23-bp ZEB1 mRNA region with seed-complementary
sequence. The lower panel shows the relative luciferase activity (Renilla luciferase activity/firefly luciferase activity) by the transfection of dsEcad215,
dsEcad302, and dsEcad640. P-values were determined by Student’s t-test (&; P,0.01, *; P,0.05).
doi:10.1371/journal.pone.0028688.g003
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Figure 4. Reporter analyses using pLuc-CDS and pLuc-39UTR. (A) Structure of pLuc-CDS. Two seed-complementary sites of the dsEcad215
antisense strand and dsEcad640 antisense strand are shown by red and dark green inverted triangles, respectively. Orange triangle indicates thecomplete-complementary target site of siZEB1_CDS. (B) Structure of the pLuc-39UTR. Seed-complementary site of dsEcad302 sense strand is shownby light blue inverted triangle. Yellow triangle indicates the complete-complementary site of siZEB1_39UTR. The silencing activities by thetransfection of each siRNA and miRNA are shown as Renilla luciferase activity/firefly luciferase activities. The numerals in triangles show thecorresponding sites shown in Figure 3. P-values were determined by Student’s t-test (&; P,0.01, *; P,0.05).
doi:10.1371/journal.pone.0028688.g004

at 2215, 2302, and 2640 bp additively. Both of them containtwo E-boxes, which are ZEB1-binding sites, situated at 224 bp
and 274 bp [39]. Furthermore, E-box-mutated constructs werealso generated by site-directed mutagenesis. E-box consensus
sequence is CANNTG. The E-box sequences of CACCTG at

cad670-Luc. proE-cad178-Luc contains E-cadherin promoter
region from 2178 to +92, which is known to be a minimum
region to exhibit promoter activity [41]. proE-cad670-Luc
contains the region from 2670 to +92, which contains
complementary sites of dsEcad215, dsEcad302, and dsEcad640
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Figure 5. Reporter analyses of pLuc-CDS containing mutations in the seed-complementary sites. (A) Structure of the pLuc-CDS, and twoseed-complementary sites of dsEcad215 antisense strand. The silencing activities by the transfection of dsEcad215 on wild-type pLuc-CDS (WT), pLucCDS-m215-1, -2, and -1+2 are shown as Renilla/firefly. (B) Structure of the pLuc-CDS and two seed-complementary sites of the dsEcad640 antisense
strand. The silencing activities by dsEcad640, miR-302a, miR-372, miR-373, miR-520c, and miR-520f on wild-type pLuc-CDS (WT), pLuc-CDS-m640-1, -2,
and -1+2 are shown as Renilla/firefly.
doi:10.1371/journal.pone.0028688.g005

224 bp and CAGGTG at 274 bp were simultaneously mutatedto AACCTA and AAGGTA, and designated as proE-cad178-LucmEbox and proE-cad670-Luc-mEbox, respectively. Then, PC-3
cell lines stably transfected with each reporter construct were
established. The stable integration of these reporters was
ascertained by genomic PCR using purified genome DNA(Figure 6A). At 3 days after transfection of dsEcad215, dsEcad302,
dsEcad640, or siZEB1 into these cells, firefly luciferase activities
were measured. In all reporter constructs, transfection of siZEB1
into the cells stably transfected with proE-cad178-Luc or proEcad670-Luc increased luciferase activities efficiently up to
approximately tenfold compared to the results of siControl(Figure 6B, and D). However, no or little increases of luciferase
activities were observed in the cells containing proE-cad178-LucmEbox or proE-cad670-Luc-mEbox (Figure 6C, and E). Similarly,
dsEcad215, dsEcad302, and dsEcad640 increased luciferase
activities in the cells containing proE-cad178-Luc or proEcad670-Luc up to 2,4-fold (Figure 6B, and D), but the increase
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of luciferase activity was not observed in the E-box-mutatedreporter-containing cells (Figure 6C, and E). These results
suggested that dsEcad215, dsEcad302, and dsEcad640 increased
E-cadherin transcription through E-box binding protein(s) withseed-complementarity. One of such candidate genes was presumed
to be ZEB1.
Meanwhile, although E-boxes are mutated in proE-cad670mEbox (Figure 6E), the perfect complementary sites of dsEcad215,
dsEcad302, and dsEcad640 are exist in this reporter. No of little
increase of luciferase activities by the transfection of dsEcads wasobserved, indicating that perfect complementary sites of dsEcads
in ZEB1 promoter are not functional in our reporter assay system.

