In: Gene Silencing: Theory, Techniques and Applications
ISBN: 978-1-61728-276-8
Editor: Anthony J. Catalano© 2010 Nova Science Publishers, Inc.
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Chapter XIII

Genome-wide Identification
and Analysis of miRNAs
Complementary
to Upstream Sequencesof mRNA Transcription Start Sites
Kenta Narikawa1, Kenji Nishi2, Yuki Naito2, Minami Mazda2
and Kumiko Ui-Tei1,2,*
1

Department of Computational Biology, Graduate School of Frontier Sciences,
University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8561, Japan2
Department of Biophysics and Biochemistry, Graduate School of Science, University ofTokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Abstract
Small regulatory RNAs including short interfering RNAs (siRNAs) and microRNAs(miRNAs) are crucial regulators of gene expression at the posttranscriptional level.
Recently, additional roles for small RNAs in gene activation and suppression at thetranscriptional level were reported; these RNAs were shown to have sequences thatclosely or completely match to their respective promoter regions. However, no global
analysis for identifying target sequences for miRNAs in the promoter region have been
carried out in the human genome.
We performed a genome-wide search for upstream sequences of mRNA transcription
start sites where miRNAs are capable of hybridizing with high complementarity. We
identified 219 sites in the 10-kb upstream regions of transcription start sites withcomplete complementarity to 94 human mature miRNAs. Furthermore, the mismatchedpositions and nucleotides in near-completely matched sites were highly biased, and most*

Email address: ktei@bi.s.u-tokyo.ac.jp

288

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
of them appear to be possible target sites of miRNAs. The expression of downstream
genes of miRNA target sites were examined following transfection of each miRNA into
three different human cell lines. The results indicate that miRNAs dynamically modulates
gene expression depending on the downstream genes and the cell type.

Introduction
Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs(miRNAs) regulate gene expression at the posttranscriptional level in various organisms [1,2].
RNA silencing encompasses a group of mechanistically related pathways. However, thebiogenesis and mechanism of action differ between siRNA and miRNA. In the siRNAmediated RNA interference (RNAi) pathway, long double-stranded RNAs (dsRNAs) areprocessed by the cytoplasmic RNase III enzyme Dicer into 21–23-nucleotide (nt) siRNAs.
The siRNA guide strand that is incorporated into the RNA-induced silencing complex (RISC)
closely or completely matches their target sequences, and a core component of RISC,
Argonaute (Ago) protein, mediates the sequence-specific cleavage of target mRNAs [3,4]. In
the miRNA pathway, miRNAs are transcribed by RNA polymerases II and III [5]. This
generates primary miRNA (pri-miRNA) transcripts, which are further processed to stemloop-structured precursor miRNAs (pre-miRNAs) by the nuclear RNase III Drosha as part ofa microprocessor complex with DGCR8 [6-8]. The pre-miRNA is exported into thecytoplasm by the Exportin-5/Ran-GTP complex [9]. Dicer cleaves the cytoplasmic premiRNA hairpin into the mature miRNA, which enters the miRNA-containing
ribonucleoprotein (miRNP) and is capable of binding to partially complementary sequences
within the 3′ UTR of the target mRNA.
In the past few years, additional roles for small non-coding RNAs to direct both geneactivation and suppression at the transcriptional level in nucleus of human cells have beenreported. Exogenous siRNAs with sequence complementarity to promoter regions have beenshown to induce transcriptional gene silencing (TGS) [10-15]. Meanwhile, siRNA sequenceswith promoter complementarity also direct transcriptional activation in a process referred toas RNA activation (RNAa) [16-18]. While little is known about the mechanisms andbiological effects of this aberrant siRNA behaviour, studies regarding the mechanisms of
TGS and RNAa have provided some insight. TGS induced by promoter-directed siRNAs has
been shown to require Ago proteins [12,13] and promoter-associated RNAs that span thetargeted loci [15,19]. RNAa was shown to be associated with the 5′ end of the siRNA guidestrand and Ago2 [17] and antisense transcripts [18,19]. However, siRNA targeted to the HIV1 LTR promoter was also demonstrated to induce gene activation via an indiscriminate offtarget effect [20].
Both TGS and RNAa have been shown to be inducible by endogenous miRNAs thatcompletely or nearly completely match promoters in human cells [21,22]. miR-373 has highly
complementary target sequences to the promoters of E-cadherin and CSDC2 (cold shock
domain-containing protein C2), and it induces the expression of both genes [22].
Furthermore, miR-320 is encoded within the promoter region of the cell cycle gene POLR3D
in the antisense orientation, and transcriptionally silences POLR3D when associated with
Ago1, Polycomb-group (PcG) component EZH2, and trimethyl histone H3 lysine 27(H3K27me3) [21]. These results suggest that endogenous miRNAs sharing complementaryGenome-wide Identification and Analysis of miRNAs Complementary…

289

sequences to promoter regions regulate gene expression at the level of transcription. In thehuman genome, ~700 miRNA genes are distributed in single or clustered fashion andcomprise independent transcription units in intergenic regions or are embedded within intronsof protein-coding genes [23]. While genome-wide computational and experimental searches
for predicting miRNA regulatory targets in the 3′ UTRs but not in the upstream regions havebecome major challenges [24], but their intended regions have been mainly 3′ UTRs. To date,
no global analyses have been carried out to identify miRNAs targeting upstream regions ofmRNA coding regions. We performed genome-wide analyses searching for upstream
sequences of transcription start sites (TSSs) where miRNAs share complementarity. Thechanges in the expression of downstream genes due to the miRNAs were examined.

Materials and Methods
Search for Upstream Regions Containing Sequences
Complementary to miRNAs
The transcriptional start sites were assumed to be the 5′-terminus of the mRNAs
registered in the RefSeq database in this study. The 10-kb sequences upstream of the TSSs
were retrieved by matching of the 5′-terminus of mRNA sequences registered in the RefSeq
release 27 database (http://www.ncbi.nlm.nih.gov/RefSeq/) [25] with human genome
sequences from the UCSC Genome Browser (http://genome.ucsc.edu) [26]. Then, the 10-kb
upstream sequences of human mRNA and the miRNA sequences retrieved from the miRBase
release 10.0 (http://microrna.sanger.ac.uk/) [27] were aligned, and regions with perfectcomplementarity in both directions were selected. In addition, 1-kb upstream regions that
contained incomplete complementary sequences (i.e., 1- or 2-bp mismatches) with miRNAs
were also selected.

Cell Culture
Human carcinoma cell line of the uterine cervix, HeLa, and prostatic carcinoma cell line,
PC-3 (Health Science Research Resources Bank, Japan), were cultured in Dulbecco‘s
Modified Eagle‘s Medium (DMEM; Invitrogen) at 37˚C. Media for both cell lines were
supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Mitsubishi Kagaku) andantibiotics (10 units/ml of penicillin (Meiji) and 50 µg/ml of streptomycin (Meiji)). Human
MCF-7 breast cancer cell line (Health Science Research Resources Bank, Japan) was cultured
in Modified Eagle‘s Medium (MEM; Invitrogen) supplemented with 10 % heat-inactivated
FBS, 100 mM sodium pyruvate (Invitrogen) and 4 mg/ml insulin (Invitrogen).

Plasmid Construction and RNA Silencing Assay
All plasmids constructed are derivatives of psiCHECK-1 (Promega, Madison,
Wisconsin). Chemically synthesized double-stranded oligonucleotides containing 81-bp were290

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.

inserted into the corresponding restriction enzyme sites of psiCHECK-1 to generate
psiCHECK-miR-target. Each oligonucleotide included three tandem repeats of an identical
25-bp containing completely matched target sequences of miR-378, miR-548c-5p, miR-548d5p, miR-572, miR-574-5p and miR-606 miRNAs and cohesive XhoI/EcoRI ends (Table 1).
The inserted miRNA targets were expressed as part of the 3 UTR of the Renilla luciferase
mRNA in the transfected cells. Cells in each well of a 24-well culture plate were transfected
simultaneously with one of the psiCHECK-miR-target (100 ng), a pGL3-Control (0.5 µg;
Promega) and synthetic miRNA (0.005, 0.05, and 0.5 µg; Sigma). Cells were harvested 24 hafter transfection and relative luc activity (Renilla luciferase activity/firefly luciferase
activity) was determined using the Dual-Luciferase Reporter Assay System (Promega).
Unrelated siRNA against the firefly luciferase gene (5′-CGUACGCGGAAUACUUCGAUU3′ and 5′-UCGAAGUAUUCCGCGUACGUG-3′) was used as a miRNA control (dsControl).
Table 1. Oligonucleotide sequences inserted
into psiCHECK-miR-target constructs
oligo（-SS）

sequence (5' ⇒ 3')378-SS

tcgaGGCCTTCTGACTCCAAGTCCAGTGCGGCCTTCTGACTCCAA
GTCCAGTGCGGCCTTCTGACTCCAAGTCCAGTGC

548c-5p-SS

tcgaTGGCAAAAACCGCAATTACTTTTGCTGGCAAAAACCGCAA
TTACTTTTGCTGGCAAAAACCGCAATTACTTTTGC

548d-5p-SS

tcgaTGGCAAAAACCACAATTACTTTTGCTGGCAAAAACCACAA
TTACTTTTGCTGGCAAAAACCACAATTACTTTTGC

572-SS

tcgaGGCTGGGCCACCGCCGAGCGGACGAGGCTGGGCCACCGCC
GAGCGGACGAGGCTGGGCCACCGCCGAGCGGACGA

574-5p-SS

tcgaCACACACTCACACACACACACTCACCACACACTCACACACACACACTCACCACACACTCACACACACACACTCAC
tcgaGTATCTTTGATTTTCAGTAGTTTACGTATCTTTGATTTTCAGTAGTTTACGTATCTTTGATTTTCAGTAGTTTAC

