Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.

Abstract

MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121-317 miRNAs) were increased over 2-fold and the levels of a small number of miRNAs (6-35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.

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Plasma MicroRNA Profiles in Rat Models of
Hepatocellular Injury, Cholestasis, and Steatosis
Yu Yamaura1, Miki Nakajima1, Shingo Takagi1, Tatsuki Fukami1, Koichi Tsuneyama2, Tsuyoshi Yokoi1*
1Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan, 2Graduate School of Medicine and
Pharmaceutical Science, University of Toyama, Sugitani, Toyama, Japan
Abstract
MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important
roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable
attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA
expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and
fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found
that the levels of a large number of miRNAs (121–317 miRNAs) were increased over 2-fold and the levels of a small number
of miRNAs (6–35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct
ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis
discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed
in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the
most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level,
reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the
types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and
identified the miRNAs that could be specific and sensitive biomarkers of liver injury.
Citation: Yamaura Y, Nakajima M, Takagi S, Fukami T, Tsuneyama K, et al. (2012) Plasma MicroRNA Profiles in Rat Models of Hepatocellular Injury, Cholestasis, and
Steatosis. PLoS ONE 7(2): e30250. doi:10.1371/journal.pone.0030250
Editor: Young Nyun Park, Yonsei University College of Medicine, Republic of Korea
Received July 28, 2011; Accepted December 16, 2011; Published February 17, 2012
Copyright: ? 2012 Yamaura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by Grant-in-Aid for Scientific Research (B) from Japan Society for the Promotion of Science (21390174) and in part by a
Health and Labor Science Research Grant from the Ministry of Health, Labor and Welfare of Japan (H20-BIO-G001). No additional external funding was received for
this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tyokoi@kenroku.kanazawa-u.ac.jp
Introduction
MicroRNAs (miRNAs) are a family of short noncoding RNA
whose final product is a 22-nucleotide functional RNA molecule
[1]. They regulate the expression of target genes by binding to
complementary regions of transcripts to repress their translation or
cause mRNA degradation. There is growing evidence that
miRNAs play a fundamental role in a variety of physiological
and pathological processes in animals [1,2]. At present, more than
1400, 720, and 400 miRNAs have been identified in human,
mouse, and rat, respectively. Many miRNAs are expressed in a
tissue or cell-specific manner. Aberrant expression of miRNAs in
tissues has been implicated in a variety of diseases including cancer
[3], viral hepatitis [4], and heart disease [5]. Recently, it was
reported that miRNAs are present in the body fluids such as
plasma [6], serum [6], urine [7], and saliva [8,9]. Their expression
patterns significantly vary in various diseases suggesting their
potential as biomarkers [6,10,11]. The first study of a plasma
miRNA profile in liver injury was from Wang et al. [12]. They
comprehensively analyzed the plasma miRNA expression in mice
with hepatocellular injury caused by acetaminophen (APAP)
overdose. Subsequently, it was reported that the plasma miR-
122 level was increased in a rat model of hepatocellular injury
caused by trichlorobromomethane or carbon tetrachloride (CCl4)
administration [13], and was increased in a mouse model of D-
galactosamine/lipopolysaccharides- or alcohol-induced liver injury
[14]. However, information on the plasma miRNA changes by
liver injury is still limited.
Acute liver injury is classified into three types: hepatocellular
injury, cholestasis, or mixed type [15,16]. Chronic liver injury is a
progressive disease showing increasing severity such as steatosis,
steatohepatitis, fibrosis, cirrhosis and cancer. For the diagnosis of
liver injury, alanine aminotransferase (ALT), aspartate amino-
transferase (AST), alkaline phosphatase (ALP) and total bilirubin
(T-Bil) in the blood are commonly used. However, these
parameters cannot thoroughly identify the type of liver injury. In
addition, these parameters may show increases with extrahepatic
injury such as muscle damage or cardiac injury [17,18]. Moreover,
ALT is not always correlated well with the histomorphologic data
of liver [19,20]. In this study, we sought to compare the plasma
miRNA expression profiles in various types of liver injury using rat
models in order to evaluate whether plasma miRNAs can be
markers that can distinguish the different types of liver injury. In
addition, we determined the time course of changes of selected
plasma miRNA levels with acute liver injury, and evaluated the
utility of miRNAs as quantitative markers of liver injury.
