Int. J. Mol. Sci. 2013, 14, 11171-11189; doi:10.3390/ijms140611171
OPEN ACCESSInternational Journal of

Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
ArticleFluctuation of Global Gene Expression by Endogenous miRNA
Response to the Introduction of an Exogenous miRNA
Yoshiro Nagata 1,†, Eigo Shimizu 2,†, Naoki Hibio 1 and Kumiko Ui-Tei 1,2,*
1

2†

Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo,
5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8561, Japan;
E-Mails: a5205075@gmail.com (Y.N.); lapcat.nh@gmail.com (N.H.)
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; E-Mail: eigo0shimizu@gmail.com
These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: ktei@bi.s.u-tokyo.ac.jp;
Tel.: +81-3-5841-3043; Fax: +81-3-5841-3044.
Received: 21 March 2013; in revised form: 15 April 2013 / Accepted: 6 May 2013 /Published: 27 May 2013

Abstract: Most of the intracellular endogenous microRNAs (endo-miRNAs) areconsidered to be saturated in Argonaute (Ago) proteins in the RNA-induced silencing
complexes (RISCs). When exogenous miRNAs (exo-miRNAs) are introduced into cells,
endo-miRNAs in the RISC may be replaced with exo-miRNAs or exo-miRNAs, andendo-miRNAs might also compete for the position in the newly synthesized RISC witheach other. This would lead to the fluctuation of global gene expression not only byrepression of exo-miRNA target gene expression, but also by the increase of theendo-miRNA target gene expression. In the present study, we quantified the changes in theexpression levels of target genes of exo-miRNA and endo-miRNA in the cells transfectedwith fifteen different exo-miRNAs by microarray experiments. Different exo-miRNAs
increased ratios of expression levels of target genes of a given endo-miRNA to different
extents, suggesting that the replacement efficiencies might differ according to theexo-miRNA types. However, the increased ratios in the expression levels of eachendo-miRNA target genes by the transfection of any particular exo-miRNA were mostly
equivalent, suggesting that the endo-miRNAs present in the RISC might be replaced withexcessive exo-miRNAs at similar levels, probably because they exist in single-stranded
forms in the RISC.

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Keywords: exogenous microRNA; endogenous microRNA; microarray; seed-matched target

1. Introduction
MicroRNAs (miRNAs) are an abundant class of non-coding RNAs, about ~22 nucleotides long,that are key posttranscriptional regulators of gene expression in various organisms, including animals,
plants and protozoa [1–3]. Most endogenous miRNAs (endo-miRNAs) actively silence target genes
mainly by contiguous and perfect Watson-Crick base-pairing between the miRNA 5'-proximal seed region(positions 2–8) and its complementary sequences in 3' untranslated regions (3' UTRs) of target
genes [4–6]. However, transfection of the small interfering RNA (siRNA)/miRNA expression
construct into cells relieves repression of the target genes of endo-miRNAs dose-dependently at low
concentrations and reaches the saturation level at high concentration [7–10].
These interfering effects are shown to be caused by competition for RNA silencing components.
One of the key components is the nuclear karyopherin Exportin-5 [11–13], which binds tosiRNA/miRNA precursors to transport them from the nucleus to the cytoplasm in the presence ofRan-GTP. The overloading of Exportin-5 by the excessive production of hairpin-structured
siRNAs/miRNAs transcribed from their expression constructs could result in a decrease of cellular
miRNA function [7,14]. However, the silencing activities of siRNA/miRNA duplexes are not affectedby Exportin-5, since they do not need to be transported from the nucleus to the cytoplasm for theirfunction [8,10,11]. Nonetheless, siRNA/miRNA duplexes have been shown to cause upregulation of
their non-target genes by competition with endo-miRNAs. They compete for another saturable
component of RNA silencing machinery, the RNA-induced silencing complex (RISC) [8,10,15]. The
displacement of endo-miRNAs from the RISC by the introduction of synthetic siRNAs/miRNA
duplexes are shown to be observed as the increase of endo-miRNA target gene expression [10].
RISC is the cytoplasmic effector machine of the miRNA silencing pathway. RISC assembly is
mediated by the RISC loading complex, which is a multi-protein complex composed of the coreprotein, Argonaute (Ago), the RNase Dicer and the double-stranded RNA-binding protein, TRBP(TAR RNA binding protein) [16–19]. Initially, miRNA duplexes and siRNAs are loaded into Agoprotein contained in the RISC loading complex [20,21]. To form the active RISC that performs genesilencing, the small RNA duplex needs to be separated or unwound into the single stranded form
guiding it to its target mRNAs, within Ago protein. Then Dicer and its interactor TRBP dissociatefrom the RISC. Generally, miRNA forms an imperfect duplex composed of a miRNA strand and an
opposite-strand miRNA. Evolutionary pressure has selected one particular strand of the duplex as themain regulator, which is preferentially loaded onto RISC, with the opposite strand being lessfunctional [22,23]. The strand choice is considered to be not random and is partly determined by the
intrinsic sequence and/or mismatched base-pairing of the miRNA duplex. The major determinants ofRISC loading have been shown to be the thermodynamic properties: the strand with the less stable 5'end is more often loaded onto active RISC [24–26]. Furthermore, central mismatches of miRNAs havealso been shown to promote RISC loading [27]. In the cells, RISC is considered to be saturated withendo-miRNAs. Because these experiments were carried out by the transfection of exogenous miRNAs