606-SS

oligo（-AS）

sequence (5' ⇒ 3')

378-AS

aattGCACTGGACTTGGAGTCAGAAGGCCGCACTGGACTTGGAGTCAGAAGGCCGCACTGGACTTGGAGTCAGAAGGCC

548c-5p-AS

aattGCAAAAGTAATTGCGGTTTTTGCCAGCAAAAGTAATTGCGG
TTTTTGCCAGCAAAAGTAATTGCGGTTTTTGCCA

548d-5p-AS

aattGCAAAAGTAATTGTGGTTTTTGCCAGCAAAAGTAATTGTGG
TTTTTGCCAGCAAAAGTAATTGTGGTTTTTGCCA
aattTCGTCCGCTCGGCGGTGGCCCAGCCTCGTCCGCTCGGCGGT
GGCCCAGCCTCGTCCGCTCGGCGGTGGCCCAGCC
aattGTGAGTGTGTGTGTGTGAGTGTGTGGTGAGTGTGTGTGTGT
GAGTGTGTGGTGAGTGTGTGTGTGTGAGTGTGTG

572-AS
574-5p-AS
606-AS

aattGTAAACTACTGAAAATCAAAGATACGTAAACTACTGAAAATCAAAGATACGTAAACTACTGAAAATCAAAGATAC

AS and SS, respectively, indicate antisense and sense strand sequences of three copies of each mRNA target
sequences. Both strands were annealed and inserted into the psiCHECK vector.

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291

Table 2. Stem–loop RT primers specific for each miRNAnamesequence (5' ⇒ 3')
st21
gtcgtatccagtgcagggtccgaggtattcgcactggatacgactcaaca
st378
gtcgtatccagtgcagggtccgaggtattcgcactggatacgacccttct
st548c-5p
gtcgtatccagtgcagggtccgaggtattcgcactggatacgacggcaaa
st548d-5p
gtcgtatccagtgcagggtccgaggtattcgcactggatacgacggcaaa
st572
gtcgtatccagtgcagggtccgaggtattcgcactggatacgactgggcc
st574-5p
gtcgtatccagtgcagggtccgaggtattcgcactggatacgacacacac
st606
gtcgtatccagtgcagggtccgaggtattcgcactggatacgacatcttt
st21, 378, 548c-5p, 548d-5p, 572, 574-5p and 606 are RT primers for miR-21, miR-378, miR-548c-5p, miR548d-5p, miR-572, miR-574-5p, and miR-606, respectively.

Table 3. PCR primers used for each miRNAnamesequence (5' ⇒ 3')
stRT-21fp
gcccgctagcttatcagactgatg
stRT-378fp
gccgccactggacttggagtc
stRT-548cfp
gccgcaaaagtaattgcggt
stRT-548dfp
gccgcgaaaagtaattgtggt
stRT-572fp
gccgcggtccgctcggcggtg
stRT-574fp
gcccgctgagtgtgtgtgtgtgagt
stRT-606fp
gcccgcaaactactgaaaatga
stRT-rpgtgcagggtccgaggt
stRT-21fp, stRT-378fp, stRT-548c-5pfp, stRT-548d-5pfp, stRT-572fp, stRT-574-5pfp and stRT-606fp are
forward primers for PCR of miR-21, miR-378, miR-548c-5p, miR-548d-5p, miR-572, miR-574-5p and miR606, respectively. stRT-rp is a universal reverse primer.

Table 4. PCR primers for downstream genes
gene namefp sequence (5' ⇒ 3')
rp sequence (5' ⇒ 3')
PCR product (bp)
OSBPL10
catgctggtggtgtactctgct
cgggagcttggagcactctt
144
RNPS1
tagggctccttcacctaccaaa
cgatccttctcactcgactcct
102
OSBPL9agcgtccatcttccctacca
tgctactcggtggtgaatgg
105
POLR3Agacctggagttgccgtgttt
tgatgtggcagcaggttttg
88
ANXA3
gcatctcatggtggcccta
cttcatttgcctgcttgtcc
136
CDC23
ctgcttattgcgggccttac
gcatccatatcctgggcatc
149
RNF6
agattatcggcttatgagagacca
ttccttgacgccatctaacc
94
C20orf43
ctcttgggaaggcagcatct
ctggaggtcatcgtgcttgt
125
actin
cacactgtgcccatctacga
gccatctcttgctcgaagtc
203
PCR primers are indicated by the gene names. fp and rp represent forward and reverse primers, respectively.
Expected size of PCR product is shown in the right colume.

Detection of Endogenous miRNAs by RT-PCR
Endogenous miRNAs were detected by RT-PCR according to the procedure reported byChen et al [28]. Briefly, 1 ml of the HeLa, PC-3, or MCF-7 cell suspensions (containing 1 x

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Kenta Narikawa, Kenji Nishi, Yuki Naito et al.

105 cells/ml) was inoculated into each well of a 24-well plate 24 h prior to transfection. Cells
were transfected without or with chemically synthesized miRNAs (500 ng/well) using
Lipofectamine 2000 (Invitrogen, Carlsbad, California). The cells transfected with synthesized
miRNAs were used as miRNA detection control. Total RNA was purified 3 days after
transfection using the mirVana miRNA Isolation Kit (Ambion, Austin, Texas) according to
the manufacturer′s protocol. The reverse transcriptase reactions contained total RNA (0.25µg), 50 nmoles of stem–loop RT primer specific for each miRNA (Table 2), 1 RT buffer(RETROscript Kit; Ambion), 0.25 mM dNTPs, 3.33 U/µl of MultiScribe reverse transcriptase(Applied Biosystems, Foster City, California), and 0.25 U/µl of RNase OUT (Invitrogen).
The mixture was incubated at 16 ºC for 30 min, 42 ºC for 30 min, 85 ºC for 5 min and thenheld at 4 ºC. The PCR reaction was performed using AmpliTaq Gold PCR Master Mix(Applied Biosystems) for each miRNA using universal reverse primers (Table 3). The
mixture was incubated at 95 ºC for 10 min, followed by 40 cycles at 95 ºC for 15 s, and 60 ºC
for 1 min. The reactions were fractionated on a 3 % agarose gel with TBE.

Detection of Downstream Gene Expression by Real Time RT-PCR
Using Lipofectamine 2000 (Invitrogen), HeLa, PC-3 and MCF-7 cells were cotransfectedwith or without 500 ng/well chemically synthesized miRNA. Total RNA was treated with
RQ1 DNase (Promega) and purified using RNeasy Mini Kit (QIAGEN) 3 days after
transfection. Reverse transcriptase reactions were carried out using total RNA (0.25 µg) with
Anchored-oligo(dT)18 primer by Transcriptor High Fidelity cDNA Synthesis Kit (Roche)
according to the manufacturer‘s protocol. Real time PCR was performed using FastStart
Universal SYBR Green Master (Roche) with forward and reverse primers specific for
OSBPL10, RNPS1, OSBPL9, POLR3A, ANXA3, CDC23, RNF6, C20orf43 and actin genes(Table 4). The mixture was incubated at 95 ºC for 10 min, followed by 40 cycles of 95 ºC for
15 sec and 60 ºC for 1 min and monitored by ABI PRISM 7000 (Applied Biosystems). The
expression level of each mRNA was first normalized to the amount of actin and then to thesiRNA against the firefly luciferase (dsControl) transfection control.

