Sensors and Materials, Vol. 24, No. 6 (2012) 303–310
MYU Tokyo
S & M 0886

Genomewide Analysis of Expression of Genes
Located near miRNAs in Humans and Mice
Hidenori Inaoka*, Yutaka Fukuoka1,** and Makoto Noshiro
School of Allied Health Sciences, Kitasato University,
1-15-1, Kitasato, Minami, Sagamihara, Kanagawa 252-0373, Japan
1Graduate School of Biomedical Science, Tokyo Medical and Dental University,
Yushima, Bunkyo, Tokyo, 113-8510, Japan(Received October 3, 2011; accepted January 24, 2012)
Key words:

MicroRNA (miRNA), transcription

MicroRNAs (miRNAs) are 20- to 22-nucleotide RNAs that regulate the transcription
and translation of genes. In our previous studies, we reported that a miRNA has thepotential to affect the transcription of genes located near it. The number of miRNAs
recorded in databases continues to increase. However, the effects of the miRNAs,
including newly found miRNAs, on the transcription of genes located near them have
not yet been analyzed. In this study, we investigated the effects in humans and mice.
The average expression levels are increased in the 104 bp vicinity of miRNA in all
the datasets investigated. Many of the newly found miRNAs are located in an intron,
exon or untranslated region (UTR) of host genes. When a host gene is transcribed, themiRNA located in such a region of the host gene is also transcribed. In addition, recent
reports indicate that some noncoding RNAs, including miRNAs, can upregulate theexpression of genes that have the complement sequences of the noncoding RNA in theirpromoter regions. Therefore, if genes having complement sequences of miRNAs arelocated around these miRNAs, the expression levels of these genes could be increased.
These phenomena may be the reasons for the increased average expression levels around
miRNAs.

1.

Introduction

MicroRNAs (miRNAs) are 20- to 22-nucleotide RNAs that regulate the transcription
and translation of genes.(1) There is mounting evidence that small noncoding RNAs
including miRNAs play an important role in the control of the dynamics of localized
gene expression through heterochromatin formation.(2) Therefore, we have hypothesized
Corresponding author: e-mail: inaoka@kitasato-u.ac.jp
Present affiliation: Department of Electrical Engineering, Kogakuin University, Tokyo, Japan

*

**

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Sensors and Materials, Vol. 24, No. 6 (2012)that miRNAs may play a role in the localized expression of genes in their neighborhood.
We defined a series of increasingly large chromosomal windows centered on eachmiRNA location. The expression levels within each window were analyzed in ourprevious studies. In those studies, we reported that messenger RNA expression levels
obtained from genes localized within a 104 bp chromosomal distance of miRNAs weredecreased in worms (103 miRNAs) and mice (228 miRNAs).(3,4) These results suggestthat a miRNA has the potential to affect the transcription of genes located near it. The
number of miRNAs found by experimental methods or computational estimations
continues to increase. Currently, over 700 and 1,400 miRNAs are known in mouse andhuman, respectively. However, the effects of the miRNAs, including newly found ones,on the transcription of genes located near them have not yet been analyzed. In this study,
we investigated the effects in humans and mice.

2.

Materials and Methods

2.1

Materials

2.2

Methods

3.

Results

The positions of miRNAs in the human and mouse genomes were obtained from themicroRNA database (miRBase) Release 17.(5) The positions of genes in the human andmouse genomes were obtained from the National Center for Biotechnology Information(NCBI) Build 37.2. Gene expression datasets were downloaded from NCBI Gene
Expression omnibus (GEO). As human data, GEO accession numbers GSE1643，
GSE3141，GSE3526，GSE4655，GSE6565，GSE8586, and GSE9520 were used.(6–12)
Each of these datasets was measured using Affymetrix Human Genome U133 Plus 2.0
Array. As mouse data, GSE1049, GSE1052, GSE1301, GSE1745, GSE3234, GSE3351,
and GSE26875 were used.(13–16) Each dataset was obtained using Affymetrix Mouse
Expression 430B Array.
Gene expression values in each dataset were normalized using the average value ofthe whole data in the dataset. A window centered at a miRNA was set, and the averageof the normalized expression levels was calculated for that window. Fifteen different
window widths were used: 104 bp, 2×104 bp, 4×104 bp, 105 bp, 2×105 bp, 4×105 bp, 106
bp, 2×106 bp, 4×106 bp, 107 bp, 2×107 bp, 4×107 bp, 108 bp, 2×108 bp, and 4×108 bp.
After this calculation, the data for all the miRNAs were merged, and the relationshipbetween the average expression level and window width was investigated. The reason
for the size of the minimum window is as follows. The average numbers of genes per
104 bp in human and mouse are 4.4 and 4.8, respectively. If the size of the minimum
window is shorter than 104 bp, the number of genes in the windows will be less than 1.
Therefore, in this study, the size of the minimum windows was determined to be 104 bp.