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Materials and Methods
Chemicals and Reagents
APAP, a-naphthyl isothiocyanate (ANIT) and CCl4 were
purchased from Wako Pure Chemicals (Osaka, Japan). Methapyr-
ilene (MP) was from Sigma-Aldrich (St. Louis, MO). Standard diets
(StdD), high fat diets (HFD) and methionine choline-deficient diet
(MCDD)wereobtainedfromOrientalYeast(Tokyo,Japan).RNAiso
was from Takara (Shiga, Japan). mirVana PARIS kit, Megaplex
pools, TaqMan microRNA Reverse Transcription kit, TaqMan
microRNA assays, TaqMan 26 Universal PCR Master Mix No
AmpErase UNG and TaqMan Rodent MicroRNA Array v2.0 were
from Applied Biosystems (Foster City, CA). All other chemicals and
solvents were of the highest grade commercially available.
Animal Models
Animal maintenance and treatment were conducted in
accordance with the National Institutes of Health Guide for
Animal Welfare of Japan, as approved by the Institutional Animal
Care and Use Committee of Kanazawa University, Japan. The
study was approved by the Animal Ethics Committee of
Kanazawa University (No. 31203). Male 5-week-old Sprague-
Dawley rats were purchased from Japan SLC (Hamamatsu,
Japan). Rats were housed in a controlled environment (temper-
ature 2561uC, humidity 50610%, and 12 h light/12 h dark
cycle) in the institutional animal facility with access to food and
water ad libitum. Rats were acclimatized before use for the
experiments. To make the hepatocellular injury models, rats
(n=6–8) were orally administered 500 mg/kg (low dose) or
1,000 mg/kg (high dose) APAP suspended in 0.5% carboxymeth-
ylcellulose (CMC) after fasting. In some experiments, rats were
administered APAP without fasting. Rats (n=6) were orally
administered 300 mg/kg MP dissolved in 0.5% CMC. These rats
were sacrificed 24 h after the administration. To make cholestasis
models, rats (n=5) were orally administered 150 mg/kg ANIT
dissolved in corn oil and were sacrificed 48 h after the
administration. Bile duct ligated (BDL) or sham operated rats
(male, 5-week-old, n=3–4) were purchased from Japan SLC, and
were sacrificed 3 days after the operations. To make steatosis and
steatohepatitis models, rats (n=5) were fed HFD and MCDD,
respectively for 8 weeks. To make a fibrosis model, rats (n=5)
were intraperitoneally administered 0.5 mg/kg CCl4 dissolved in
olive oil twice a week for 10 weeks, and were sacrificed 3 days after
the last administration. After sacrifice, blood and liver were
collected. EDTA was added as an anticoagulant to the blood and
kept on ice for 30 min. After centrifugation, plasma was collected
and kept at 280uC until use. A part of the liver was fixed in
buffered neutral 10% formalin.
Biochemical Assay and Pathological Examination
Plasma ALT, AST and T-Bil levels were determined by using
the Dri-Chem 4000 (FUJIFILM, Saitama, Japan) according to the
manufacturer’s instructions. The formalin fixed samples were
embedded in paraffin and sectioned, and then stained with
hematoxylin-eosin for microscopic examination. A part of the
frozen liver was embedded in optimal cutting temperature (O.C.T)
compound (Sakura Finetek Japan, Tokyo, Japan) and sectioned,
and then stained with Oil red O and hematoxylin.
Evaluation of Stability of Plasma miRNAs
The study using human samples was approved by the Ethics
Committee of Kanazawa University, Japan (No. 203). Written
informed consent was obtained from all subjects. Blood samples
were collected from 9 healthy male subjects (22 to 27-year-old) or
2 non-treated male rats (6-week-old). EDTA was added as an
anticoagulant to the blood and kept on ice for 30 min. After
centrifugation, plasma was collected and pooled. The pooled
samples were divided into each 30 mL, and were kept at 4uC, room
temperature or 37uC. After 3, 6, 12 and 24 h, total RNAs were
prepared and individual miRNAs were measured as described
below.
TaqMan MicroRNA Assay
Total RNAs including small RNAs from the plasmawereisolated
using RNAiso according to the manufacturer’s instructions. Dr.