Results
Search for miRNAs Having Perfect Complementarity within
Upstream Sequences of mRNA Coding Regions
We identified 219 sites in the 10-kb upstream regions of TSSs with complete
complementarity to 94 human mature miRNAs (Tables 5 and 6). In this study, the 5′-end ofmRNA was assumed to be TSS (position +1). More than one site was included in some of the
upstream sequences, and overlapping transcriptional variants were counted at several sites;the 219 sites corresponded to the upstream sequences of 155 genes (Table 5). Among thesesites, 153 sites were complementary to pre-miRNAs which generate 88 mature miRNAs
registered in the miRBase, indicating that these miRNAs are transcribed from the upstream
regions as origins. As shown for miR-320 [21], these miRNAs may have multiple regulatory

Genome-wide Identification and Analysis of miRNAs Complementary…

293

functions if the antisense transcript is transcribed at the same position. Except for these cases,
the 66 sites of 50 genes (labelled with blue in Table 6) appeared perfect complementarity to
the miRNAs (miR-378, miR-548c-5p, miR-548d-5p, miR-572, miR-574-5p and miR-606)
transcribed from the different location other than the 10-kb upstream regions.
Table 5. Results of the search for miRNAs with complementarity to upstream sequencesof transcription start sites
miRNAsTotal number
Perfect match containing
miRNA transcription sites
Perfect match
1-bp mismatch
2-bp mismatch722
94* (20**)

Complementarysites***
24,854219* (36**)

Genes
18,578155* (26**)

6* (4**)
10**
44**

66* (13**)69**4453**

50* (10**)
53**
763**

The search was carried out using 722 mature miRNA sequences against 24,854 sequences 10-kb upstream of
18,578 genes transcription start sites. * indicates the outcome of the 10-kb upstream sequences; ** denotesthe 1-kb upstream sequences. *** Note that some of the upstream sequences have multiple complementarysites. Details of completely complementary and 1-bp mismatched sites are shown in Tables 6 and 7.

Table 6. List of miRNAs and genes with perfectly matched sequences in the 10-kb
upstream regions
let-7igene

ID

UGAGGUAGUAGUUUGUGCUGUUC12orf61

NM_175895

position

let-7i*gene

ID

CUGCGCAAGCUACUGCCUUGCUC12orf61

NM_175895

miR-1

gene

ID

positionUGGAAUGUAAAGAAGUAUGUAU

C20orf200

NM_152757

-2827

miR-7

gene

ID

positionUGGAAGACUAGUGAUUUUGUUGUISG20L1

NM_022767-9502

miR-7-2*gene

ID

position

CAACAAAUCCCAGUCUACCUAA

ISG20L1

NM_022767-9462

miR-10agene

ID

position

UACCCUGUAGAUCCGAAUUUGUG

HOXB3
HOXB4

NM_002146
NM_024015

-5478
-1545

-257

+

origin+origin+origin-origin-origin-originoriginposition
-313

294

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 6. (Continued)

miR-10a*gene

ID

position

CAAAUUCGUAUCUAGGGGAAUA

HOXB3
HOXB4

NM_002146
NM_024015

-5437
-1504

miR-10bgene

ID

position

UACCCUGUAGAACCGAAUUUGUG

HOXD4

NM_014621-1056

miR-10b*gene

ID

position

ACAGAUUCGAUUCUAGGGGAAU

HOXD4

NM_014621-1017

miR-15bgene

IDposition

UAGCAGCACAUCAUGGUUUACA

IFT80

NM_020800-5075

miR-15b*gene

IDposition

CGAAUCAUUAUUUGCUGCUCUA

IFT80

NM_020800-5113

miR-16gene

ID

position

UAGCAGCACGUAAAUAUUGGCG

IFT80

NM_020800-5222

miR-16-2*

gene

ID

position

CCAAUAUUACUGUGCUGCUUUA

IFT80

NM_020800-5265

miR-22gene

ID

position

AAGCUGCCAGUUGAAGAACUGU

WDR81

NM_152348-2601

miR-22*gene

ID

position

AGUUCUUCAGUGGCAAGCUUUA

WDR81

NM_152348-2563

miR-25gene

ID

positionCAUUGCACUUGUCUCGGUCUGA

AP4M1

NM_004722-7915

miR-25*gene

ID

position

AGGCGGAGACUUGGGCAAUUG

AP4M1

NM_004722-7877

miR-26agene

IDposition

UUCAAGUAAUCCAGGAUAGGCU

AVIL

NM_006576-8610

miR-26a-2*

gene

ID

position

CCUAUUCUUGAUUACUUGUUUC

AVIL

NM_006576

-8572

-

originorigin-origin-origin+origin+origin+origin+origin+origin+origin+origin+origin-origin-originGenome-wide Identification and Analysis of miRNAs Complementary…

295

Table 6. (Continued)
miR-34bgene

ID

position

CAAUCACUAACUCCACUGCCAU

BTG4
FLJ46266
FLJ46266

NM_017589
NM_001100388
NM_207430

-648
-1798
-1798

miR-34b*gene

ID

position

UAGGCAGUGUCAUUAGCUGAUUG

BTG4FLJ46266
FLJ46266

NM_017589
NM_001100388
NM_207430

-611
-1835
-1835

miR-34c-3p

gene

ID

position

AAUCACUAACCACACGGCCAGG

BTG4
FLJ46266
FLJ46266

NM_017589
NM_001100388
NM_207430

-1145
-1301
-1301

miR-34c-5p

gene

ID

position

AGGCAGUGUAGUUAGCUGAUUGC

BTG4
FLJ46266
FLJ46266

NM_017589
NM_001100388
NM_207430

-1112
-1334
-1334

miR-92bgene

ID

positionUAUUGCACUCGUCCCGGCCUCC

MUC1
MUC1
MUC1
MUC1
MUC1
MUC1
MUC1

NM_001018016
NM_001018017
NM_001044390
NM_001044391
NM_001044392
NM_001044393
NM_002456

-2328
-2328
-2328
-2328
-2328
-2328
-2328

miR-92b*gene

ID

position

AGGGACGGGACGCGGUGCAGUG

MUC1
MUC1
MUC1
MUC1
MUC1
MUC1
MUC1

NM_001018016
NM_001018017
NM_001044390
NM_001044391
NM_001044392
NM_001044393
NM_002456

-2287
-2287
-2287
-2287
-2287
-2287
-2287

miR-93geneID

position

CAAAGUGCUGUUCGUGCAGGUAG

AP4M1

NM_004722-7670

miR-93*geneID

position

ACUGCUGAGCUAGCACUUCCCG

AP4M1

NM_004722

-7709

+
-

originoriginorigin+-originoriginorigin+-originoriginorigin+-originoriginorigin

+
+
+
+
+
+
+

originoriginoriginoriginoriginoriginorigin

+
+
+
+
+
+
+

originoriginoriginoriginoriginoriginorigin+origin+origin296

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 6. (Continued)

miR-106b

geneID

position

UAAAGUGCUGACAGUGCAGAU

AP4M1

NM_004722-7444

miR-106b*

gene

ID

position

CCGCACUGUGGGUACUUGCUGC

AP4M1

NM_004722-7484

miR-132

gene

ID

position

UAACAGUCUACAGCCAUGGUCG

HIC1
HIC1

NM_001098202
NM_006497

-6360
-5149

miR-132*

gene

ID

position

ACCGUGGCUUUCGAUUGUUACU

HIC1
HIC1

NM_001098202
NM_006497

-6324
-5113

miR-138

gene

ID

position

AGCUGGUGUUGUGAAUCAGGCCG

SLC12A3

NM_000339-6703

miR-138-2*

gene

ID

position

GCUAUUUCACGACACCAGGGUUSLC12A3

NM_000339-6656

miR-141gene

ID

position

UAACACUGUCUGGUAAAGAUGG

EMG1

NM_006331-6676

miR-141*gene

ID

position

CAUCUUCCAGUACAGUGUUGGA

EMG1

NM_006331-6718

miR-142-3p

gene

ID

positionUGUAGUGUUUCCUACUUUAUGGABZRAP1

NM_004758-3150

miR-142-5p

gene

ID

position

CAUAAAGUAGAAAGCACUACUBZRAP1

NM_004758-3186

miR-146b-3p

gene

ID

position

UGCCCUGUGGACUCAGUUCUGG

CUEDC2

NM_024040-3890

miR-146b-5p

gene

ID

positionUGAGAACUGAAUUCCAUAGGCU

CUEDC2

NM_024040-3854

miR-181cgene

IDposition

AACAUUCAACCUGUCGGUGAGUNANOS3

NM_001098622-2411

miR-181c*

gene

ID

position

AACCAUCGACCGUUGAGUGGACNANOS3

NM_001098622

-2373

+

origin+origin++originorigin++originorigin-origin-origin-origin-origin-origin-origin+origin+origin-origin-originGenome-wide Identification and Analysis of miRNAs Complementary…