Increase of E-cadherin expression via suppression of the
ZEB1 transcription factor by miR-302/372/373/520 family
miRNAs
The seed sequence of dsEcad640 antisense strand (AAGUGCU)
is same as those of the miR-302/372/373/520 miRNA family
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Figure 6. Promoter assay of E-cadherin. (A) Establishment of stable transfectants of control pGL3-Basic (lane1), proE-cad178-Luc (lane2), proEcad178-Luc-mEbox (lane3), proE-cad670-Luc (lane4), and proE-cad670-Luc-mEbox (lane5) confirmed by genomic PCR. PCR primer set of primer-F1
and primer-R1 (see B and C) detects 1970 bp of proE-cad178-Luc, proE-cad178-Luc-mEbox, proE-cad670-Luc, and proE-cad670-Luc-mEbox. The
primer set of primer-F2 and primer-R1 (see D and E) can detect 2462 bp of proE-cad670-Luc and proE-cad670-Luc-mEbox. (B–D) Structures of
luciferase reporters containing E-cadherin promoters, and the effects of siRNA/miRNA for each reporter. Structures of proE-cad178-Luc containing theE-cadherin promoter region from 2178 to +92 (B), proE-cad178-Luc-mEbox, in which the two upstream E-boxes were mutated (C), proE-cad670-Luc
containing the E-cadherin promoter region from 2670 to +92 (D), proE-cad670-Luc-mEbox, in which the two upstream E-boxes were mutated (E).
The effects of dsEcad215, dsEcad302, dsEcad640, miR-302a, miR-372, miR-373, miR-520c, miR-520f, and siZEB1_CDS were determined using stablytransfected cells with each luciferase reporter, and shown as Renilla/firefly. Note that perfect-complementary sites of dsEcad215, dsEcad302, anddsEcad640 were located at 2215 (red), 2302 (blue), and 2640 bp (green), respectively in proE-cad670-Luc (D) and proE-cad670-Luc-mEbox (E) but
not in proE-cad178-Luc and proE-cad178-Luc-mEbox. Black boxes indicate wild-type E-boxes, which nucleotide sequences are described below the Eboxes. White boxes represent mutated E-boxes, in which the substituted nucleotides are shown in red characters.
doi:10.1371/journal.pone.0028688.g006
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members, miR-302a, miR-372, miR-373, miR-520a-3p, and miR520f, although the seed sequence of miR-520f is shifted by 1 nt to
1–7 nt from 2–8 nt (Figure 1B). As shown above, the seedcomplementary sites of dsEcad640 were found in ZEB1 CDS, andexpected to regulate E-cadherin expression. Then, the effects on
ZEB1 CDS were determined by the transfection of miR-302a,
miR-372, miR-373, miR-520c, and miR-520f duplexes along with
pLuc-CDS and pGL3-Control into PC-3 cells (Figure 1). In thesimilar fashion as shown by dsEcad640, the transfection of thesemiRNAs showed apparent silencing activities on Renilla luciferase
gene fused with ZEB1 CDS (Figure 4A), but no or little silencing
activities were observed when pLuc-39UTR was co-transfected(Figure 4B). Furthermore, the reduction of relative luciferase
activities by the transfection of these miRNAs with pLuc-CDS
were partially repressed by introducing mutations into each of two
seed-matched sites of dsEcad640 antisense strand, and thesilencing activity was almost perfectly eliminated when both sites
were simultaneously mutated (Figure 5B). Thus, the members ofthe miR-302/372/373/520 miRNA family were revealed to beable to reduce the expression of ZEB1 by targeting its CDS.
In the promoter analyses, miR-302a, miR-372, miR-373, miR520c, and miR-520f duplexes showed the effects to increase theexpression of proE-cad178-Luc and proE-cad670-Luc (Figure 6B,
and D). However, no or little increases in luciferase activities wereobserved when the reporters with mutated E-boxes were used
(Figure 6C, and E). These results suggest that members of themiR-302/372/373/520 miRNA family activate E-cadherin transcription via repression of ZEB1 transcriptional factor in an
analogous fashion to dsEcad640.