GenTLE Precipitation Carrier (Takara) was used as a carrier. The
cDNAs were synthesized using TaqMan microRNA Reverse
Transcription kit with TaqMan microRNA assays 56 reaction
mix according to the manufacturer’s protocol. To the cDNA
sample, TaqMan 26Universal PCR Master Mix (No AmpErase
UNG) and TaqMan microRNA assays 56 reaction mix were
added, and the real-time PCR was performed using MP3000P
(Stratagene, La Jolla, CA) with the MxPro QPCR software.
TaqMan MicroRNA Array Analysis
Total RNAs including small RNAs were prepared from 600 ml
pooled rat plasma (n=3–8) using the mirVana PARIS kit
according to the manufacturer’s protocol except that the acid/
phenol/chloroform extraction was repeated two times. The
cDNAs were synthesized from the total RNA using Megaplex
pools according to the manufacturer’s protocol. Primers used in
the reverse transcription reaction were TaqMan stem-loop primers
for 365 individual miRNAs and 3 endogenous controls. Pre-
amplification was performed by adding of 26 TaqMan PreAmp
Master Mix and 106 Megaplex PreAmp Primers to the cDNA
sample. To the preamplification products, TaqMan 26Universal
PCR Master Mix (No AmpErase UNG) were added. The entire
mixture was applied to individual ports of TaqMan Array Rodent
MicroRNA A+B Cards Sets v2.0, 384-well microfluidics cards
containing 375 (A array) or 210 (B array) primer-probe sets for
individual miRNAs. Quantitative real-time PCR was performed
using a 7900HT Fast Real-Time PCR system (Applied Biosystems)
with an SDS software v.2.4. Expression levels were evaluated using
comparative cycle threshold (Ct) method. Ct values ranged from 0
to 40. miRNAs giving Ct values .32 in all groups were omitted
from data analysis because this cut-off value was recommended by
the manufacturer. The data were presented as (40 - Ct). DCt
values [DCt=(40 - Ct model)2(40 - Ct control)] were calculated
as fold changes. The hierarchical clustering was performed using
Cluster 3.0 software (complete linkage) and mapletree.
Statistical Analysis
Data are expressed as mean 6 SD. Comparison of two groups
was made with a Mann-Whitney’s U-test. Comparison of multiple
groups was made with Kruskal-Wallis followed by Dunn’s test. A
value of P,0.05 was considered statistically significant.
Results
Plasma biochemistry and histopathology of rat models of
acute and chronic liver injury
The plasma ALT and AST levels were significantly elevated by
the administration of APAP (Fig. 1A). The elevation of the ALT
and AST levels in high dose of APAP-treated rats was considerably
higher than that in low dose APAP-treated rats. The hepatocel-
lular injury was observed at the pericentral regions. Thus, the
APAP-induced hepatocellular injury model was established. Next,
we sought to establish another hepatocellular injury model by the
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administration of MP. In the treated rats, the plasma ALT and
AST levels were significantly elevated and the hepatocellular
injury was observed at the periportal regions (Fig. 1A). Thus, we
established two types of hepatocellular injury model. In the ANIT-
treated rats, the plasma ALT and T-Bil levels were considerably
elevated (Fig. 1B). Degenerating and vanishing bile ducts as well as
mild necrosis or inflammation were observed. Thus, the ANIT-
induced cholestasis model was established. Next, we evaluated
another cholestasis model by BDL. In the treated rats, the plasma
ALT and AST levels were considerably elevated (Fig. 1B).
Enlargement of bile ducts as well as mild necrosis or inflammation
were observed. Thus, we established two types of cholestasis
model. In the HFD-fed rats, the plasma ALT and AST levels were
not changed, whereas in the MCDD-fed rats, the ALT level
tended to be higher than that in StdD-fed rats (Fig. 1C). Cytosolic
hypertrophy and clear vacuoles containing lipid were observed in
HFD-fed rats. Diffuse macrovesicular steatosis and infiltration of
inflammatory cells were observed in MCDD-fed rats. Accumula-
tion of fat was observed in both groups by Oil red O staining.
Thus, we considered that the steatosis and steatohepatitis models
were established. In the CCl4-treated rats, the plasma ALT and
AST levels were considerably elevated (Fig. 1D). Fibrosis, adipose
degeneration, and infiltration of inflammatory cells were observed
at the periportal region. Accumulation of fat was also observed by
Oil red O staining. Thus, we established a fibrosis model.