Int. J. Mol. Sci. 2013, 1411173(exo-miRNAs), exo-miRNAs are anticipated to exclude the endo-miRNAs from RISC or compete withendo-miRNAs for RISC with each other.
In this study, to investigate the mechanism to perturb the endo-miRNA function caused by the
introduction of exo-miRNA, we performed microarray profiling to quantify changes in the expression
levels of endo-miRNA target genes following the transfection of fifteen different synthetic
exo-miRNA duplexes. Such competition among cellular miRNAs is considered to have a role innormal biological and disease-related cellular processes. The exo-miRNAs, in addition to silencing
their own target genes, clearly increased the expression of endo-miRNA target genes. The increased
levels of endo-miRNA target gene expression were varied according to the types of the transfected
exo-miRNA duplexes, suggesting each exo-miRNA duplex is presumed to have distinct
characteristics, such as structures and sequences, which affect its incorporation into the RISC.
However, the increased levels were mostly equivalent according to the types of endo-miRNAs after
transfection of any particular exo-miRNA duplex. These results might propose the possibility that theendo-miRNAs present in the RISC in single-stranded form, but not in duplex form, nor intarget-pairing form, were replaced with exo-miRNA duplexes, because double-stranded endo-miRNAs
with different characteristics might not be replaced with exo-miRNAs in the similar levels. A part ofexo-miRNAs transfected might compete with endo-miRNAs for newly synthesized Ago proteins, butsuch effects might be negligible, because the transfected exo-miRNAs are sufficiently abundant.
Furthermore, the endo-miRNA target genes with a large number of target sites in their 3' UTRs are
revealed to be strongly repressed by endo-miRNAs in normal conditions. So, the genes with short
3' UTRs, which have a small number of miRNA target sites, were weakly repressed by endo-miRNAs
in the normal condition, then efficiently repressed when exo-miRNAs were transfected.
2. Results and Discussion
2.1. Microarray Profiling of the Reduced Expression of Exo-miRNA Target GenesTo quantify global changes in the expression levels of endo-miRNA target genes resulting from thetransfection of various exo-miRNA duplexes, microarray experiments were carried out using fifteen
different exo-miRNA duplexes (let-7b, miR-1, miR-21, miR-22, miR-28, miR-30c-1, miR-186,
miR-199b, miR-200b, miR-330, miR-335, miR-346, miR-466, miR-574 and miR-3126), which werechemically synthesized to form the duplex structures the same as those shown in miRBase [28]. At
first, 24 h following the transfection of each exo-miRNA duplexes into human HeLa cells, the effects
on own target genes of either of both miRNA strand were examined. We chose this time point, because
it is reported that RNA silencing is generally maximal ~24 h post-transfection and that protein
silencing varies depending on the target, but is generally maximal ~48–72 h post-transfection [29].
Thus, the result at 24 h post-transfection may largely reflect the direct effects without downstream
effects of exo-miRNAs, since major downstream effects should be observed after 48 h
post-transfection. It has been reported that miRNA target genes mainly contain sequencescomplementary to miRNA seed regions at positions 2–8 from the 5' terminus [4–6], then theexpression patterns of these genes were analyzed. The expression patterns of each miRNA target genes
containing seed-complementary sequences in HeLa cells transfected with fifteen exo-miRNAs areInt. J. Mol. Sci. 2013, 14

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shown as MA (M = intensity ratio, A = average intensity) plots (Figure S1A-1~O-1 and S1A-3~O-3)
and cumulative distributions (Figure S1A-2~O-2 and S1A-4~O-4). The changes in expression were
calculated as the difference values subtracting the average fold changes (log2) for exo-miRNAseed-matched target genes (blue line in Figure S1A-2~O-2 and S1A-4~O-4) from the average value forthe genes without seed-matched sequence (black line in Figure S1A-2~O-2 and S1A-4~O-4). The
differential fold change values were between 0.02 and −0.17, as summarized in Figure 1, indicatingthat all of the exo-miRNAs used in this study could decrease their own target genes at the different
levels, due to the miRNA species. However, these decreases in exo-miRNA target gene expression, asa result of competition, with the corresponding endo-miRNAs might be underestimated inthis experiment.
Figure 1. Microarray analysis of exo-miRNA target gene expression. HeLa cells
transfected with each of exo-miRNA duplexes were subjected to microarray profiling.
Differential fold changes in gene expression caused by the transfection of either strand of
fifteen different exo-miRNAs. The results of the opposite strands of miR-1, miR-346 andmiR-466 were not shown, because these miRNAs were not registered in miRBase. The
MA plots and cumulative distribution patterns are shown in Figure S1.