0.55 (Figure 7). The downregulated levels of transcripts with thecommon seed-complementarities were low (P#10214); however,those with own seed sequence-complementary sequence, AAGCACU, was high (P#10257), indicating that the target recognitionof miR-520f is slightly fluctuated.
To confirm the reliability of the microarray data, we analyzed
total of 48 transcripts, 8 transcripts from each of 6 transfectants, by
qRT-PCR (Figure S3). The expression levels estimated by qRTPCR were essentially identical to those obtained in the microarray
analysis, with an estimated correlation coefficient of 0.89.

Discussion
E-cadherin is reported to be transcriptionally activated by thedsEcad215, dsEcad302, and dsEcad640 by targeting to thepromoter directly [30], [36]. However, in this study, we revealed
that E-cadherin is also activated by these dsEcads as a result of the
suppression of ZEB1 transcriptional repressor by siRNA seeddependent off-target effects (Figure 8) based on the following
results: (1) dsEcad215, dsEcad302, and dsEcad640 reduced theexpression of the ZEB1 transcriptional repressor (Figure 2); (2) the
functional seed-complementary sites of dsEcad215 and dsEcad640
were observed in the CDS of the ZEB1 mRNA, and those ofdsEcad302 were found in the 39UTR (Figure 3); (3) reporter
analyses indicated that seed-complementary sites in the CDS
function as seed-dependent silencing sites of dsEcad215 anddsEcad640, and that in the 39UTR functions as a target site ofdsEcad302 (Figures 4 and 5); (4) E-boxes in the E-cadherin
promoter were indispensable for activation of E-cadherin by
dsEcad215, dsEcad302, and dsEcad640 in our reporter assays(Figure 6). In our study, dsEcad215, dsEcad302, and dsEcad640
could not enhance the transcription of luciferase reporter gene,proE-cad670-Luc-mEbox, containing their perfect-complementary sites (Figure 6E), indicating that promoter-directed RNAa was
not represented in our reporter analyses. However, our result
might demonstrate discriminate seed-dependent function ofsiRNAs from their direct function on E-cadherin promoter, sinceparticipation of the region other than 2670 to +92 could not be
evaluated in our promoter assay. Thus, RNAa and siRNA seeddependent silencing effect might be induced simultaneously but
independently.
It is likely that transcriptional regulators other than ZEB1 canplay the equivalent role in transcriptional activation of endogenous
E-cadherin as shown in this study. For example, ZEB2 is also
known to be a transcriptional repressor of E-cadherin. ZEB2
represses E-cadherin transcription by binding to E-boxes [40], andhas 4 seed-complementary sites of dsEcad215 antisense strand, 2
sites of dsEcad302 sense strands, 1 site of dsEcad640 sense strand,and 1 site of dsEcad640 antisense strand (Table S1). In our study,
the expression of ZEB2 was not detected by qRT-PCR in PC-3
cells, probably because its expression level is very low in the cells.
However, if ZEB2 expression is sufficiently high, E-cadherin
activation is likely to be induced via repression of ZEB2 by seeddependent off-target effects. Thus, the transcriptional regulationby the siRNA/miRNA seed-dependent silencing effects might bedifferent among the cell types.
We previously reported that thermodynamic stability between
the siRNA seed sequence and target mRNA determines theefficiency of off-target effects [14]. The thermodynamic stability
could be described as the melting temperature (Tm) of the seed
duplex; siRNA with a high seed Tm shows strong off-target
silencing effect, while that with a low Tm has weak off-target
activity. The seed-dependent silencing efficiencies of the siRNAs
studied here are essentially correlated with the Tms in their seed