Stability of plasma miRNAs
It has been reported that the miRNAs in human plasma are
stable [6], but there is no information on the stability of miRNAs
in rat plasma. We investigated the stability of miRNAs in rat
plasma compared to those in human plasma. We chose miR-16,
miR-21 and miR-122 because their sequences are identical in rat
and human and these miRNAs are substantially expressed in
plasma. We found that these miRNAs in rat plasma were unstable
at 37uC, whereas those in human plasma were stable, supporting
the previous study (Fig. 2). The extent of degradation varied
depending on the kinds of rat miRNAs. The miR-21 and miR-122
were decreased to 2% and 1% of the control, respectively, after
6 h incubation at 37uC, whereas the miR-16 was sustained at 30%
of the control. The level of miR-122 was 10-fold higher than the
levels of miR-16 and miR-21, which showed similar levels. Thus, it
appeared that the extent of degradation was not associated with
the expression levels. When the plasma samples were incubated at
room temperature (,25uC), the degradation of the miRNAs was
attenuated. Moreover, the degradation of miRNAs was consider-
ably repressed when the miRNAs were kept at 4uC. Accordingly,
for the subsequent study, we kept the rat plasma samples on ice
until the RNA extraction.
Plasma miRNAs expression profiles in rat models of liver
injury
The expression profiles of the plasma miRNAs in the rat models
of liver injury were determined by TaqMan microRNA array
analysis. The global normalization method (correction with the
sum of the expression levels of detected miRNAs) is generally used
for normalization in array analyses. The numbers of miRNAs that
were detected, or the Ct values of which were ,32, in the liver
injury groups except the BDL group tended to be larger than those
in the control groups (Table 1). Accordingly, we considered that
the global normalization method would be inappropriate.
Alternatively, we confirmed by the measurement of the cel-miR-
39 levels (a miRNA in C. elegans) that the efficiencies of extraction
and detection of miRNAs were almost equal in all groups. The
number of miRNAs, which gave Ct values ,32 in at least one
group, was 433. We performed a clustering analysis for the
expression levels of the 433 miRNA. As shown in Fig. 3A, all
groups were roughly divided into three, 1) APAP (high), MP, and
ANIT groups, 2) HFD, APAP (low), CCl4, MCDD groups and the
controls, and 3) sham and BDL groups. Next, we examined the
fold changes in the miRNA expression. The numbers of miRNAs
showing .2-fold increases or ,0.5-fold decreases in the liver
injury groups in comparison with a control groups are shown in
Table 1. In most groups, the numbers of increased miRNAs were
larger than those of decreased miRNAs whereas, in the BDL
group, the numbers of decreased miRNAs were larger than those
of increased miRNAs. We performed clustering analysis for the
fold changes of the miRNAs (Fig. 3B). It was demonstrated that
the profiles of the APAP (high) and MP groups were similar. The
profile of the ANIT group was similar to the above two groups, but
that of the BDL group was quite different. The profiles of the
CCl4, HFD, and MCDD groups were roughly close to each other,
and the CCl4 group with an increased ALT level close to the
groups with acute liver injury. Thus, it was demonstrated that the
changes of miRNA expression were different between the acute
and chronic liver injuries.
Among the up-regulated miRNAs in the APAP (high) group
(317 miRNAs) and MP group (295 miRNAs), 283 miRNAs were
common (Fig. 4A). Among the 6 down-regulated miRNAs in the
APAP (high) group and 10 down-regulated miRNAs in the MP
group, only 2 miRNAs were common. Among the 280 up-
regulated miRNAs in the ANIT group and 60 up-regulated
miRNAs in the BDL group, 57 miRNAs were common (Fig. 4B).
Among the 10 down-regulated miRNAs in the ANIT group and
130 down-regulated miRNAs in the BDL group, 2 miRNAs were
common. Among the 121 up-regulated miRNAs in the HFD
group, 186 up-regulated miRNAs in the MCDD group and 225
up-regulated miRNAs in the CCl4 groups, 63 miRNAs were
common. Among the 16 down-regulated miRNAs in the HFD
group, 35 miRNAs in the MCDD group and 27 miRNAs in the
CCl4 groups, only 3 miRNAs were common. To find potential
biomarkers for hepatocellular injury, cholestasis, and steatosis, the
miRNAs whose expression was commonly changed in the same
type of liver injury were compared across the different types of
liver injury. As the results, we found that 16 miRNAs were
specifically up-regulated in hepatocellular injury model, 2
miRNAs and 3 miRNAs were specifically down-regulated in
cholestasis and steatosis models, respectively (Fig. 4, Table 2). It
was suggested that these miRNAs would be a novel biomarker for
hepatocellular injury, cholestasis, and steatosis.