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2.2. Microarray Profiling of the Increased Expression of Endo-miRNA Target Genes Resulting fromthe Introduction of Exo-miRNAs
Having found reduced expression of exo-miRNA target genes, we next analyzed the expression ofendo-miRNA target genes. As let-7b, miR-21, miR-27a, miR-17, miR-26a, miR-24, miR-30a,
miR-92a, miR-19a, miR-15a, miR-22, miR-29a, miR-125a, miR-93, miR-191, miR-103a, miR-143,
miR-100, miR-23a and miR-186 are reported to be the top 20 most highly expressed miRNA families
in HeLa cells [30], changes in the target gene expression of these representative miRNAs were
calculated (Figures 2 and 3). In this analysis, “endo-miRNA target genes” were defined as genes withsequences complementary to endo-miRNA seed sequences (positions 2–8) in their 3' UTRs, but not toboth strands of exo-miRNA seed sequences. The “endo-miRNA non-target genes” were genes with nosequences complementary to endo-miRNA or both strands of exo-miRNA seed sequences. The MA
plots and cumulative distributions were shown in Figures S2–S21. The difference values were
calculated by subtracting the average fold changes (log2) for endo-miRNA target genes (blue line in
Figures S2–S21 A~O-2) from the average value for endo-miRNA non-target genes (black line in
Figures S2–S21 A-2~O-2). The expression of most of the endo-miRNA target genes was increased by
the transfection of exo-miRNAs (Figures 2 and 3, Figures S2–S21).
Next, we compared changes in the expression of endo-miRNA target genes according to the typesof exo-miRNAs (Figure 2). Expression changes were determined by subtracting the average log2 foldchange for endo-miRNA target genes containing more than one target site(s) from the average value
for endo-miRNA non-target genes and shown as a differential fold change (log2). The increases intarget gene expression of endogenous top 20 miRNAs varied, but showed similar tendencies according
to the kind of exo-miRNA transfected (Figure 2). Of the fifteen transfected exo-miRNAs, theexogenously transfected miR-466 duplex increased the expression levels of target genes of at least 16
endo-miRNAs (let-7b-5p, miR-21-5p, miR-27a-3p, miR-17-5p, miR-26a-5p, miR-24-3p, miR-30a-5p,
miR-92a-3p, miR-19a-3p, miR-15a-5p, miR-22a-3p, miR-29a-3p, miR-93a-5p, miR-143-3p,
miR-23a-3p and miR-186-5p) significantly (Figure 2A–T), and their averaged expression level was thehighest (Figure 2U). The exo-miRNA duplexes, miR-21, miR-22, miR-28, miR-30c-1, miR-200b,
miR-346, miR574 and miR-3126, produced significant, but modest levels of increases in theendo-miRNA target genes (Figure 2U), while exo-miRNA duplexes, let-7b, miR-1, miR-28, miR-199b
and miR-335, showed low values on average (Figure 2U).
Considering another dimension of the results, changes in the target gene expression of top 20
endo-miRNAs were examined. The results clearly show that the increased ratios of target genes of any
endo-miRNAs were roughly equivalent when a particular exo-miRNA was transfected (Figure 3). The
average expression of seed-matched target genes of these 20 endo-miRNAs for each of 15 transfected
exo-miRNA are shown in Figure 3. These results indicated that the increased fold changes of
expression levels of endo-miRNA target genes were essentially determined by the kind of exo-miRNA andnot by the type of endo-miRNA, although the increase of target genes of a few types of endo-miRNAs,
such as miR-1 (Figure 3B), miR-28 (Figure 3E), miR-199b (Figure 3H) and miR-335 (Figure 3K), by
the transfection of a given miRNA were not equivalent, and averaged values after transfection of theseexo-miRNAs became low due to combining the up- and down-regulated targets (Figure 2U). The
similar results, indicating the equivalent expression levels of endo-miRNA targets by most of

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exo-miRNAs, except for miR-1, miR-28, miR-199b and miR-335, were also observed when theincreased levels were calculated for top 383 endo-miRNAs (Figure S22).
Figure 2. Mean fold changes of the expression levels of endo-miRNA target transcripts by
the transfection of different exo-miRNAs. HeLa cells were transfected with each of fifteen
exo-miRNA duplexes, log2 mean differential fold changes of the expression levels of
seed-matched target genes of each of the top 20 endo-miRNAs were calculated. Data wereshown with respect to endo-miRNAs, let-7b-5p (A); miR-21 (B); mR-27a-3p (C);miR-17-5p (D); miR-26a-5p (E); miR-24-3p (F); miR-30a-5p (G); miR-92a-5p (H);miR-19a-5p (I); miR-15a-5p (J); miR-22-3p (K); miR-29a-3p (L); miR-125a-5p (M);miR-93-5p (N); miR-191-5p (O); miR-103a-3p (P); miR-143-3p (Q); miR-100-5p (R);miR-23a-3p (S); and mR-186-5p (T); and their averaged values were shown in (U). Notethat each exo-miRNA increased the endo-miRNA target genes in different degrees. The
individual data are shown in Figures S2–S21. Data represent the mean ± SE (* p < 0.05).