Microarray analyses of gene expression profiles by thetransfection of dsEcad640 and members of miR-302/372/
373/520 family miRNAs
To assess the parallel genome-wide regulation by dsEcad640
and the members of miR-302/372/373/520 family, microarray
analyses were performed using PC-3 cells transfected with each of
the miR-302a, miR-372, miR-373, miR-520c, and miR-520f
duplexes, as well as with dsEcad640 at 24 hour. The mean
expression levels of the transcripts that have common seedcomplementary sequences, AGCACUU, to dsEcad640 and miR302/372/373/520 miRNA family members in their 39UTR wereapparently reduced by the transfection with dsEcad640, miR302a, miR-372, miR-373, and miR-520c duplexes (Figure S1).
Among the 903 genes with the seed complementarities, theexpression levels of 324 genes were commonly decreased (Figure 7,
Table S2), while those of 114 genes increased (Figure 7, Table S2).
The correlation coefficients of the expression levels of thesecommon seed-complementary target genes were high between
0.62 and 0.86 each other (Figure 7). The increase of E-cadherin
mRNA was not detected by microarray analyses using the cells at
24 hour after transfection, consistent with the result shown in
Figure 2A. The seed sequences of dsEcad640 sense strand, miR302*, the opposite strand of miR-372, miR-373*, miR-520c-5p,
and the opposite strand of miR-520f, are different (Figure 1B).
Microarray profiles showed that these variable seed sequences ofthe opposite strands exhibit no or little silencing activities (FigureS2). These results indicate that miR-302/372/373/520 family
members and dsEcad640 show analogous genome-wide gene
regulation due to the common seed sequence. The complete
dataset is available at the NCBI GEO database (http://www.ncbi.
nlm.nih.gov/projects/geo, accession number GSE32999).
The microarray profile of miR-520f showed relatively low
correlation coefficients with the other miRNAs ranging from 0.34
to 0.41, although its correlation coefficient with dsEcad640 was
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E-Cadherin Activation via Small RNAs

Figure 7. Microarray profiles of gene expression by the transfection of miR-302/372/373/520 family members and dsEcad640. In
each square panel, the abscissa and longitudinal axes indicate the fold changes (log2) by the transfection of miRNAs shown in upper side and rightside, respectively. The number of genes containing the common seed sequences in their 39UTRs was 903. Among them, the number ofdownregulated genes was 324 (blue), and that of upregulated was 114 (red). The correlation coefficient (r) is indicated at the upper left side in eachsquare panel.
doi:10.1371/journal.pone.0028688.g007

duplexes as shown previously [14]. The calculated Tm in the seed
duplex of dsEcad215 antisense strand is the highest at 32.1uC(Figure 1), and showed the strongest silencing effect on the target
genes (Figure 3B). The seed Tm s are also high in dsEcad640
antisense strand (29.5uC) and dsEcad302 sense strand (26.5uC),
and showed sufficient silencing activities. However, no or weaksilencing activities of dsEcad302 antisense strand and dsEcad640
sense strand were detected, probably because their Tm s are low at
19.1uC and 12.1uC, respectively.
The seed complementary sites in the ZEB1 CDS were found to betarget sites of seed-dependent silencing by dsEcad215, dsEcad640,
and miR-302/372/373/520 family members (Figure 4A). Most of
all the identified target sites of endogenous miRNAs are known to belocated in the 39UTRs of transcripts [13], [38], [44] with few
exceptions [45]–[47]. The biological basis suggested for thisobservation is that ribosomal complexes override on the CDS andPLoS ONE | www.plosone.org