Next, we looked at the miRNAs whose expression was
specifically changed in each model. By the comparison across
the all models, we found that 12 out of 38 miRNAs (sum of up-
regulated and down-regulated miRNAs) and 11 out of 20 miRNA
were specifically changed in the APAP and MP groups,
respectively (Fig. 4A, Table 3). It was considered that these
miRNAs could be used to know the damaged area, pericentral or
periportal region. Since the 223 miRNAs and 3 miRNAs that were
up-regulated in ANIT and BDL groups, respectively were also up-
regulated in the hepatocellular injury models, no miRNAs were
found to be specific for these models. However, 8 out of 8 miRNAs
and 101 out of 208 miRNAs were specifically down-regulated in
ANIT and BDL groups, respectively (Fig. 4B, Table 3). It was
suggested that these miRNAs would be candidate biomarkers of
intrahepatic and extrahepatic cholestasis. We found that 3 out of
28 miRNAs, 17 out of 50 miRNAs, and 11 out of 92 miRNA were
specifically changed in the HFD, MCDD, and CCl4 groups,
respectively (Fig. 4C, Table 3). It was suggested that these miRNAs
would be candidate biomarkers of steatosis, steatohepatitis and
Plasma MicroRNA Profiles in Rats with Liver Injury
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Figure 1. Plasma ALT, AST or T-Bil levels and histopathological changes of liver in rat models of hepatocellular injury induced by
the administration of APAP (n=6–8) or MP (n=6) (A); cholestasis induced by the administration of ANIT, or BDL (n=5) (B); steatosis
or steatohepatitis induced by feeding of HFD (n=5) or MCDD (n=5) (C); and fibrosis induced by the administration of CCl4 (n=6)
(D). Data are mean 6 SD. Significantly different from control group (*P,0.05 and **P,0.01). Liver sections were stained with HE for all models
(original magnification6200) and Oil red O for the chronic liver injury models (original magnification6400). CV: Central vein; P: Portal region; BD: Bile
duct.
doi:10.1371/journal.pone.0030250.g001
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Figure 2. Stability of miR-16, miR-122 or miR-21 in rat (A) or human (B) plasma. Plasma samples from 2 non-treated male rats or 9 male
healthy subjects were pooled and incubated at 4uC, room temperature (RT) or 37uC. Data represent copy numbers per one mL of plasma. Data are
mean 6 SD of triplicate determination (n=3).
doi:10.1371/journal.pone.0030250.g002
Table 1. Number of miRNAs whose expressions were detected and changed with liver injury in rat plasma.
Drug or dietType DetectableCt, ,32Up (. .2.0-fold) Down (, ,0.5-fold)
CMC (fasted) 277181--
APAP (Low)Hepatocellular 32823123718
APAP (High)Hepatocellular400 3133176
CMC284 179--
MP Hepatocellular 381282 29510
Corn oil 286177--
ANITCholestasis 352259 28010
Sham 241137--
BDLCholestasis207 12660130
StdD 269175--
HFD Steatosis294 206121 16
MCDDSteatohepatitis305214 18635
Olive oil248152--
CCl4Fibrosis289 201 225 27
The total number of miRNAs on the array system is 585.
APAP: acetaminophen; MP: methapyrilene; ANIT: a-naphthyl isothiocyanate; BDL: bile duct ligation; StdD: standard diet; HFD: high fat diet; MCDD: methionine choline-
deficient diet.
doi:10.1371/journal.pone.0030250.t001
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fibrosis, respectively. Especially, the miRNAs whose expressions
changed only in the HFD group might be associated with the fat
accumulation without inflammation or fibrosis.
All groups except the BDL and HFD groups showed necrosis
and inflammation. Based on the fact, we sought to identify the
miRNAs whose expressions were changed in response to necrosis
and inflammation. By searching miRNAs whose expressions were
commonly increased in the APAP, MP, ANIT, MCDD, and CCl4
groups, but not in BDL and HFD groups, we found that 67
miRNAs might reflect necrosis and inflammation (Fig. 4D).