The average expression level and standard error in each window in a human dataset,
GSE1643, are plotted as a function of the window width (Fig. 1). Figure 2 indicatesthe average expression levels in all the human data. The average expression level and

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2

normalized expression

normalized expression

Sensors and Materials, Vol. 24, No. 6 (2012)

1

0

4

10

6

10
window width (bp)

10

8

2

GSE1643
GSE3141
GSE3526
GSE4655

1.5

1

4

10

6

10
window width (bp)Fig. 1 (left). The average expression level of GSE1643 is plotted as a function of chromosomal
distance to the miRNA in base pairs.
Fig. 2 (right). The average expression levels of all human datasets are plotted as a function ofchromosomal distance to the miRNA in base pairs.

standard error in each window in a mouse dataset, GSE1049, are plotted as a function of
the window width in Fig. 3. For all the mouse data, the average expression level in eachwindow is plotted in Fig. 4. In both humans and mice, the average expression levels are
increased in the 104 bp vicinity of miRNA in all the datasets. The average expression
values in the 104 and 106 bp were analyzed for statistical significance using Student’s
t-test. A p-value < 0.05 was considered statistically significant. All the datasets except
GSE1301 and GSE3351 showed that the average expression level was significantly
higher in 104 bp than in 106 bp.

4.

Discussion

The average expression levels of all the datasets investigated in this study are
increased in the 104 bp vicinity of miRNA in both humans and mice. However, ourprevious results showed that the average expression levels are decreased in the 104 bp
vicinity of miRNA in worms(3) and mice.(4) The new analysis yields results that are
different those of previous analyses. In what follows, we will discuss the possible
reasons for the differences.

4.1

Worm analysis

GSE6565
GSE8586
GSE9520

The average expression levels around miRNAs were also analysed in worms using
the information obtained from miRBase Release 17. Although the number of miRNAs
in worms was increased from 103 to 207 in the new analysis, the result showed thesame tendency as the previous one.(3) Figure 5 illustrates the average expression levelas a function of the window width in each group in two worm datasets (growth anddevelopmental,(17) germline(18)). The average expression levels are decreased in the 104

108normalized expression

Sensors and Materials, Vol. 24, No. 6 (2012)

3
2
1
0

4

10

6

8

10
window width (bp)

GSE1049
GSE1052
GSE1301
GSE1745

3
2
1
4

10

GSE3234
GSE3351
GSE26875

6

10

10
window width (bp)8

10

Fig. 3 (left). The average expression level of GSE1049 is plotted as a function of chromosomal
distance to the miRNA in base pairs.
Fig. 4 (right). The average expression levels of all mouse datasets are plotted as a function ofchromosomal distance to the miRNA in base pairs.

normalized expression

normalized expression

306

1.1

growth and devlopmental
germline

1
0.9
0.8

4

10

6

10
window width (bp)10

8Fig. 5. The average expression level of all worm datasets are plotted as a function ofchromosomal distance to the miRNA in base pairs.

bp vicinity of miRNAs. These findings indicate that the average expression levels ofhumans and mice tend to be different from those of worms. The gene expression levelsin the former species increased in the 104 bp vicinity of miRNAs.
Many miRNAs and their targets are conserved among species. It is considered thatthe expression levels around the conserved miRNAs show the same tendency. Using thefamily gene information obtained from miRBase Release 17, the numbers of miRNAs
conserved among worm, mouse, and human were counted. Only 5 out of 207 worm
miRNAs were conserved in mouse and human miRNAs. These conserved miRNAs

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were let-7, mir-9, mir-124, mir-1, and mir-34. These miRNAs are conserved among over
80 species. This finding indicates that most of the worm miRNAs are different frommouse and human miRNAs. It is considered that the target genes of the worm miRNAs
are also different from those of mouse and human miRNAs. Therefore, the expression
levels around miRNAs showed a different tendency.

4.2

Mouse analyses

4.3

Effects of miRNA location

As described above, the results obtained from new analysis of mice are different
from those of previous analysis. The different results could be partly explained by thedifferent numbers of miRNAs used in the two analyses. The number of mouse miRNAs
obtained from miRBase Release 17 was 715 in this study, whereas that in the previous
study was 228 (miRBase Release 7.2). Therefore, the number of mouse miRNAs usedin this study was over three times higher than that in the previous study. The numberof miRNAs that have genes in their 104 bp vicinity was increased from 62 in Rel. 7.2
to 420 in Rel. 17. A recent change in the miRNA detection approach has considerablyincreased the number of miRNAs detected. In previous miRNA detection approaches,
putative miRNA sequences that were conserved in other species (e.g., worm) weresearched. However, in the current miRNA detection approach, the entire genome wasscanned for hairpin structures, and the thermodynamic stability and structural features of
the hairpins were calculated using a computational method and then putative miRNAs
were determined.(19) Therefore, the types of miRNAs newly added in this study have thepotential to be functionally different from those of miRNAs used in the previous study.
As the numbers of different types of miRNAs were increased in the new analysis, theeffects of those miRNAs on the expressions of the genes located around them may differ
from those in the previous studies.
Another reason for the different results in mice could be the different information on
the chromosomal positions of genes in the two analyses. The chromosomal positions ofgenes were obtained from NCBI Build 37.2 in this study, whereas NCBI Build 34.1 wasused in the previous study. The number of genes whose chromosomal positions were
determined was higher in B37.2 than in B34.1. The numbers of genes located in the 104
bp vicinity of miRNAs were 442 in B37.2 and 50 in B34.1. Therefore, the number ofgenes located around miRNAs was considerably increased in this study.
Some miRNAs are located in an intron, exon or untranslated region (UTR) of hostgenes and some miRNAs are located between genes (intergenes). Using miRBase
information, miRNAs were clustered into five groups: intronic, exonic, 3’-UTR, 5’-UTR,
and intergenic. The number of miRNAs in each group is shown in Table 1. The average
expression level in each miRNA group in a human dataset, GSE1643, is plotted as afunction of the window width in Fig. 6. Figure 7 illustrates the average expression levelas a function of the window width in each group in a mouse dataset, GSE1049.
In GSE1643, all the miRNA groups except the intronic group showed increasing
average expression levels near miRNAs. In the intronic group, there was no relationshipbetween the average expression level and window width. In the other human datasets,