inhibit the miRNA-programmed RISC from attaching to the target
site; when the translational process is slowed, less physical constraint
is imposed by the ribosomes, thus allowing the RISC to attach to thetarget [48]. Because the expression level of ZEB1 is not so high in
PC-3 cells, siRNA/miRNA might easily accessible to ZEB1 CDS.
However, the seed-complementary sites in pLuc-CDS, which isdriven by SV40 promoter, were also functional (Figure 4A). Thus,
additive factor(s), such as thermodynamic stability in the seed duplex,
might also account for regulation of the silencing efficiencies on
CDS. The siRNA with the higher seed duplex stability might easily
access to CDS compared to that with low stability.
In this study, the members of the endogenous miR-302/372/
373/520 family were found to suppress ZEB1 transcription factor
in PC-3 cells and finally activate the expression of E-cadherin
through a similar regulatory pathway induced by dsEcad640. This
regulatory pathway might be functional in some situations such as
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E-Cadherin Activation via Small RNAs

chimeric mice. Our result might account for a pathway to gain
pluripotency by linking the mirPS induction pathway by miR-302
and that to induce pluripotent state of FAB-SCs by E-cadherin
upregulation.

Materials and Methods
Preparation of siRNAs and miRNAs
DsEcad215, dsEcad302, and dsEcad640 were chemicallysynthesized (Sigma). MiR-302a, miR-372, miR-373, miR-520c,
miR-520f miRNA duplexes were synthesized to form the same
sequences and structures described in miRBase [51]. SiRNAs
against ZEB1 CDS and 39UTR, SNAIL, SLUG, and E12/E47
were designed using our algorithms for selecting highly functional
siRNAs [14], [52]. SiRNA which has no seed-complementary
sequence against ZEB1, firefly and Renilla luciferase genes wasused as siControl. MiRNA and siRNA sequences used in this study
were shown in Figure 1 and Table S3.

Construction of luciferase reporters
The silencing efficacies due to seed-complementary regions in
ZEB1 mRNA were analyzed by psiCHECK-SM reporters(Figure 3B). Both strands of 23-nt oligonucleotides containing
the seed-complementary regions of dsEcad215, dsEcad302, anddsEcad640 in ZEB1 mRNA with overhang sequence of either
EcoRI or NotI were chemically synthesized (Table S4). Afterannealing them, the double-stranded oligonucleotides wereinserted into the 39UTR of the Renilla luciferase gene in thepsiCHECK-1 vector digested with the same restriction enzymes togenerate psiCHECK-SM.
The ZEB1 CDS was amplified from cDNAs synthesized from
PC-3 mRNAs using primers with restriction enzyme sites of SalI
and NotI on each end (Table S5). psiCHECK-1 vector was also
amplified using primers with same enzyme sites. Both theamplified ZEB1 CDS and psiCHECK-1 were digested with SalI
and NotI, and ligated to generate pLuc-CDS. Then, ZEB1 CDS
was expressed as a fusion protein with Renilla luciferase in pLucCDS by eliminating the stop codon by using primers, pLuc-ZEB1CDS-F and -R (Table S5). The ZEB1 39UTR was amplified from
cDNAs from PC-3 mRNAs using primers with restriction enzyme
sites of EcoRI and NotI (Table S5) on each end and inserted into
psiCHECK-1 digested with the same restriction enzymes togenerate pLuc-39UTR. The site-directed mutagenesis at eachseed-complementary site in dsEcad215, dsEcad302, or dsEcad640
was carried out using the PCR primers shown in Table S5.
For promoter analysis, the E-cadherin promoter region from2178 to +92 and 2670 to +92 were amplified using primers withrestriction enzyme sites of BglII or HindIII at each end (Table S5)
from genomic DNA of HeLa cells. Amplified PCR products wereinserted into the upstream region of the firefly luciferase gene ofthe pGL3-Basic vector (Promega) and named proE-cad178-Luc
and proE-cad670-Luc, respectively (see Figure 6). Two E-boxes
were mutated by site-directed mutagenesis using the PCR primers
shown in Table S5 and designated as E-cad178-Luc-mEbox andproE-cad670-Luc-mEbox, respectively.
The constructs were purified with the Genopure Plasmid Midi
Kit (Roche) and sequenced.