297

Table 6. (Continued)
miR-181dgene

IDposition

AACAUUCAUUGUUGUCGGUGGGUNANOS3

NM_001098622-2226

miR-190b

gene

ID

positionUGAUAUGUUUGAUAUUGGGUU

TPM3

NM_152263-1600

miR-191geneID

position

CAACGGAAUCCCAAAAGCAGCUG

C3orf60
C3orf60
C3orf60
C3orf60
DALRD3

NM_199069
NM_199070
NM_199073
NM_199417
NM_001009996

-947
-947
-491
-947
-2110

miR-191*geneID

position

GCUGCGCUUGGAUUUCGUCCCC

C3orf60
C3orf60
C3orf60
C3orf60
DALRD3

NM_199069
NM_199070
NM_199073
NM_199417
NM_001009996

-989
-989
-533
-989
-2068

miR-195

gene

ID

position

UAGCAGCACAGAAAUAUUGGC

BCL6B

NM_181844-5363

miR-195*

gene

ID

position

CCAAUAUUGGCUGUGCUGCUCC

BCL6B

NM_181844-5401

miR-196agene

ID

positionUAGGUAGUUUCAUGUUGUUGGG

HOXB9
HOXC9

NM_024017
NM_006897

-6080
-8331

miR-196a*

gene

ID

position

CGGCAACAAGAAACUGCCUGAG

HOXC9

NM_006897-8294

miR-196bgene

ID

positionUAGGUAGUUUCCUGUUGUUGGG

HOXA9

NM_152739-4019

miR-200c

geneID

positionUAAUACUGCCGGGUAAUGAUGGA

EMG1

NM_006331-7089

miR-200c*

gene

ID

position

CGUCUUACCCAGCAGUGUUUGG

EMG1

NM_006331

-7128

-

origin-origin++++-originoriginoriginoriginorigin++++-originoriginoriginoriginorigin+origin+origin-originorigin-origin-origin-origin-origin298

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 6. (Continued)

miR-208b

geneID

position

AUAAGACGAACAAAAGGUUUGU

MYH6

NM_002471-9745

miR-210gene

ID

position

CUGUGCGUGUGACAGCGGCUGA

C11orf35
KIAA1542

NM_173573
NM_020901

-7354
-8353

miR-212gene

ID

position

UAACAGUCUCCAGUCACGGCC

HIC1
HIC1

NM_001098202
NM_006497

-6000
-4789

miR-219-1-3p

gene

ID

position

AGAGUUGAGUCUGGACGUCCCG

RING1
RXRB
RXRB
RXRB

NM_002931
NM_021976
NM_021976
NM_021976

-613
-7237
-7241
-7245

miR-219-5p

gene

ID

positionUGAUUGUCCAAACGCAAUUCU

RING1
RXRB
RXRB
RXRB

NM_002931
NM_021976
NM_021976
NM_021976

-654
-7196
-7200
-7204

miR-301agene

ID

position

CAGUGCAAUAGUAUUGUCAAAGC

PRR11

NM_018304-4561

miR-320gene

ID

position

AAAAGCUGGGUUGAGAGGGCGAPOLR3D

NM_001722

miR-324-3p

gene

ID

position

ACUGCCCCAGGUGCUGCUGG

DLG4

NM_001365-3589

miR-324-5p

gene

ID

position

CGCAUCCCCUAGGGCAUUGGUGU

DLG4

NM_001365-3626

miR-345

gene

ID

position

GCUGACUCCUAGUCCAGGGCUCSLC25A29

NM_001039355-1353

miR-375

geneID

positionUUUGUUCGUUCGGCUCGCGUGACRYBA2CRYBA2CRYBA2

NM_005209
NM_057093
NM_057094

-8270
-8264
-8264

-110

-

origin+originorigin++originorigin+++originoriginoriginorigin+++originoriginoriginorigin+origin+origin-origin-origin+origin-originoriginoriginGenome-wide Identification and Analysis of miRNAs Complementary…

299

Table 6. (Continued)
miR-378

gene

ID

positionACUGGACUUGGAGUCAGAAGGOSBPL10

NM_017784-4582

miR-425

gene

ID

position

AAUGACACGAUCACUCCCGUUGA

C3orf60
C3orf60
C3orf60
C3orf60
C3orf60
DALRD3

NM_199069
NM_199070
NM_199073
NM_199074
NM_199417
NM_001009996

-1420
-1420
-964
-254
-1420
-1637

miR-425*

gene

ID

position

AUCGGGAAUGUCGUGUCCGCCC

C3orf60
C3orf60
C3orf60
C3orf60
C3orf60
DALRD3

NM_199069
NM_199070
NM_199073
NM_199074
NM_199417
NM_001009996

-1461
-1461
-1005
-295
-1461
-1596

miR-483-3p

gene

ID

position

UCACUCCUCUCCUCCCGUCUU

IGF2AS

NM_016412-6345

miR-483-5p

gene

ID

position

AAGACGGGAGGAAAGAAGGGAG

IGF2AS

NM_016412-6305

miR-484

gene

ID

position

UCAGGCUCAGUCCCCUCCCGAUKIAA0430
NDE1

NM_014647
NM_017668

-149
-6946

miR-497

gene

ID

position

CAGCAGCACACUGUGGUUUGU

BCL6B

NM_181844-5051

miR-497*

gene

ID

position

CAAACCACACUGUGGUGUUAGA

BCL6B

NM_181844-5091

miR-548c-5p

gene

ID

position

AAAAGUAAUUGCGGUUUUUGCC

ABTB1
ABTB1
ABTB1
ANXA3
C1orf94
C20orf43
CDC23
CDC42EP4
EBI2

NM_032548
NM_172027
NM_172028
NM_005139
NM_032884
NM_016407
NM_004661
NM_012121
NM_004951

-8217
-8217
-8217
-248
-1735
-8342
-6363
-3863
-8486

-

+
+
+
+
+
-

originoriginoriginoriginoriginorigin

+
+
+
+
+
-

originoriginoriginoriginoriginorigin+origin+origin+-originorigin+origin+origin

+
+
+
+

300

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 6. (Continued)

miR-548c-5p

gene

ID

position

FGF23
FOXC2
GNB5
GPR141LBP
MANBALMANBALMTHFSDNLGN4XNLGN4X
NR0B1
NSUN5
NSUN5
OR2T8
PDE6C
PLAC1
PSTPIP2
RBP4
RNF6
RNF6
RNF6
UBQLN2ZNF331ZNF418

NM_020638
NM_005251
NM_006578
NM_181791
NM_004139
NM_001003897
NM_022077
NM_022764
NM_020742
NM_181332
NM_000475
NM_018044
NM_148956
NM_001005522
NM_006204
NM_021796
NM_024430
NM_006744
NM_005977
NM_183043
NM_183044
NM_013444
NM_001079907
NM_133460

-9861
-8776
-3189
-8087
-6809
-4841
-4841
-3325
-9292
-8474
-5731
-6978
-6978
-7046
-3974
-1244
-798
-7378
-7624
-8154
-8292
-1751
-9126
-732

miR-548d-5p

gene

ID

position

AAAAGUAAUUGUGGUUUUUGCCADAM23
ALDH1L2
C1orf94
C8B
CLEC12A
CLEC12A
FSTL5
GC
GJB3
GJB3
MAPKAPK
5
MAPKAPK
5
NANOS1NLRP4POLR3ASOX30SOX30SPAG11B
SPAG11B
TAS2R39
WNT5A
WWP2

NM_003812
NM_001034173
NM_032884
NM_000066
NM_138337
NM_201623
NM_020116
NM_000583
NM_001005752
NM_024009

-7057
-914
-1790
-333
-525
-525
-9873
-6013
-3678
-2557

+
+
+
+
+
+
+

NM_003668

-2843

+

NM_139078

-2843

+

NM_199461
NM_134444
NM_007055
NM_007017
NM_178424
NM_058206
NM_058207
NM_176881
NM_003392
NM_199424

-9261
-8220
-8654
-621
-621
-5668
-5668
-2594
-7212
-2131

+
+
+
+
+
-

+
+
+
+
+
+
+
+
+
+
+
+

Genome-wide Identification and Analysis of miRNAs Complementary…

301

miR-565

gene

ID

GGCUGGCUCGCGAUGUCUGUUUSACM1L

NM_014016

miR-572

gene

ID

GUCCGCUCGGCGGUGGCCCA

RNPS1

NM_006711

miR-574-5p

gene

ID

positionUGAGUGUGUGUGUGUGAGUGUGU

JPH2
JPH2
LOC645037(GAGE2C)
OPN4
OPN4
PRKCE

NM_020433
NM_175913

-1016
-1016

+
+

NM_001098411

-6003

+

NM_001030015
NM_033282
NM_005400

-283
-283
-815

+
+
+

miR-580gene

ID

positionUUGAGAAUGAUGAAUCAUUAGG

SKP2
SKP2

NM_005983
NM_032637

-4159
-4159

miR-584

gene
NM_001101
342

ID

positionmiR-596gene

ID

position

AAGCCUGCCCGGCUCCUCGGG

ARHGEF10

NM_014629-6737

miR-606

geneID

position

AAACUACUGAAAAUCAAAGAUOSBPL9OSBPL9OSBPL9NM_148904
NM_148905
NM_148907

-5101
-5101
-5131

miR-607

geneID

position

GUUCAAAUCCAGAUCUAUAAC

LCOR

NM_032440-4276

miR-611gene

ID

position

GCGAGGACCCCUCGGGGUCUGAC

FEN1

NM_004111

miR-616

gene

ID

position

AGUCAUUGGAGGGUUUGAGCAG

MBD6

NM_052897-3672

miR-616*

gene

ID

position

ACUCAAAACCCUUCAGUGACUU

MBD6

NM_052897-3632

UUAUGGUUUGCCUGGGACUGAG

NM_001101342

position-443+

originposition
-24

-923

-156

-

+
+

originorigin+origin-origin++++origin+origin+origin+origin302

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 6. (Continued)