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Figure 2. Cont.

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Figure 2. Cont.

Our results were obtained from our own microarray experiments using one human cell line; itshould reduce variables compared to the previous studies. To confirm the reliability of the microarray
data, we analyzed 28 transcripts by quantitative RT-PCR (Figure S23). The expression levels estimatedby quantitative RT-PCR were essentially identical to those obtained in the microarray analysis, with anestimated correlation coefficient of 0.83.
Figure 3. Mean fold changes of the expression levels of endo-miRNA target transcripts
following the transfection of exo-miRNAs. HeLa cells were transfected with each offifteen exo-miRNA duplexes, log2 mean differential fold changes of the expression levelsof seed-matched target genes of each of the top 20 endo-miRNAs were calculated. Data
were shown with respect to transfected exo-miRNAs, miR-7b (A); miR-1 (B); miR-21 (C);miR-22 (D); miR-28 (E); miR-30c-1 (F); miR-186 (G); miR-199b (H); miR-200b (I);miR-330 (J); miR-335 (K); miR-346 (L); miR-466 (M); miR-574 (N); miR-3126 (O); andtheir averaged values were shown in (P). Target genes of different endo-miRNAs are
increased by exo-miRNAs in approximately comparable levels. The individual data areshown in Figures S2–S21. Data represent the mean ± SE (* p < 0.05).

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Figure 3. Cont.

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Figure 3. Cont.

2.3. Reporter Analysis of Exo-miRNA and Endo-miRNA Target Expression in Cells Transfectedwith Exo-miRNA
A part of the target genes with 3' UTRs complementary to endo-miRNA seed sequences is notnecessarily downregulated by endo-miRNAs in normal conditions. The accessibilities of endo-miRNAs tosuch genes are speculated to be interfered by some causes, such as binding of specific RNA binding
proteins or RNA secondary structures. Thus, the changes in the expression levels of seed-complementarytarget genes of endo-miRNAs shown in Figures 2 and 3 might be modest or low (generally +/− 2 to10%). To confirm the downregulation of exo-miRNA target gene expression and upregulation ofendo-miRNA target gene expression resulting from transfection of exo-miRNA, we carried out
reporter assays using luciferase expression constructs carrying sequences perfectly complementary
to exo-miRNAs or endo-miRNAs in the 3' UTR of the Renilla luciferase gene in psiCHECK-1(Figure 4A). Even when the changes in the expression levels of seed-complementary endogenous
target genes of exo-miRNAs or endo-miRNAs are small, the expression levels of luciferase reporter

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with perfect complementary sequences of miRNAs are expected to show remarkable effects, although
the effect is not biologically relevant. Exo-miR-200b or exo-miR-330 was transfected into HeLa cellswith each reporter construct and a firefly luciferase expression construct (pGL3-Cont) (internal
control). A double-stranded DNA (miDNA), which mimics the miRNA structure, was used as amiRNA control. At 24 h post-transfection, the relative luciferase activity was calculated (Figure 4).
Both exo-miRNAs silenced their own targets (Figure 4B,E). In contrast, the luciferase activities ofpsiCHECK-1 constructs containing the endo-miRNA target sequences apparently increased(Figure 4C,D,F,G). These results suggest that exo-miRNAs interfered with endo-miRNA silencing
activity by competing for the RNA silencing machinery downstream of Exportin-5 probably replacingwith endo-miRNAs in the RISC, thereby leading to increased expression of endo-miRNA target genes.
Figure 4. Luciferase reporter analyses of changes in the expression of exo-miRNA andendo-miRNA targets. (A) Schematic structure of a luciferase reporter containing a miRNA
completely matched target sequence in the 3' UTR and mRNA transcribed from thereporter. The exo-miRNAs, miR-200b (B–D) and miR-330 (E–G), respectively, weretransfected into HeLa cells at 50 nM. At 24 h post-transfection, luciferase activities were
measured. The luciferase activities derived from the constructs carrying exo-miRNA target
sequences were decreased by the transfection of respective exo-miRNA (B,E). In contrast,
the luciferase activities from the constructs carrying endo-let-7b-5p (C), endo-miR-21-5p(D,G) and endo-miR-7b-5p (F) target sequences were increased, indicating that exo-miRNAs
repressed endo-miRNA silencing activities.

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Figure 5. Mean fold changes of target genes of exo-miRNAs and endo-miRNAs according
to the 3' UTR lengths. Differential fold changes (log2) of expression levels of exo-miRNAtargets (A); and endo-miRNA targets (B); and the number of top 20 endo-miRNA target
sites (C) according to the lengths of 3' UTRs. The averaged numbers of exo-miRNA target
sites are 53, 137, 210, 250 and 149 (A), and those of endo-miRNA target sites are 53, 134,
185, 184 and 84 (B) in the 3' UTRs of 0–284, 285–647, 648–1236, 1237–2392 and2393~nucleotides (nts). Each fraction contains the same number of mRNAs registered in
the RefSeq database.