Among them, we focued on miR-122, which is the most abundant
miRNA in liver [21], in the next experiment.
Time course of plasma miRNA change in rats with acute
liver injury
We examined the time course of the plasma miRNA changes in
rats with acute liver injury, focusing on miR-122. We measured
the plasma ALT and miR-122 levels over time in six rats
administered 1000 mg/kg APAP. The ALT levels began to
increase 6 h after the administration, reached a peak at 24 h
and then decreased (Fig. 5A). In contrast, the plasma miR-122
levels began to increase 3 h after the administration, reached a peak
at 12–36 h and then decreased. Although there were large
interindividual differences in the ALT levels, the interindividual
differences in plasma miR-122 level were relatively small. It should
be noted that the change of plasma miR-122 was more dynamic
than that of ALT. Next, we looked at five rats treated with 300 mg/
kgMP.TheALTand miR-122levelsbegantoincrease3 hafterthe
administration, but the extent and rate of the increase of miR-122
was more dramatic than those of the ALT levels (Fig. 5B). These
results suggest that the plasma miRNA level changed more
sensitively than the ALT level did in response to acute liver injury.
Association of the plasma miR-122 increase with
hepatocellular injury
We examined whether the increase of plasma miR-122 was due
to liver injury or the administered chemicals. To address this issue,
500 mg/kg of APAP were orally administered to rats without
Figure 3. Hierarchical clustering of plasma miRNA expression profiles in rats with liver injury (A) and the fold changes between the
injury model and control (B). The levels were clustered by using Cluster 3.0 software (complete linkage) and visualized by using MapleTree
software. Data are presented as 40-Ct (A) and log2(B) value.
doi:10.1371/journal.pone.0030250.g003
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fasting. In contrast to the administration of APAP with fasting,
which decreased the hepatic glutathione level, the treatment
neither caused histopathological changes (data not shown) nor an
elevation of ALT in rats (4164 U/L versus 3466 U/L in control)
(Fig. 6). In these rats, the plasma miR-122 levels were almost the
same as those in control rats (40-Ct value: 7.162.6 versus 7.161.6
in control). Therefore, we could conclude that the increase of the
plasma miR-122 was due to liver injury, but not to APAP itself.
When 500 mg/kg APAP were administered with fasting, hepato-
cellular necrosis and inflammation were observed in all 12 rats,
with scores of + (7 rats, closed circle), ++ (1 rat, closed triangle),
and +++ (4 rats, closed square). In the 7 rats showing mild (+)
histopathological changes, the increase of the ALT levels was not
significant (1046107, 3.1 fold of control), but the increase of the
miR-122 level was remarkable (10.661.0, 11.3 fold of control). In
addition, the extent of the increase of miR-122 mirrored the
Figure 4. Up- or down-regulated miRNAs in hepatocellular injury models (A), cholestasis models (B), and chronic liver injury models
(C). Venn diagram shows the number of changed miRNAs. The numbers in the parenthesis are the numbers of miRNAs whose expressions were
specifically changed only in the given models. Heat map of 67 miRNAs in all models, which were commonly up-regulated with necrosis and
inflammation (D).
doi:10.1371/journal.pone.0030250.g004
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histopathological changes (++ 15.2 and +++ 20.061.5). In the rats
administered 1000 mg/kg APAP with fasting, the interindividual
variability of miRNA (20.761.7) was less than that of ALT
(696065615). Thus, it is suggested that miRNA would be a more
sensitive and quantitative biomarker of liver injury, with low
interindividual variability, than a conventional biomarker, ALT.
Discussion
It was reported in 2008 that miRNAs stably exist in human
plasma or serum [6,10]. After these reports, miRNAs in body
fluids have been investigated in a wide variety of patient samples
and animal models and have been revealed as potential
biomarkers of various diseases [22]. In contrast to human
miRNAs, there was no report on whether the miRNAs in rodent
plasma are also stable. Therefore, we first determined the stability
of miRNAs in rat plasma and found that the miRNAs in rat
plasma were less stable than those in human plasma. The extent of
degradation was different among the miRNAs, but was indepen-
dent of the expression levels of these miRNAs. We could provide
important information for dealing with rat plasma miRNAs.