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Table 1
Number of miRNAs in each group.
Intronic
847
392

Exonic169
140

intronic
exonic33'-UTR
66
34

3'−UTR
5'−UTR
intergenic

2
1
0

4

10

6

10
window width (bp)8

105'-UTR
20
8

normalized expression

normalized expression

Human
Mouse

Intergenic
484
226

intronic
exonic33'−UTR
5'−UTR
intergenic

2
1
0

4

10

6

10
window width (bp)8

10

Fig. 6 (left). The average expression levels obtained from miRNA groups (intronic, exonic,
3'-UTR, 5'-UTR, and intergenic) in GSE1643 are plotted as a function of chromosomal distance tothe miRNA in base pairs.
Fig. 7 (right). The average expression levels obtained from miRNA groups (intronic, exonic,
3'-UTR, 5'-UTR, and intergenic) in GSE1049 are plotted as a function of chromosomal distance tothe miRNA in base pairs.

all the miRNA groups showed increasing average expression levels near miRNAs. In
mouse data GSE1049, in contrast, the 5’-UTR and intergenic groups showed decreasing
average expression levels near miRNAs, whereas the other groups showed increasing
average expression levels near miRNAs. In the 3’-UTR group, the average expression
level tends to increase in the 105 to 106 bp width and decrease in the 104 bp width. In
addition, the decreasing average expression levels near miRNAs were found in theintergenic group in all the other mouse datasets. Many mouse datasets showed that the
average expression levels were decreased in the 104 bp width in the 5’-UTR group andthe average expression levels were increased from 105 to 106 bp in the 3’-UTR group.
These findings indicate that the average expression levels near miRNAs are generally
increased, whereas those of some miRNA groups are decreased in the mouse datasets.
As described above, the average expression levels near intergenic miRNAs weredecreased in the 104 bp width, whereas the average expression levels near intronic/
exonic miRNAs were increased in mice. Therefore, the reason for the decreasing
average expression levels near miRNAs in the previous study could be partly explainedon the basis of the ratio of the number of miRNAs in each group. Table 2 compares309

Sensors and Materials, Vol. 24, No. 6 (2012)

Table 2Differences in the number of miRNAs in each group between miRBase Release 7.2 and Release
17.
Rel. 7.2
Rel. 17

Intronic
94
392

Exonic54
140

3'-UTR
534

5'-UTR
0
8Intergenic
77
226

the number of miRNAs in each group obtained from miRBase Release 7.2 and Release
17. As shown in Table 2, the number of intergenic groups was relatively high in Rel.
7.2. Therefore, there could be a stronger tendency of the average expression levels todecrease in the 104 bp vicinity of miRNAs in the previous analysis.
From the results described above, it is suggested that the average expression levelsof the intronic and exonic groups strongly affect the overall trend of the average
expression levels. The reason for the increasing average expression levels near miRNAs
in the intronic and exonic groups could be explained as follows. When a host gene istranscribed, a miRNA located in an intron or exon of the host gene is also transcribed.
In other words, the expression of miRNAs located in the host genes is linked to theexpression of the host genes.(20,21)
Recent reports indicate that some noncoding RNAs, including miRNAs, canupregulate the expression of genes that have the complement sequences of the noncoding
RNA in their promoter regions, although the exact mechanism is still unknown.(22,23)
Therefore, if genes having complement sequences of miRNAs are located around thesemiRNAs, the expression levels of these genes could be increased because of the reason
described above. This phenomenon may be one of the reasons for the increased average
expression levels.

5.

Conclusion

The results of this study indicate that the average expression levels near miRNAs
obtained from mammals (humans and mice) and worms are different. These suggestthat there exist some classes of miRNAs, i.e., those conserved over various species andthose that evolved in higher organisms. The different tendencies in mammals and worms
imply that different classes of miRNAs have different functionalities. In the future, therelationship between the chromosomal location of the miRNA and the function of thetarget gene will be investigated using gene functional analysis.

Acknowledgements
This study was financially supported in part by the regular budget of Kitasato
University School of Allied Health Sciences, and a scientific research grant from the
Ministry of Education, Culture, Sports, Science and Technology of Japan (#20510184) to
Yutaka Fukuoka.

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