2.4. Exo-miRNA Targets with Short 3' UTRs Are Efficiently Downregulated and Endo-miRNA Targets
with Long 3'UTRs Are Efficiently Upregulated by the Introduction of Exo-miRNA Duplex
Among exo-miRNA target genes, the genes with short 3' UTRs were more efficiently
downregulated than those with long 3' UTRs by the transfection of exo-miRNAs (Figure 5A),

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consistent with the previous report [31]. In this analysis, we used the fold change values of
exo-miRNA targets with 3' UTRs complementary to seed sequence of either one strand of exo-miRNA
duplex: let-7b-5p, miR-1, miR-21-5p, miR-22-5p, miR-28-5p, miR-30c-5p, miR-186-5p, miR-199b-5p,
miR-200b-3p, miR-330-5p, miR-335-5p, miR-346, miR-466, miR-574-5p and miR-3126-5p. In
contrast, the top 20 endo-miRNA targets, which have no exo-miRNA target sites, were found to be
more strongly upregulated, according to the increase of the 3' UTR lengths (Figure 5B). It waspresumed that a large number of endo-miRNA target sites are situated in the long 3' UTRs, but small
in the short 3' UTRs; then, the genes with long 3' UTRs might be strongly repressed by endo-miRNAs
in the normal condition, but those with short 3' UTRs might be repressed weakly. So, we calculated thenumber of top 20 endo-miRNA target sites in the various length of mRNA 3' UTRs. As expected, alarge number of target sites of top 20 endo-miRNAs were found in the long 3' UTRs, but a small
number of them were in the short 3' UTRs (Figure 5C), indicating that the genes with long 3' UTRs
should be strongly repressed by endo-miRNAs compared to those with short 3' UTRs in the normal
condition. However, once exo-miRNAs are transfected into the cells, a part of the endo-miRNAs
loaded on the RISCs should be replaced with exo-miRNAs. As a result, the constant downregulation ofendo-miRNA targets by endo-miRNAs might be cancelled, and the expression of these genes are
upregulated. Thus, the changes of expression levels of the exo-miRNA target genes might beelaborately regulated by the endo-miRNAs pre-situated in the RISCs.
2.5. Highly Expressed Exo-miRNA Targets Are Efficiently Downregulated and Endo-miRNA Targets
with Low Expression Levels Are Efficiently Upregulated by the Transfection of the Exo-miRNA Duplex
Among exo-miRNA targets, the genes with high expression levels in the normal conditions weremore efficiently downregulated by the transfection of exo-miRNAs compared to those with low
expression levels (Figure 6A). The results showed good agreement with the previous study [31]. In
contrast, the endo-miRNA targets with low expression levels in the normal conditions were uncovered
to be intensively upregulated by the exo-miRNA transfection, and those with high expression levels
were not upregulated efficiently (Figure 6B). So, we investigated the number of endo-miRNA target
sites in gene with different expression levels. As a result, a large number of target sites were found in
the mRNAs with low expression levels, but a small number of the sites were detected in the mRNAs
with high expression levels (Figure 6C), suggesting that many genes with low expression levels might
be downregulated by endo-miRNAs, but those with high expression levels are not repressed byendo-miRNAs in the normal condition. Thus, the changes of expression levels of the exo-miRNAtarget genes might be also suitably regulated by the endo-miRNAs pre-situated in the RISCs.

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Figure 6. Mean fold changes of target genes of exo-miRNAs and endo-miRNAs,
according to the expression levels. Differential fold changes (log2) of target gene
expression levels of exo-miRNAs (A) and endo-miRNAs (B) and the number of the top 20
endo-miRNA target sites (C), according to the expression levels of exo-miRNA targets.
The averaged numbers of exo-miRNA target sites are 130, 217, 237 and 215 (A), and thoseof endo-miRNA target sites are 92, 164, 176 and 202 (B) in the 3' UTRs of genes withdifferential fold changes of 0~7, 7~9, 9~11 and 11~.