miR-632

gene

ID

position

GUGUCUGCUUCCUGUGGGA

C17orf75

NM_022344-7999

miR-636

gene

ID

positionUGUGCUUGCUCGUCCCGCCCGCA

JMJD6
JMJD6
MFSD11

NM_001081461
NM_015167
NM_024311

-9689
-9689
-1213

miR-649

gene

ID

position

AAACCUGUGUUGUUCAAGAGUCSLC7A4

NM_004173-1654

miR-658
GGCGGAGGGAAGUAGGUCCGUUG
GU

gene

ID

position

EIF3EIP

NM_016091-5052

miR-659

gene

ID

position

CUUGGUUCAGGGAGGGUCCCCAANKRD54
EIF3EIP

NM_138797
NM_016091

-3418
-1649

miR-661

gene

ID

position

UGCCUGGGUCUCUGGCCUGCGCGU

PLEC1
PLEC1
PLEC1
PLEC1

NM_201381
NM_201382
NM_201383
NM_201384

-492
-1332
-2705
-5639

miR-801

geneID

positionGAUUGCUCUGCGUGCGGAAUCGAC

TAF12

NM_005644-5568

miR-923

gene

ID

position

GUCAGCGGAGGAAAAGAAACU

NLE1
NLE1

NM_001014445
NM_018096

-8906
-8906

miR-935

gene

ID

position

CCAGUUACCGCUUCCGCUACCGC

CACNG6CACNG6CACNG6

NM_031897
NM_145814
NM_145815

-9926
-9926
-9926

miR-940

gene

ID

position

AAGGCAGGGCCCCCGCUCCCC+

origin+originoriginorigin-origin+origin+originorigin-originoriginoriginorigin+origin-originorigin-originoriginorigin

RNPS1
NM_006711-4010+ origin
RNPS1
NM_080594-3693+ origin
Mature miRNAs and genes with perfectly matched sequences in regions 10-kb upstream of the TSSs. The
nucleotide sequence below each miRNA designation shows the mature miRNA sequence. The gene nameindicates the gene located downstream of the miRNA complementary site. ID indicates the accession ID ofeach transcript. Position indicates the nucleotide position relative to the 5′-end of the miRNA counted fromthe transcription start site (+1). The +/– shows the orientation of the miRNA target sequences; this symbol
indicates the relative direction with respect to downstream mRNA transcription (+/– = same/opposite). In theGenome-wide Identification and Analysis of miRNAs Complementary…

303

most right colume, ―origin‖ indicates the locus from which own miRNA is originally transcribed. Blue
indicates the sites appearing perfectly complementarity to the miRNAs transcribed from the different
locations.

Figure 1. Positions containing 1-bp mismatches in 1-kb upstream regions of mRNA coding regions.
The mismatched positions in miR-548b-5p (A), miR-548c-5p (B), miR-548d-5p (C), miR-566 (D) and miR574-5p (E) with target genes within the 1-kb upstream regions of the mRNA coding regions are shown fromthe 5′-end (+1) of the guide strand. The red bar indicates miRNA that is perfectly complementary to the target
sequence when the nucleotide is changed to A. Green bar, U; Yellow, G; Blue, C.

Search for and Analysis of miRNAs with Complementary
Sequences Containing 1- and 2-bp Mismatches within 1-kb of theUpstream Sequences
In a study on RNAi, target mRNAs carrying a few base mismatches with siRNAs havebeen demonstrated to be silenced to various degrees according to the position and the identity

304

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.

of the mismatched nucleotide [29, 30]. Furthermore, miRNAs recognize target mRNA by the―seed sequence‖ positioned between nucleotides 2–8 from the miRNA 5′-end [31-35], but notby the entire sequence. In RNAi, the identities of off-targets are also determined bycomplementarity to the seed regions of the introduced siRNA guide strand [36-38]. In a study
on RNAa, a sequence located at position –645 relative to the TSS in the E-cadherin promoter
was reported to function, although it has a few base mismatches with miR-373 [22]. Then, we
searched for 1- and 2-bp mismatched positions and nucleotides of human miRNAs with the 1kb of the upstream sequences.
We found 69 sites that had 1-bp mismatched sequences to 10 miRNAs in the 1-kb
upstream regions of 53 genes (Tables 5 and 7). The complementary sites of five miRNAs(miR-548b-5p, miR-548c-5p, miR-548d-5p, miR-566 and miR-574-5p) recognized multiple
1-bp mismatched sites; the mismatched positions and nucleotides of these miRNAs are
summarized in Figure 1. The major mismatched sites of miR-548b-5p and miR-574-5p wereidentical at nucleotide positions 19 and 17, respectively, from the 5′-end of each miRNA(Figure 1A and E). When G is changed to U at position 19 in miR-548b-5p or A is changed to
U at position 17 in miR-574-5p, the majority of these miRNAs can pair with perfectcomplementarity, indicating mismatched nucleotides are remarkably characteristic. The major
mismatched nucleotides in miR-548c-5p, miR-548d-5p and miR-566 were found at position
11 or 12 corresponding to the central region of the miRNAs (Figure 1B–D). In total, 87 % of
1-bp mismatched nucleotides were found in the non-seed region, and the remaining 13 % ofthem were mismatched in the seed region. Thus, the mismatched positions and nucleotides
were highly biased. Total of 4453 target sites containing 2-bp mismatches with 44 miRNAs
were found (Table 5). Among them, miR-548c-3p, miR-548d-5p, miR-566 and miR-574-5p
had more than ten mismatched target sites (Figure 2). The major mismatched positions in thetarget sequences were unevenly positioned at 11 and 18 in miR-566 (212 of 276 sites (77 %),Figure 2A). In the cases of miR-548d-5p and miR-548c-3p, the mismatched nucleotides wereconcentrated at position 12 and 7, respectively (Figure 2B and C). The miR-574-5p (5′UGAGUGUGUGUGUGUGAGUGUGU-3′) is highly GU repeated sequence without thenucleotides at positions 3 and 17 where Us are changed to As. The 3753 of 4060 mismatchedtarget sites (92 %) of miR-574-5p showed simultaneous nucleotide changes from As to Us atpositions 3 and 17 (Figure 2D), suggesting that these sites might be highly distributed GU
repeated sequences. In total, 2-bp mismatched positions and nucleotides are also remarkably
biased similar to the 1-bp mismatched sites. In total, only 12.0 % of 2-bp mismatchednucleotide positions were found in the seed region, and the remaining 88.0 % of them weremismatched in the non-seed region, except for the miR-574-5p mismatched sites.

Changes in Gene Expression by Targeting Upstream Sequenceswith miRNASix miRNAs, which transcribed from the different locations other than the upstream
regions, were found to be completely complementary at 66 sites in the 10-kb upstream
regions of 50 genes (Tables 5 and 6). To investigate whether miRNAs complementary to
upstream sequences of mRNAs are general regulators of downstream genes, chemicallysynthesized miRNAs were transfected into the cells and changes in the expression ofdownstream genes were measured by real-time PCR. Similar to endogenous miRNAs, theGenome-wide Identification and Analysis of miRNAs Complementary…