Although the relative instability of miRNAs in rat plasma was
revealed, our finding does not hinder the potential as a biomarker,
because the degradation can be suppressed if the samples are kept
below 4uC or stored in freezer, and it is speculated that the
circulating miRNAs in body would be stable at similar extent
between human and rat, based on the fact encompassing in vesicle
or binding to proteins. Thus, we consider that the miRNAs in rat
plasma could also be useful as a biomarker.
In this study, we established various rat models with liver injury
including hepatocellular injury (APAP or MP), cholestasis (ANIT
or BDL), steatosis (HFD), steatohepatitis (MCDD), and fibrosis
(CCl4), and determined the plasma miRNA expression profiles. In
the hepatocellular injury models caused by 500 mg/kg (low dose)
or 1000 mg/kg (high dose) of APAP, 231 and 313 miRNAs out of
328 and 400 detectable miRNAs were up-regulated in the plasma,
respectively. In a previous study by Wang et al. [12] using a mouse
model that was administered 300 mg/kg APAP, it was demon-
strated that 25 miRNAs out of 53 detectable miRNAs were up-
regulated (.1.5 fold). Almost all the up-regulated miRNAs (22 our
of 25 miRNAs) such as miR-122, miR-192, miR-685, miR-193,
and miR-29c were also up-regulated in our rat model, suggesting
that common plasma miRNAs seem to be up-regulated in APAP-
induced liver injury independent of species. It should be noted that
the miRNAs that were detected or up-regulated in their study were
considerably fewer than those in our study, although the number
of probes were comparable between the studies (the hybridization-
based array they used contained probes representing 576 mouse
miRNAs, whereas the TaqMan real time-PCR based-array we
used contained probes representing 585 rodent miRNAs). The
differences may possibly be due to species differences in the
expression level of miRNAs in plasma or differences in the
sensitivity of the platforms used for array analysis.
Wang et al. [12] also reported using a mouse model with APAP
that the levels of the miRNAs, which were increased in plasma,
were decreased in liver. They described that the cellular damage in
the liver tissue resulted in the transport or release of cellular
miRNAs into the plasma, which may be a similar process by which
cellular enzymes are released after cellular damage. Although we
have not confirmed yet whether such an inverse correlation also
exists in rat models, it might be true because the miRNAs whose
expressions in plasma were increased were tended to be common
in the models representing hepatocellular damage (APAP, MP,
ANIT, MCDD, and CCl4 groups). Additionally, we found
miRNAs whose expressions were specifically changed in the
APAP and MP group. APAP and MP induced hepatocellular
Table 2. The miRNAs whose expressions were changed in hepatocellular injury, cholestasis and steatosis.
Type of liver injury
Fold change (log2) Fold change (log2)
Up-regulated
miRNAsAPAP MP
Down-regulated
miRNAsAPAP MP
Hepatocellular injurymiR-200a*13.2 8.7
let-7c-1*10.8 3.0
miR-50310.2 9.0
miR-337-3p9.67.1
miR-10b9.22.2
miR-34c8.66.3-
miR-3278.58.8
miR-3518.25.5
miR-7047.89.7
miR-4107.61.7
Top 10 out of 16 miRNAs
ANIT BDLANITBDL
Cholestasis-miR-190
25.9
26.0
miR-743b
23.3
28.3
HFDMCDDCCl4 HFDMCDD CCl4
Steatosis- miR-449c
27.0
26.0
22.6
miR-410
24.4
24.4
22.8
miR-10b*
22.5
25.0
25.1
doi:10.1371/journal.pone.0030250.t002
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Table 3. The miRNAs whose expressions were changed only in the given model of liver injury.