3. Experimental Section
3.1. Cell Culture and miRNA Synthesis
Human HeLa cells were cultured and used in reporter assays and microarray analyses. Cells were
cultured at 37 °C in Dulbecco’s modified Eagle’s Medium (Invitrogen, Carlsbad, NM, USA),supplemented with 10% heat-inactivated fetal bovine serum (Sigma, St. Louis, MO, USA). They wereplated on 24-well culture plates (1 × 105 cells/mL/well) 24 h prior to transfection. Transfection was
carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, NM, USA). Each RNA strand of themiRNA duplex was chemically synthesized (Sigma, St. Louis, MO, USA) and annealed to form theduplex structures the same as those shown in miRBase [28]. The sequences of the synthetic miRNAs

Int. J. Mol. Sci. 2013, 14

11185(let-7b, miR-1, miR-21, miR-22, miR-28, miR-30c-1, miR-186, miR-199b, miR-200b, miR-330,
miR-335, miR-346, miR-466, miR-574 and miR-3126) are listed in Table S1.
3.2. Construction of Luciferase Reporters
All of the reporter plasmids constructed were derivatives of psiCHECK-1 (Promega, Fitchburg, WI,
USA). Oligonucleotides with target sequences completely matched to each miRNA strand (cm-target)
were chemically synthesized with cohesive XhoI/EcoRI ends (Table S2). They were then inserted into
the corresponding restriction sites of psiCHECK-1 to generate miRNA cm-targets (miR-200b-3p
target, miR-7b-5p target, miR-21-5p target and miR-330-5p target). Each of the inserted targets was
expressed as part of the 3' UTR region of Renilla luciferase mRNA in transfected cells.
HeLa cells growing in 24-well plates were transfected simultaneously with miRNA target (100 ng),
pGL3-Control (Promega, 0.5 µg) and miRNA (50 nM). The cells were harvested 24 h post-transfection
and the relative luciferase activity (Renilla luc activity/firefly luc activity) was determined using a
Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA). The pGL3-Control encoding
firefly luciferase served as a control for the calculation of relative luciferase activity for miRNAs.
3.3. Microarray Analysis
HeLa cells (1 × 105 cells/mL) were transfected with 50 nM of each of 15 miRNA duplexes. At 24 hpost-transfection, total RNA was purified using an RNeasy Kit (Qiagen, Hilden, Germany). The steps
were repeated four times and RNA quality assessed using a NanoDrop 2000 spectrophotometer(Thermo Scientific, Waltham, MA, USA) and a Bioanalyzer (Agilent, Santa Clara, CA, USA). RNAs
recovered independently were mixed equally for cDNA synthesis using an Agilent One Color SpikeMix Kit (Agilent, Santa Clara, CA, USA). Cy3-labeled cRNA was synthesized using a Quick Amp
Labeling Kit (Agilent, Santa Clara, CA, USA) and was hybridized to an Agilent Whole Human
Genome Microarray (4 × 44 K multi-pack format), according to the manufacturer’s protocol. RNA
from mock-transfected cells treated with transfection reagent in the absence of miRNA was used as acontrol. Transcript expression values were calculated using Microarray Suite 5.0 (MAS5: Affymetrix,
Santa Clara, CA, USA) [32] with quantile normalization [33]. To identify transcripts whose expression
was upregulated or downregulated, the cumulative distribution of expression changes for transcripts
containing the site was compared with that for transcripts with no canonical site. NCBI’s Reference
Sequence (RefSeq) was used to identify mRNAs with sequences complementary to the seed regions of
the transfected miRNAs. Data are presented as an MA plot (M = intensity ratio, A = average intensity)and a cumulative frequency distribution. Changes in expression are shown as fold changes (log2).
3.4. Quantitative RT-PCR
Total RNA was reverse-transcribed using a Transcriptor High Fidelity cDNA Synthesis kit (Roche,
Basel, Switzerland). The resultant cDNA samples were incubated with FastStart Universal SYBRGreen Master (Roche, Basel, Switzerland) at 95 °C for 10 min, followed by PCR amplification. PCR
product levels were monitored using an ABI PRISM 7000 sequence detection system and analyzed
with ABI PRISM 7000 SDS software (Applied Biosystems). The expression of each target gene wasInt. J. Mol. Sci. 2013, 14

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first normalized to that of β-actin and then to the mock-transfection control. The primer sets used arelisted in Table S3.
4. Conclusions
In this study, we quantified the changes in expression levels of endo-miRNA target genes resulting
from the transfection of exo-miRNA duplexes. The expression levels of endo-miRNA target genes
with seed-complementary sequences were increased by the transfection of exo-miRNA duplexes,
while exo-miRNA target gene expression was reduced (Figures 1–3 and Figures S1–S3). These results
suggest that exo-miRNA duplex transfected into the cells may compete with endo-miRNAs for the
RISC, which may be saturated with endo-miRNAs under normal conditions in HeLa cells.
In miRNA-mediated gene silencing, the structure of the RNA-protein complex is known to bealtered [23]. The miRNA duplex in RISC loading complex is unwound, yielding single-stranded RNA,
which is loaded onto the RISC and recognizes target mRNAs through base-pairing in the seedregion [4–6]. Our results indicate that the expression of the target genes of a given endo-miRNA
differed according to the exo-miRNA duplex that was transfected (Figures 2 and 7A), whereas with agiven exo-miRNA duplex, the fold changes in the target gene expression of the endo-miRNAs
examined were mostly equivalent, except for a limited types of exo-miRNAs (miR-1, miR-28,
miR-199b and miR-335), despite differences in the structures and sequences of the endo-miRNA
duplexes (Figure 3 and Figure 7B). One of the possible explanations of these results is that the RISC
exchange reaction might be occurred associated with single-stranded endo-miRNAs and not endo-miRNA
duplexes or target-paired endo-miRNAs (Figure 7), because double-stranded endo-miRNAs in the
RISC might not be replaced with exo-miRNAs at similar levels, due to their different structures andsequences. Most miRNA-RISCs might be in the form of single-stranded miRNAs in RISCs, so as to bereadily replaced by double-stranded miRNAs.
Figure 7. Predicted model for the RNA-induced silencing complexes (RISCs) replacement.
Different types of exo-miRNAs transfected into cells may be replaced with single-stranded
endo-miRNAs loaded on the RISC with different efficiencies (A); however, different typesof endo-miRNAs may be replaced with a given exo-miRNAs with similar efficiencies (B).