305

synthetic mature miRNAs were annealed with opposite strand RNAs to form duplex RNAs
(Figure 3A).
To investigate the effects of synthetic miRNAs against the completely matchedsequences in the 3′ UTRs, psiCHECK-miR-target constructs containing three copies ofcompletely matched target sequences in the 3′ UTR of the Renilla luciferase gene were made(Figure 3B). The synthetic miRNA and the corresponding psiCHECK-miR-target constructwere transfected simultaneously into HeLa cells, and luciferase activity was measured. All of
six miRNAs (miR-378, miR-548c-5p, miR-548d-5p, miR-572, miR-574-5p, and miR-606)
reduced Renilla luciferase activity (Figure 3C), indicating that these synthetic miRNAs canrepress gene expression when completely matched sequences are contained in the 3′ UTRs.
Next, the endogenous expression of six miRNAs labelled with blue in Table 6 were
examined by RT-PCR in human HeLa, PC-3, and MCF-7 cells. For detection of miRNAs by
RT-PCR, we used miRNA-specific stem–loop primers for reverse transcription according to
the method reported by Chen et al. [28]. The synthetic miRNAs transfected into HeLa cells
were detected using stem–loop RT primers and PCR primers specific for each miRNA.
Endogenous miR-378 and miR-574-5p were detected in all three cell lines, but miR-548c-5p,
miR-548d-5p, miR-572 and miR-606 were not detected in any of the cell lines tested (Figure
4).
We examined the downstream gene expression changes caused by the introduction of
miRNAs; the expression of 8 genes that had sequences corresponding to the transfected
miRNAs in their upstream regions were determined by real-time RT-PCR (Figure 5). The
expression of OSBPL10 was reduced significantly in two of three cell lines by transfection
with miR-378 (Figure 5A). The expression levels of four genes, RNF6, RNPS1, C20orf43
and ANXA3 were lowered in one of three or two cell lines by miR-548c-5p, miR-572, miR548c-5p and miR-548c-5p, respectively (Figure 5B-E). The expression levels of OSBPL9 and
CDC23 genes were significantly increased by transfection of miR-606 and miR-548c-5p,
respectively, in one of three cell lines (Figure 5F,G). Thus, the mRNA expression was
differentially regulated in each cell type. Furthermore, the effects of miRNA transfection
varied depending on the downstream genes. In PC-3 cells, RNF6, C20orf43 were reduced bymiR-548c-5p transfection, but ANXA3 and the other genes (GNB5, MANBAL, and RBP4)
containing perfect complementary sites with miR-548c-5p in the upstream regions were not
reduced (Figure 5, data not shown). POLR3A was decreased with miR-548d-5p, but theexpression levels of the other genes (MAPKAPK5 and GJB3) were not changed in PC-3 cells(Figure 5, data not shown). In this study, when mRNA levels were significantly increased, thechanges were not greater than ~30 % in any instances. Similarly, miRNA transfection
resulted in ~40 % downregulation of mRNAs in all cases of reduction. Although the rate ofchange of gene expression was not so abundant, the effect of miRNA was obviously dosedependent (Figure 6). Note that miR-378 and miR-574-5p are endogenously expressed in all
three cell lines, which could lead to an underestimation of their effects on gene expressions.

Figure 2
306

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
C

Number of complementarysites with 2-bp mismatch

A

UG

GC

AC

G

U
A

miR-566
100
90
80
70
60
50
40
30
20
10 A UC UA
0 1 2 3 4 5

A

G

G
UC
6

7 8

G

9 10 11 12 13 14 15 16 17 18 19

Nucleotide position
AB

miR-548d-5p

Number of complementarysites with 2-bp mismatch

U

11
C
10
9
8
7
6
5
4
U
3
A A
2U G
U G C C A CG
G U
1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Nucleotide position
AC

A

C

miR-548c-3p

Number of complementarysites with 2-bp mismatch

G

11
C
10
9
8
7
A
6
5
4
G
3
2
C
A
C
1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

U

D

miR-574-5p

Number of complementarysites with 2-bp mismatch

Nucleotide position

4500
U
U
4000
3500
3000
2500
2000
1500
1000
500
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Nucleotide position

Figure 2. Positions containing 2-bp mismatches in 1-kb upstream regions of mRNA coding regions.
The mismatched positions in miR-566 (A), miR-548d-5p (B), miR-548c-3p (C) and miR-574-5p (D) withtarget sites within the 1-kb upstream sequences of the mRNA coding regions are shown from the 5′-end (+1)
of the guide strand. The red bar indicates miRNA that is perfectly complementary to the target sequencewhen the nucleotide is changed to A. Green bar, U; Yellow, G; Blue, C.

Genome-wide
Figure3 Identification and Analysis of miRNAs Complementary…

307

A
miR-378

miR-548c-5p

miR-548d-5p

miR-572

miR-574-5p

miR-606

C
UGU
CU
5’-CU CUGACUCCAGGUCC GU-3’
5’-AAAAGUAAUUG GGUUUUUGCC-3’ 5’-AAAAG AAUUGUGGUUUUUGCC-3’|| |||||||||:|||| ||||||||||||| |:||||||||||| ||:|||||||||||GA GACUGAGGUUCAGG CA-5’ 3’-CGUUUUCAUUAAC CUAAAAAC-5’ 3’-CGUUUUC UUGACACCAAAAAC-5’
3’-G A
--U
U
U
C
U
-A
A U
U-3’
5’-C UUGGUUUUUAGUAGUUU A-3’
5’-GGCCGCUGC GGGCGGA GG-3’ 5’-UG G GUGUGUGUGUGAGUGUG| |||:||||:||||||||||||||:|| |:||||||| | |||||||::||||:||
3’-UAGAACUAAAAGUCAUCAAA-5’
CCCGGUGGCG CUCGCCU
3’-ACAC C CACACACGUACUCGCA-5’
A
3’-A
G
G-5’
- -

B
SV40
enhancer/promoter

Renilla Luciferase

Synthetic poly (A)Target site (x3)

XXXXXXXXXXXXXXXXXXXXXX

Complete match miRNA target| | | | | | | | | | | | | | | | | | | | | |XXXXXXXXXXXXXXXXXXXXXX

miRNA

60
40
20
0

Relative luc activity (%)

5

120

50

100
80
60
40
20
5

50

80
60
40
20
0

500 (ng)

dsControl miR-572

0100

500 (ng)

5

120

50

Relative luc activity (%)

80

dsControl miR-548c-5p
120dsControl miR-548d-5p
120
100
80
60
40
20
0

500 (ng)

dsControl miR-574-5p

100

5Relative luc activity (%)

100

Relative luc activity (%)

dsControl miR-378
120

Relative luc activity (%)

Relative luc activity (%)

C

120

50

500 (ng)

dsControl miR-606

100

80
60
40
20
0
5

50

500 (ng)

80
60
40
20
0
5

50

500 (ng)

Figure 3. Effects of miRNAs against completely matched targets in 3′ UTRs.
(A) Chemically synthesized miRNAs; the red characters indicate mature miRNAs. (B) The schematic
structure of the target sequence expression construct, psiCHECK-miR-target, which contains three copies ofmatched target sequences in the 3′ UTR of the Renilla luciferase gene. (C) Silencing activity of synthetic
miRNAs; the activity was monitored using psiCHECK-miR-target constructs. The silencing activity is shownas the percentage of relative luc activity normalized to the dsControl transfection (100 %).

Figure 4308

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.

miR-548c-5p

dsControl

miR-548c-5pMarkermiR-574-5p

dsControl

miR-574-5p

dsControl

dsControlmiR miR-21 -548c-5p

miR miR-21 -574-5p

MarkermiR-606

dsControl

miR-606

dsControlmiR miR-21 -606

miR-572

dsControl

miR-572

miR-548d-5p

dsControl

dsControlRT

dsControlmiR miR-21 -572

non-RT

dsControl

miR-548d-5p

miR
miR-21 -548d-5p

miR-378

miR-378

miR-378

MarkermiR-21

dsControl

Transf ected Detected
dsRNA
miRNA

A

(bp)
2000
1500
1000

RT

non-RT

B

RT

non-RT

500
400
300
200
100

miR-21

miR-378
miR-548c-5p
miR-548d-5p
miR-572
miR-574-5p

miR-606
HeLa

PC-3

MCF-7

Figure 4. Detection of endogenous miRNAs by RT-PCR.
(A) Detection of miRNAs by stem-loop RT primers specific for each miRNA. The synthetic miRNAs weretransfected into HeLa cells, and each miRNA was detected with specific stem–loop RT primers. miR-21 wasused as transfection and detection control. Expression of each endogenous miRNA was detected whendsControl alone was transfected and intended miRNA was detected by specific primers for RT-PCR.
Expected sizes of each miRNA is 64, 61, 60, 61, 61, 65 and 62 bp for miR-21, miR-378, miR-548c-5p, miR548d-5p, miR-572, miR-574-5p and miR-606, respectively. (B) Endogenous miRNAs detected using stemloop RT-PCR in HeLa, PC-3, and MCF-7 cells.