Figure 8. Possible mechanism of E-cadherin activation by small
RNAs. The transcription of E-cadherin is suppressed by ZEB1transcriptional repressor in PC-3 cells (upper panel). However, whendsEcad215, dsEcad302, dsEcad640, or members of miR-302/372/373/
520 family act on ZEB1 mRNA and suppress the expression of ZEB1, thetranscriptional repression of E-cadherin is alleviated (lower panel).
doi:10.1371/journal.pone.0028688.g008

the induction of pluripotency in vivo. A noncoding RNA cluster
containing mir-302b, mir-302c, mir-302a, and mir-367 are known
to be expressed most abundantly in human ES cells, and quickly
decrease after cell differentiation and proliferation [49]. The
transfection of the mir-302 cluster into human Colo and PC-3 cells
has been shown to generate ES-like cells, known as miRNAinduced pluripotent stem (mirPS) cells [50]. In the unrelated
studies, E-cadherin expression is shown to be profoundly linked tothe induction of pluripotency in bFGF, Activin, and BIO-derived
mouse stem cells (FAB-SCs) [34]. FAB-SCs are blastocyst-derived
stem cells, but have no potency for embryoid body and teratoma
formation, or embryonic development upon blastocyst transplantation. However, brief stimulation by Leukemia Inhibitory Factor(LIF) and Bone Morphogenetic Protein 4 (BMP4) upregulates Ecadherin expression to levels comparable to those observed inmouse ES cells, and induces the pluripotent state to allow thegeneration of teratoma and give germ line contribution in
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Cell culture and transfection
Human prostate cancer-derived PC-3 cells and humanembryonic kidney-derived HEK293 cells were cultured in
RPMI-1640 medium (Wako Pure Chemical Industries) andDulbecco’s modified Eagle’s medium (Invitrogen), respectively,
with 10% fetal bovine serum (Sigma) at 37uC. The day before
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E-Cadherin Activation via Small RNAstransfection, the cells were inoculated at 56104 cells/mL/well ineach well of a 24-well cell culture plate (diameter, 15 mm).
To investigate the changes in mRNA levels of E-cadherin,
ZEB1, SNAIL, SLUG, and E12/E47, PC-3 cells were transfectedwith each dsEcad or siRNA against ZEB1, SNAIL, SLUG, or
E12/E47 (5 nM) using Lipofectamine 2000 (Invitrogen) according
to the manufacture’s instruction. At the indicated times after
transfection, mRNA levels were measured by qRT-PCR.
In the luciferase reporter assay of dsEcads and miRNAs for theirseed-matched sequences in ZEB1 CDS and 39UTR, HEK293
cells were transfected with each dsEcad or miRNA (5 nM) andpGL3-Control, which is firefly luciferase expression vector (0.5 mg;
Promega) used as an internal control, along with psiCHECK-SM,
pLuc-CDS, or pLuc-39UTR (0.1 mg) simultaneously. At 1 day
after transfection, firefly and Renilla luciferase activities were
measured using the Dual Luciferase Reporter Assay System(Promega) with ARVO SX 1420 Multilabel Counter (PerkinElmer
Life Sciences), and the Renilla luciferase activities relative to thefirefly luciferase activities were calculated.
For promoter analysis, PC-3 cells stably transfected with proEcad178-Luc, proE-cad670-Luc, proE-cad178-Luc-mEbox, or
proE-cad670-Luc-mEbox were established. Each promoter-containing construct (0.5 mg) was transfected into PC-3 cells with
pSilencerPuro (0.5 mg; Ambion), which expresses Puromycin
resistant gene. The cells were selected with Puromycin (2 mg/ml)
for about 3 weeks. Then, each dsEcad or miRNA (5 nM) wastransfected into the cells. At 3 days after transfection, firefly
luciferase activity was measured. Establishment of stable cell lines
was confirmed by genomic PCR using primers shown in Table S6.