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11187

Furthermore, it was apparently revealed that endo-miRNAs constantly repress the expression ofendogenous mRNAs with endo-miRNA target sites, and their repression is probably relieved by the
replacement of endo-miRNAs on the RISC by the exo-miRNAs transfected (Figures 5 and 6). Thus,
global gene expression by endogenous miRNAs might be fluctuated by the transfection of
exo-miRNAs or the increase of expression levels of endo-miRNAs. Competition similar to that shown
here between exo-miRNA duplexes and endo-miRNAs may also occur among newly transcribedendo-miRNAs and may provide a mechanism for orchestrating cellular programs.
Acknowledgments
We thank Yuki Naito for helpful discussions about the bioinformatics analysis and Kenji Nishi fortechnical advises on microarray experiments. This work was supported by a Grant-in-Aid for Scientific
Research (grant numbers 21115004, 21310123), the Cell Innovation Project from the Ministry of
Education, Culture, Sports, Science and Technology of Japan and the Core Research Project forPrivate University matching fund subsidy to K.U.-T.
Conflict of Interest
The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.

7.

8.

9.

Rana, T.M. Illuminating the silence: Understanding the structure and function of small RNAs.
Nat. Rev. Mol. Cell Biol. 2007, 8, 23–36.
Filipowicz, W.; Bhattacharyya, S.N.; Sonenberg, N. Mechanisms of post-transcriptional
regulation by microRNAs: Are the answers in sight? Nat. Rev. Genet. 2008, 9, 102–114.
Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004, 116,
281–297.
Grimson, A.; Farh, K.K.; Johnston, W.K.; Garrett-Engele, P.; Lim, L.P.; Bartel, D.P. MicroRNAtargeting specificity in mammals: Determinants beyond seed pairing. Mol. Cell 2007, 27, 91–105.
Lewis, B.P.; Burge, C.B.; Bartel, D.P. Conserved seed pairing, often flanked by adenosines,
indicates that thousands of human genes are microRNA targets. Cell 2005, 120, 15–20.
Lim, L.P.; Lau, N.C.; Garrett-Engele, P.; Grimson, A.; Schelter, J.M.; Castle, J.; Bartel, D.P.;
Linsley, P.S.; Johnson, J.M. Microarray analysis shows that some microRNAs downregulate large
numbers of target mRNAs. Nature 2005, 433, 769–773.
Grimm, D.; Streetz, K.L.; Jopling, C.L.; Storm, T.A.; Pandey, K.; Davis, C.R.; Marion, P.;
Salazar, F.; Kay, M.A. Fatality in mice due to oversaturation of cellular microRNA/short hairpin
RNA pathways. Nature 2006, 441, 537–541.
Castanotto, D.; Sakurai, K.; Lingeman, R.; Li, H.; Shively, L.; Aagaard, L.; Soifer, H.;
Gatignol, A.; Riggs, A.; Rossi, J.J. Combinatorial delivery of small interfering RNAs reduces
RNAi efficacy by selective incorporation into RISC. Nucleic Acids Res. 2007, 35, 5154–5164.
Stewart, C.K.; Li, J.; Golovan, S.P. Adverse effects induced by short hairpin RNA expression in
porcine fetal fibroblasts. Biochem. Biophys. Res. Commun. 2008, 370, 113–117.