Table 7. List of miRNAs and genes with 1-bp mismatched complementary sequences
within the 1-kb upstream regions
miR-190UGAUAUGUUUGAUAUAUUAGGU

gene

ID

RNF212

NM_194439

miR-297
AUGUAUGUGUG
CAUGUGCAUG

gene

ID

C12orf54

NM_152319

position

sub

-974

7-U

position

sub

-151

15-A

sequence (5' ⇒ 3')
UGAUAUUUUUGAUAUAUUAGGU
sequence (5' ⇒ 3')
AUGUAUGUGUGCAUAUGCAUG++

Genome-wide Identification and Analysis of miRNAs Complementary…
positionsubsequence (5' ⇒ 3')

3

-914

19-U

AAAAGUAAUUGUGGUUUUUGCC

C8B

NM_000066

-333

19-U

CLEC12A

NM_138337

-525

19-U

CLEC12A

NM_201623

-525

19-U

SOX30

NM_007017

-621

19-U

SOX30

NM_178424

-621

19-U

gene

ID

positionsub

ACTR10

NM_018477

-735

13-U

miR-548b-5p

gene

ID
NM_00103417

AAAAGUAAUUGUGGUUUUGGCC

ALDH1L2

miR-548c-5p
AAAAGUAAUUGCGGUUUUUGCC

NM_00103417
ALDH1L2

3

-914

12-U

AOX1

NM_001159

-468

20-A

C5orf32

NM_032412

-715

13-U

C8B

NM_000066

-333

12-U

CCDC90

NM_00103171

A

3

-913

8-G

CLEC12A

NM_138337

-525

12-U

CLEC12A

NM_201623

-525

12-U

LPO

NM_006151

-167

22-A

MS4A4A

NM_148975

-446

13-A

OR10W1

NM_207374

-104

13-A

PGM2L1

NM_173582

-789

13-U

PSMA1

NM_002786

-454

9-C

NM_00100148
PTER

4

-887

2-G

PTER

NM_030664

-887

2-G

AAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCC
sequence (5' ⇒ 3')
AAAAGUAAUUGCUGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGC
GGUUUUUACC
AAAAGUAAUUGCUGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAGUUGC
GGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGC
GGUUUUUGCA
AAAAGUAAUUGC
AGUUUUUGCCAAAAGUAAUUGC
AGUUUUUGCCAAAAGUAAUUGCUGUUUUUGCCAAAAGUAACUGCGGUUUUUGCCAGAAGUAAUUGC
GGUUUUUGCCAGAAGUAAUUGC
GGUUUUUGCC

309

+
+
+
+
+

+

+
+
-

+
+
+
+
+

-

310

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 7. (Continued)

miR-548c-5p

miR-548d-5p
AAAAGUAAUUGUGGUUUUUGCC

miR-566
GGGCGCCUGUG
AUCCCAAC

gene

ID

SOX30

sub

NM_007017

-621

12-U

SOX30

NM_178424

-621

12-U

gene

ID

position

sub

ANXA3

NM_005139

-248

12-C

C5orf32

NM_032412

-658

22-A

PSTPIP2

NM_024430

-798

12-C

ZNF418

NM_133460

-732

12-C

gene

ID

positionsub

C14orf2

NM_004894

-979

11-A

C20orf24

NM_018840

-980

18-G

C20orf24

NM_199483

-980

18-G

-267

12-G

GGGCGCCUGUGGUCCCAAC+GGGCGCCUGUAAUCCCAAC+

NM_00101741
CT45-1

7
NM_00103172

miR-574-5p
UGAGUGUGUGUGUGUGAGUGUG
U

sequence (5' ⇒ 3')
AAAAGUAAUUGUGGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCC

positionsequence (5' ⇒ 3')
AAAAGUAAUUGC
GGUUUUUGCCAAAAGUAAUUGUGGUUUUUGCA
AAAAGUAAUUGC
GGUUUUUGCCAAAAGUAAUUGC
GGUUUUUGCC
sequence (5' ⇒ 3')
GGGCGCCUGUAAUCCCAACGGGCGCCUGUGAU
CCCAGCGGGCGCCUGUGAU
CCCAGC

GSTCD

0

-444

11-A

PRRG2

NM_000951

-788

11-A

RASL11A

NM_206827

-104

11-A

RLN3

NM_080864

-591

11-A

ZNF594

NM_032530

-466

11-A

gene

ID
NM_00108051

positionsubsequence (5' ⇒ 3')

BAHCC1

9

-40

17-U

UGAGUGUGUGUGUGUGUGUGUGU

C4orf31

NM_024574

-206

17-U

CALN1

NM_031468

-899

21-G

CD300A

NM_007261

-496

12-A

CD300A

NM_007261

-556

10-U

EMP2

NM_001424

-645

17-U

GGGCGCCUGUAAUCCCAACGGGCGCCUGUAAUCCCAACGGGCGCCUGUAAUCCCAACGGGCGCCUGUAAUCCCAACUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGAGUGGGUUGAGUGUGUGUAUGUGAGUGUGUUGAGUGUGUUUGUGUGAGUGUGUUGAGUGUGUGUGUGUGUGUGUGU

+
+

+
+
+
+

+
-

+
+
-

-

Genome-wide Identification and Analysis of miRNAs Complementary…

311

Table 7. (Continued)
miR-574-5p

gene

ID

position

sub

ERC1

NM_015064

-302

3-U

ERC1

NM_178037

-302

3-U

ERC1

NM_178038

-302

3-U

ERC1

NM_178039

-302

3-U

ERC1

NM_178040

-302

3-U

GDF5

NM_000557

-159

17-U

GLT25D2

NM_015101

-23

17-U

MAP3K13

NM_004721

-197

17-U

MUC16

NM_024690

-194

17-U

NM_00103001
OPN4

5

-154

17-U

OPN4

NM_033282

-154

17-U

NM_00100445
OR1N2

7

-423

17-U

PI3

NM_002638

-801

3-U

NM_00101303

miR-646
AAGCAGCUGCCUCUGAGGCSORCS1

1

-756

17-U

SORCS1

NM_052918

-756

17-U

SYT14

NM_153262

-659

17-U

TRPA1

NM_007332

-273

17-U

VCX

NM_013452

-143

17-U

VCX3A

NM_016379

-142

3-U

gene

ID

positionsub

PRELID2

NM_138492

-419

2-G

PRELID2

NM_182960

-451

2-G

PRELID2

NM_205846

-451

2-G

sequence (5' ⇒ 3')
UGUGUGUGUGUGUGUGAGUGUGUUGUGUGUGUGUGUGUGAGUGUGUUGUGUGUGUGUGUGUGAGUGUGUUGUGUGUGUGUGUGUGAGUGUGUUGUGUGUGUGUGUGUGAGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGUGUGUGUGUGUGUGAGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGAGUGUGUGUGUGUGUGUGUGUUGUGUGUGUGUGUGUGAGUGUGU
sequence (5' ⇒ 3')
AGGCAGCUGCCUCUGAGGC
AGGCAGCUGCCUCUGAGGC
AGGCAGCUGCCUCUGAGGC

+
+
+

+
+

+

+
+
+
+
+

+
+
+

312

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.
Table 7. (Continued)

miR-649
AAACCUGUGUUGUUCAAGAGUC

Figure 5

miR-940
AAGGCAGGGCCC
CCGCUCCCC

gene

ID

positionsub

ZNF211

NM_006385

-289

19-U

ZNF211

NM_198855

-289

19-U

gene

ID

positionsub

GABRA2

NM_000807

-249

15-C

sequence (5' ⇒ 3')
AAACCUGUGUUGUUCAAGUGUCAAACCUGUGUUGUUCAAGUGUC
sequence (5' ⇒ 3')
AAGGCAGGGCCC
CCCCUCCCC-+

Mature miRNAs and genes with complementary sequences containing 1-bp mismatches in the 1-kb
upstream sequences of the TSSs. The nucleotide sequence below each miRNA designation shows themature miRNA sequence. The gene name indicates the gene located downstream of the miRNA
complementary site. ID indicates the accession ID of each transcript. Position indicates the nucleotide
position relative to the 5′-end of the miRNA counted from the transcription start site (+1). ―Sub‖
represents the mismatched position and nucleotide in miRNA. The red characters in the miRNA
sequences show sequences mismatched. The +/– shows the orientation of the miRNA target sequences;
this symbol indicates the relative direction with respect to downstream mRNA transcription (+/– =same/opposite).

60

*

40
20

60
40

20

100

*

80
60
40
20

ND
HeLa PC-3 MCF-7

F

dsControl miR-606

140

*

120
100
80
60

40
20
0

HeLa

PC-3 MCF-7

*

80
60
40
20
0

HeLa PC-3 MCF-7

OSBPL9 mRNA level (%)

dsControl miR-548c-5p

ANXA3 mRNA level (%)

*

0

120

0

100

80

HeLa PC-3 MCF-7

E

dsControl miR-572

120

100

0

C

D
C20orf43 mRNA level (%)*

dsControl miR-548c-5p

120dsControl miR-548c-5p
140
120
100

G

dsControl miR-548c-5p
140

120
100

*

80
60

40
20
0

HeLa PC-3 MCF-7

*

80
60
40
20
0

HeLa PC-3 MCF-7

HeLa PC-3 MCF-7

H
POLR3A mRNA level (%)

RNF6 mRNA level (%)

100
80

B

RNPS1 mRNA level (%)

dsControl miR-378

120

CDC23 mRNA level (%)

OSBPL10 mRNA level (%)

A

120

dsControl miR-548d-5p

100

*

80
60
40
20
0

ND

ND

HeLa PC-3 MCF-7

Figure 5. Changes in mRNA expression levels after transfection of miRNAs.
Downstream mRNAs were detected by real-time RT-PCR in HeLa, PC-3, and MCF-7 cells. The
combinations of downstream mRNA/miRNA were as follow: (A) OSBPL10/miR-378, (B) RNF6/miR-548c5p, (C) RNPS1/miR-572, (D) C20orf43/miR-548c-5p, (E) ANXA3/miR-548c-5p, (F) OSBPL9/miR-606, (G)CDC23/miR-548c-5p, and (H) POLR3A/miR-548d-5p. The expression level of each mRNA was normalized
to the dsControl, which is presented as 100 %. The values are shown as the means ± SD from at least three
independent experiments. Statistical significance was calculated using paired two-tailed Student's t-test; *P <0.05. ND, not determined.