Microarray analysis
Chemically synthesized dsEcad640, miR-302a, miR-372, miR373, miR-520c, and miR-520f duplexes were transfected into PC3 cells. At 24 hours after transfection, the total RNA was extractedusing the RNeasy Mini Kit. As a control, mock transfected cells
treated with transfection reagent without siRNA and miRNA wereused. Transfection was carried out four times independently, andthe RNA quality was examined using Bioanalyzer (Agilent). Mixed
RNA samples (150 ng total) of four experiments were used for the
microarray analysis. Cy-3 labeled cRNA was prepared using the
Quick Amp Labeling Kit (Agilent), and purified with the RNeasy
Mini Kit. The cRNA was hybridized against the Agilent Whole
Human Genome Microarray (4644 K multipack format) at 65uC
for 17 hours, then washed with Gene Expression Wash Buffer 1
and Wash Buffer 2 (Agilent) in 0.005% Triton X-102. The
microarray slide was scanned using the DNA Microarray Scanner(Agilent) and quantified using Feature Extraction Software(Agilent). To confirm the results of the microarray, qRT-PCR
was carried out using the PCR primers shown in Table S6.

Accession number
The microarray data reported herein are available at the NCBIGEO database (http://www.ncbi.nlm.nih.gov/projects/geo, accession number GSE32999).

Statistical analysis
Differences of significance in the expression levels of mRNAs
and luciferase activities of reporter analyses were analyzed by the
unpaired Student’s t-test. Microarray data were analyzed by the
one-sided Kolmogorov–Smirnov (K-S) test.

Quantitative RT-PCR
Total RNA was purified using the RNeasy Mini Kit (Qiagen)according to manufacture’s instruction. cDNA was synthesized byreverse transcription using the Anchored-oligo (dT) 18 primer with
the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). The
qRT-PCR was performed with primers specific for each gene
(Table S6) using the FastStart Universal SYBR Green Master(Roche) with ABI PRISM 7000 (Applied Biosystems). The reaction
was carried out at 95uC for 10 min, followed by 40 cycles of 95uC
for 15 seconds and 60uC for 1 min.

Supporting Information
Figure S1 Microarray profiles of transcripts containing
common seed-complementary sequences in their
39UTRs. Microarray profiles of transcripts containing common
seed-complementary sequences of dsEcad640 and members of
miR-302/372/373/520 family by the transfection of (A) dsEcad640, (B) miR-302a duplex, (C) miR-372 duplex, (D) miR-373
duplex, (E) miR-520c duplex, and (F) miR-520f duplex. The left
panels represent MA plots. The changes in gene expression are
shown as log2 of the fold change ratio (ordinate), relative to mocktransfection. The abscissa is the signal intensity of the transcript(log2 scale). Red and gray dots, respectively, represent transcripts
complementary to the seed sequences and those with no seed
complementarity. The right panels indicate the cumulative
fraction of transcripts with one or more sequences complementary
to the seed sequences of each siRNA and miRNA. The red andgray lines indicate the cumulative fraction of transcripts with andwithout seed complementarity, respectively. Results of a one-sided
K-S test for seed-dependent off-target effects are as follows:transcripts with seed-complementary sequences of dsEcad640,
P#10259; those of miR-302a, P#10245; those of miR-372,
P#10220; those of miR-373, P#10239; those of miR-520c,
P#10240; those of miR-520f using common seed sequence,P#10214; miR-520f using own seed sequence, P#10257.
(TIF)

Western blot
The cells transfected with dsEcads were recovered with Passive
Lysis Buffer (Promega). Each of 5 mg of total protein was mixed
with the equal volume of 26SDS PAGE Sample Buffer (4% SDS,
0.1 M Tris-HCl (pH 6.8), 12% 2-mercaptoethanol, 20% glycerol,
0.01% bromophenol blue). After boiling for five minutes, the lysate
was separated by SDS-PAGE and transferred to the BioTraceTM
PVDF membrane (Pall Corporation) using iBrotTM Dry Blotting
System (Invitrogen). The membrane was blocked for 1 hour in
TBS-T (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Triton
X-100) supplemented with 5% DifcoTM Skim Milk (Becton,
Dickinson and Company), and incubated with 61,000-diluted
anti-ZEB1 antibody (SANTA CRUZ BIOTECHNOLOGY,
INC.) or 62,000-diluted anti-GAPDH antibody (Ambion) at
4uC overnight. The membrane was washed three times with TBST, and reacted with 610,000-diluted alkaline phosphatase-labeledanti-rabbit or anti-mouse antibody (Cappel) at 4uC overnight.
After being washed three times with TBS-T, the membrane wastransferred into alkaline phosphatase development solution(100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2),
and reacted with CDP-StarTM detection reagent (GE Healthcare), and visualized with LAS3000 (FUJIFILM).
PLoS ONE | www.plosone.org