Int. J. Mol. Sci. 2013, 1411188

10. Khan, A.A.; Betel, D.; Miller, M.L.; Sander, C.; Leslie, C.S.; Marks, D.S. Transfection of small
RNAs globally perturbs gene regulation by endogenous microRNAs. Nat. Biotechnol. 2009, 27,
549–555.
11. Yi, R.; Qin, Y.; Macara, J.G.; Cullen, B.R. Exportin-5 mediates the nuclear export of pre-microRNAs
and short hairpin RNAs. Genes Dev. 2003, 17, 3011–3016.
12. Lund, E.; Guttinger, S.; Calado, A.; Dahlberg, J.E.; Kutay, U. Nuclear export of microRNA
precursors. Science 2004, 303, 95–98.
13. Bohnsack, M.T.; Czaplinski, K.; Gorlich, D. Exportin-5 is a RanGTP-dependent dsRNA-binding
protein that mediates nuclear export of pre-miRNAs. RNA 2004, 10, 185–191.
14. Kim, V.N. MicroRNA precursors in motion; exportin-5 mediates their nuclear export. Trends Cell
Biol. 2004, 14, 156–159.
15. Vankoningsloo, S.; de Longueville, F.; Evrard, S.; Rahier, P.; Houbion, A.; Fattaccioli, A.;
Gastellier, M.; Remacle, J.; Raes, M.; Ranard, P.; et al. Gene expression silencing with “specific”
small interfering RNA goes beyond specificity–a study of key parameters to take into account in
the onset of small interfering RNA off-target effects. FEBS J. 2008, 275, 2738–2753.
16. Gregory, R.I.; Chendrimada, T.P.; Cooch, N.; Shienkhattar, R. Human RISC couples microRNAbiogenesis and posttranscriptional gene silencing. Cell 2005, 123, 631–540.
17. Haase, A.D.; Jaskiewicz, L.; Zhang, H.; Lainé, S.; Sack, R.; Gatignol, A.; Filipowicz, S. TRBP, aregulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functionas in RNA
silencing. EMBO J. 2005, 6, 961–967.
18. Macrae, I.J.; Ma, E.; Zhou, M.; Robinson, C.V.; Doudna, J.A. In vitro reconstitution of the human
RISC-loading complex. Proc. Natl. Acad. Sci. USA 2008, 105, 512–517.
19. Winter, J.; Jung, S.; Keller, S.; Gregory, R.; Diederichs, S. Many roads to maturity: microRNAbiogenesis pathways and their regulation. Nat. Cell Biol. 2009, 11, 228–234.
20. Matranga, C.; Tomari, Y.; Shin, C.; Bartal, D.P.; Zamore, P.D. Passenger-strand cleavage
facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 2005, 123,
607–620.
21. Leuschner, P.J.; Ameres, S.L.; Kueng, S.; Martinez, J. Cleavage of the siRNA passenger strand
during RISC assembly in human cells. EMBO Rep. 2006, 7, 314–320.
22. Tolia, N.H.; Joshua-Tor, L. Slicer and the Argonaute. Nat. Chem. Biol. 2006, 3, 36–43.
23. Czech, B.; Hannon, G.J. Small RNA sorting: Matchmaking for Argonautes. Nat. Rev. Genetics
2011, 12, 19–31.
24. Khvorova, A.; Reynolds, A.; Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias.
Cell 2003, 115, 209–215.
25. Schwarz, D.S.; Hutvágner, G.; Du, T.; Xu, A.; Aronin, N.; Zamore, P.D. Asymmetry in theassembly of the RNAi enzyme complex. Cell 2003, 115, 199–205.
26. Ui-Tei, K.; Naito, Y.; Takahashi, F.; Haraguchi, T.; Ohki-Hamazaki, H.; Juni, A.; Ueda, R.;
Saigo, K. Guidelines for the selection of highly effective siRNA sequences for mammalian andchick RNA interference. Nucleic Acids Res. 2004, 32, 936–948.
27. Yoda, M.; Kawamata, T.; Paroo, Z.; Ye, X.; Iwasaki, S.; Liu, Q.; Tomari, Y. ATP-dependent
human RISC assembly pathways. Nat. Struct. Mol. Biol. 2010, 17, 17–23.

Int. J. Mol. Sci. 2013, 1411189

28. Griffiths-Jones, S.; Saini, H.K.; van Dongen, S.; Enright, A.J. MiRBase tools for microRNA
genomics. Nucleic Acids Res. 2008, 36, D154–D158.
29. Jackson, A.L.; Burchard, J.; Schelter, J.; Chau, B.N.; Cleary, M.; Lim, L.; Linsley, P.S.
Widespread siRNA “off-target” transcript silencing mediated by seed region sequence
complementarity. RNA 2006, 12, 1179–1187.
30. Mayr, C.; Bartel, D.P. Widespread shortening of 3’UTRs by alternative cleavage andpolyadenylation activates oncogenes in cancer cells. Cell 2009, 138, 673–684.
31. Saito, T.; Sætrom, P. Target gene expression levels and competition between transfected andendogenous microRNAs are strong confounding factors in microRNA high-throughput experiments.
Silence 2012, 3, 3.
32. Hubbell, E.; Liu, W.M.; Mei, R. Robust estimation for expression analysis. Bioinformatics 2002,
18, 1585–1592.
33. Bolstad, B.M.; Irizarry, R.A.; Gautier, L.; Wu, Z. Preprocessing High-Density Oligonucleotide
Arrays. In Bioinformatics and Computational Biology Solutions Using R and Bioconductor;
Gentleman, R., Carey, V.J., Huber, W., Irizarry, R.A., Dudoit, S., Eds.; Springer: New York, NY,
USA, 2005; pp. 13–32.
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