Genome-wide Identification and Analysis of miRNAs Complementary…

313

Figure 6. Dose-dependent effect of miRNA on gene expression. (A) Downstream RNPS1 mRNA wasdetected by real-time RT-PCR in HeLa cells transfected with miR-572 (5, 50 and 500 ng/well). (B)Downstream OSBPL10 mRNA was detected by real-time RT-PCR in PC-3 cells transfected with miR-378(5, 50 and 500 ng/well). The expression level of each mRNA was normalized to the dsControl, which ispresented as 100 %. The values are shown as the means ± SD.

Discussion
This study is the first report of global identification of miRNAs that contain completely
or near-completely (i.e., 1- or 2-bp mismatched) complementary sequences to sites in the 10kb or 1-kb upstream regions of human mRNAs. In total, 66 sites within 10-kb upstream of thecoding regions were found to be completely complementary with miRNAs transcribed fromthe different locations (Tables 5 and 6). Although each of miR-378, miR-572, and miR-606
has only one complementary site, miR-548c-5p, miR-548d-5p and miR-574-5p appeared tobe complementary with the upstream sequences of 26, 17, and 4 genes, respectively. The
miR-548c-5p and miR-548d-5p miRNAs are members of a family of human miRNA genes
derived from short miniature inverted-repeat transposable elements (MITEs) duplicated inmany genomic regions, and are capable of forming highly stable hairpin loop recognized by
the RNAi enzymatic machinery and processed to form 22 bp mature miRNA sequences [39].
Thus, the many complementary sites of miR-548 family in the upstream regions might
represent evolutionary specific regulation of gene expression related to species
diversification.
The mismatched positions and types of nucleotides spanning the miRNAs appeared to behighly biased based on searches for sequences containing 1- and 2-bp mismatches withmature miRNAs in the 1-kb upstream region (Figure 1 and 2). The seed sequences ofmiRNAs are the primary region for 3′ UTR target recognition [31-35]. In RNAi, the identities
of off-targets are determined by complementarity to the seed regions of the introduced siRNA[36-38]. In this study, among human miRNAs, 10 and 44 miRNAs were revealed to have 1and 2-bp mismatched sites, respectively, in the 1-kb upstream region of TSSs (Table 5). The
search result of 1-bp mismatched target sites of miRNAs showed that 87 % of 1-bp
mismatched sites and 88 % of 2-bp mismatched sites were located in the non-seed regions,314

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.

except for the target sites of miR-574-5p. The results suggest that these miRNAs are able toact on their upstream target sites with perfect complementarity in the seed region, if themechanism of transcriptional regulation on the upstream region is similar to that of the known
miRNA function. In RNAi, mismatches at the central region of the siRNA guide strand havebeen shown to be highly sensitive to knockdown efficiency, but the mismatches in the othersite are the most tolerable [29, 30]. In both 1-bp and 2-bp mismatched site analyses, miR548d-5p and miR-566 showed major mismatched site at positions 12 and 11 (Figs. 1 and 2),
respectively, indicating that these miRNAs can act on the upstream regions through the
known miRNA machinery but not RNAi machinery. In an analogous fashion, miR-548c-5palso has major mismatched site at position 12 (Figure 1B). In contract, major mismatched siteof miR-548c-3p was position 7, and the miR-574-5p has mismatches at positions 3 and 17(Figure 2). The positions 7 and 3 are involved in the seed region, suggesting that thesemiRNAs can not act through a known miRNA machinery.
We next evaluated the effects of the miRNAs on gene expression. Eight combinations ofmiRNAs that matched the upstream sites of mRNAs positioned –24 to –8654-bp relative to
the TSS were used. Reductions in mRNA levels were observed in 6 of 8 combinations(Figure 5). In most cases, the reduction was observed in one of three cell lines. In only one
combination, reduction was observed in two cell lines simultaneously (Figure 5A). The result
indicates that the reducing effect of each miRNA is different depending on cell type. None of
the miRNA sequences completely matched the mRNA coding regions. However, twocomplementary sites of the seed sequence of miR-548d-3p, an opposite strand of miR-548d5p, were found in the 3′ UTR and CDS of POLR3A gene (Table 8). This suggests that thedecrease in POLR3A mRNA by miR-548d-5p is a seed-dependent silencing activity.
Furthermore, two seed sequence complementary sites of miR-378 were found in the CDS ofOSBPL10 mRNA (Table 8). Recently, the CDS was reported to comprise miRNA and siRNAtargets [38,40,41]. Thus, the decrease in OSBPL10 mRNA after transfection of miR-378
could also be a seed-dependent silencing effect that targets the OSBPL10 CDS in HeLa andPC-3 cells, although such reduction was not observed in MCF-7 cells (Figure 5A). The
increases in mRNA levels were observed in two of the miRNA/mRNA combinations in only
one of three cell lines (Figure 5F, G), indicating that the machinery for increasing mRNA
expression is also highly cell type-specific. These results show that miRNA can both silence
and activate gene expression; these effects were largely dependent on cell type anddownstream gene. In this study, the indirect interaction of miRNAs through the regulation ofsome transcription factor(s) that control the expression of the downstream genes is hardly
distinguishable from the direct interaction of miRNAs with the upstream regions. Further
detailed analyses might reveal the mechanism of transcriptional regulation (e.g., RNAa, TGS)
targeted the upstream region of mRNAs.
Table 8. Seed complementary sites in the mRNA coding regions
A

gene / miRNA

Region in mRNA
5'UTR

CDS

3'UTR

OSBPL10 / miR-378

01), 2)

0

RNF6 / miR-548c-5p

0

0

0

CDC23 / miR-548c-5p

0

0

0

2

Genome-wide Identification and Analysis of miRNAs Complementary…
A

315

Region in mRNA

gene / miRNA

5'UTR

CDS

3'UTR

OSBPL9 / miR-606

0

0

0

RNPS1 / miR-572

0

0

0

C20orf43 / miR-548c-5p

0

0

0

ANXA3 / miR-548c-5p

0

0

0

P OLR3A / miR-548d-5p

0

0

0

1)
5’-ACCAUGGAGAGGGCAGUCCAGGGC-3’|| |:|||||||
miRNA 3’-GGAAGACUGAGGUUCAGGUCA-5’mRNA

2)mRNA 5’-AGCAGGGACCCAUGGAGUCCAGGA-3’|| : :|||||||miRNA
3’-GGAAGACUGAGGUUCAGGUCA-5’

B

Region in mRNA

gene / miRNA

5'UTR

CDS

3'UTR

OSBPL10 / miR-378*

0

0

0

RNF6 / miR-548c-3p

0

0

0

CDC23 / miR-548c-3p

0

0

0

OSBPL9 / miR-606 opposite strand

0

0

0

RNPS1 / miR-572 opposite strand

0

0

0

C20orf43 / miR-548c-3p

0

0

0

ANXA3 / miR-548c-3p

0

0

0

POLR3A / miR-548d-3p

0

11)

12)

1)
mRNA
miRNA

5’-AUACGCAGAUGAAAAGGUUUUUGA-3’: ||| :||||||||
3’-CGUUUUCUUUGACACCAAAAAC-5’2)5’-CAAUGGGGAUGAGAUGGUUUUUUG-3’:::|| :||||||||
miRNA 3’-CGUUUUCUUUGACACCAAAAAC-5’
mRNA

The number of seed complementary sites of the mature miRNA guide strand (A) and opposite strand (B) in
the 5′ UTR, CDS and 3′ UTR of each mRNA which expression level was changed by the corresponding
miRNA. Two seed complementary sites were observed in CDS of OSBPL10 (A). The opposite strand of
miR-548c-5p, miR-548c-3p, showed seed complementarity in CDS and 3′ UTR of POLR3A (B). The pairing
pattern of miRNA and mRNA in the seed complementary regions are shown below each table. Seven
nucleotides at nucleotide positions 2-8 from 5′ end of each miRNA were perfectly matched.

316

Kenta Narikawa, Kenji Nishi, Yuki Naito et al.

Conclusion
In the present study, we identified miRNAs and their complementary sites in upstream
sequences of mRNA coding regions. Based on our results, perfectly matched complementarysites and 1- and 2-bp mismatched sites appear to be potential targets for miRNA. Using 8
combinations of miRNAs and downstream mRNAs, we found that miRNAs are able to bothsilence and promote the expression of downstream genes and that these changes are largely
gene- and cell type-dependent.

Acknowledgments
This work was partially supported by grants from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and Core Research Project for Private University:
matching fund subsidy to K.U.-T.

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