Figure S2 Microarray profiles of transcripts containing
variable seed-complementary sequences in their
39UTRs. Microarray profiles of transcripts containing variable
seed-complementary sequences of the opposite strands in their
39UTRs by the transfection of (A) dsEcad640, (B) miR-302a
duplex, (C) miR-372 duplex, (D) miR-373 duplex, (E) miR-520c
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E-Cadherin Activation via Small RNAs

Table S1 Complementary sites of dsEcad215, dsEcad302, and dsEcad640 seed regions in the negative
regulators of E-cadherin.
(PDF)

duplex, and (F) miR-520f duplex. The left panels show MA plots.
Blue and gray dots, respectively, represent transcripts complementary to the seed of the opposite strands and those with no seed
complementarity. The right panels indicate the cumulative
fraction of transcripts with one or more sequences complementary
to the opposite strand seed sequences of each siRNA and miRNA.
The blue line indicates the cumulative fraction of transcripts with
one or more sequences complementary to the siRNA and miRNA
guide strand seeds. The gray line shows transcripts with no seed
complementarity. Results of a one-sided K-S test for seeddependent off-target effects is as follows: transcripts withcomplementary seed sequences of the opposite strand of
dsEcad640, P = 0.999; those of miR-302a, P = 0.266; those of
miR-372, P = 0.449; those of miR-373, P = 0.953; those of miR520c, P = 0.031; those of miR-520f, P = 0.998. Note that no
significant silencing effects were detected for transcripts with seedcomplementary sequences of the opposite strands.
(TIF)

Table S2 List of the increased and decreased genes thathave common seed-complementary sequences to dsEcad640 and miR-302/372/373/520 miRNA family members.
(XLS)
Table S3 SiRNA sequences used in this study.

(PDF)
Table S4 Oligonucleotides for construction of psiCHECK-SM.
(PDF)
Table S5 Oligonucleotides for construction of pLucCDS, pLuc-39UTR, proE-cad-178-Luc, and proE-cad670Luc reporters, and site-directed mutagenesis of seedcomplementary sites of dsEcad215 and dsEcad640 andE-boxes in E-cadherin promoters.
(PDF)

Figure S3 Comparison of microarray data with those ofqRT-PCR. (A) Eight genes (ZEB1, MED8, MTPN, LATS2,
RAB31, GAPDH, HINT1, PLEKHC1) were arbitrarily chosen,
and changes in mRNA expression level compared with mocktransfection were examined by qRT-PCR (abscissa) and microarray (ordinate). Note that the results of microarray are almost
linearly correlated with those of qRT-PCR. The correlation
coefficient was estimated at 0.89. Comparison at the level ofindividual gene is shown in (B–I); (B) ZEB1, (C) MED8, (D)MTPN, (E) LATS2, (F) RAB31, (G) GAPDH, (H) HINT1, and (I)
PLEKHC1. The transcripts of ZEB1, MED8, MTPN, LATS2,
and RAB31 possess seed-complementarities to either of dsEcad640, miR-302a, miR-372, miR-373, miR-520c, and miR-520f.
(TIF)

Table S6 PCR primers used in this study.

(PDF)

Acknowledgments
We thank Naoki Hibio for providing a psiCHECK-SM plasmid, and Eigo
Shimizu for assistance in reposition of microarray data.

Author Contributions
Conceived and designed the experiments: MM KN KUT. Performed theexperiments: MM KN KUT. Analyzed the data: MM KN YN KUT.
Contributed reagents/materials/analysis tools: MM KN YN KUT. Wrotethe paper: MM KUT. Analyzed microarray data: YN.

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December 2011 | Volume 6 | Issue 12 | e28688