Type of liver injury
Up-regulated
miRNAsFold change (log2)Down-regulated miRNAsFold change (log2)
Hepatocellular injury by APAP (high) miR-59210.0miR-103
22.7
miR-29b-2*8.8miR-141*
21.9
miR-3678.7 miR-764-5p
21.9
miR-19a* 8.7miR-132
21.5
miR-344-3p8.3
miR-218-1*8.1
miR-10a*8.0
miR-2175.9
Hepatocellular injury by MP miR-697 9.6miR-687
29.0
miR-200c* 8.1miR-30a*
28.3
miR-879* 5.9miR-29b
25.8
miR-30c-1* 4.3miR-744*
25.4
miR-1492.3miR-181c
24.8
miR-29b* 1.0
Cholestasis by ANIT- miR-704
27.3
miR-875-5p
27.0
miR-218-1*
26.4
miR-337-3p
25.6
miR-411
25.0
miR-351
23.4
miR-24-1*
22.8
miR-699
22.6
Cholestasis by BDL- miR-377
211.8
miR-27b
210.0
miR-872
29.7
miR-130b
29.7
miR-185
28.7
miR-361
28.1
let-7i
28.0
let-7b
27.8
miR-99a
27.7
miR-17-3p
27.6
Top 10 out of 101 miRNAs
Steatosis by HFD-miR-219-1-3p
22.5
miR-463
21.1
miR-183
21.0
Steatohepatitis by MCDDmiR-1549.6miR-7a*
210.5
miR-503*4.8 miR-181a
28.6
miR-139-3p 1.4 miR-150
27.3
miR-384-5p
26.8
miR-17*
25.6
miR-197
25.4
miR-134
22.4
miR-542-3p
22.0
miR-706
21.8
miR-148b-5p
21.7
Top 10 out of 14 miRNAs
Fibrosis by CCl4 miR-764-5p 3.3miR-30c-1*
215.3
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necrosis at the pericentral and periportal region, respectively.
Thus, such specific miRNAs would be useful to determine the
damaged area. A likely explanation for the finding that different
miRNAs were altered in plasma between the APAP and MP
groups might be that the miRNAs which are highly expressed at
the pericentral region may be selectively released to plasma in
APAP group and the miRNAs which are highly expressed at the
periportral region may be selectively released to plasma in MP
group, although it remains to be clarified whether the miRNAs in
liver may be differently expressed at pericentral and periportal
regions. It would be quite possible that the miRNA expression
profiles of the pericentral and periportal regions are different,
because it is well known that some proteins show zonal expression,
which would be due to differences in transcriptional regulation
[23,24]. We now determine the expression of miRNAs in liver at
different zones, and will investigate the relationship between the
changes of miRNA in the liver and plasma in rat models of liver
injury in the future.
We found that the changes in plasma miRNA in the
hepatocellular injury models were more dynamic than those in
ALT (Fig. 5). One of the reasons would be the difference in the
type of detection (real-time RT-PCR for miRNAs versus
colorimetric assay for ALT activity). In addition, it has been
reported that ALT is mainly expressed in the portal vein area [19],
whereas our preliminarily study revealed that the miR-122
uniformly shows high expression in liver at the pericentral and
periportal regions (unpublished data). That might be another
reason for the more sensitive response of miR-122 than ALT
toward the liver injury which would be a benefit as a biomarker.
Moreover, we showed that the plasma miR-122 level was
quantitatively correlated with the extent of histopathologic
changes (Fig. 6). Thus, as represented by miR-122, the profile of
miRNA expression could serve as a tool for understanding the
onset and progression of liver injury.
In this study, ANIT-administration or BDL was used to make
the cholestasis model. ANIT causes intrahepatic cholestasis by
damaging the cholangiocytes lining the bile ducts [25], whereas
BDL causes extrahepatic cholestasis by blocking the drainage of
bile from the liver to the duodenum. As shown in Fig. 3 and
Table 1, the plasma miRNA profiles in the two models were quite
different, which may be due to differences in the mechanisms
causing cholestasis. Since the up-regulated miRNAs were almost
common with those in the hepatocellular injury model as
described above, these miRNAs cannot be biomarkers of
cholestasis. Instead, we could identify the miRNAs that can be
biomarkers of cholestasis, among the down-regulated miRNAs.
Why was such a large number of miRNAs decreased in plasma of
BDL group? One may consider that the miRNAs may be instable
in plasma with high levels of bile acids. However, the possibility
may be denied because a recent study reported that miRNAs are
present in bile that is abundant in bile acids and bilirubin [26].
Another possibility is that the bile acids accumulated in
hepatocytes inhibit the secretion of miRNAs. To obtain the
answer, the determination of hepatic miRNA expression profiles in
ANIT and BDL groups might be useful.
In the chronic liver injury models including the HFD, MCDD,
and CCl4groups, we found that 3 miRNAs (miR-10b*, miR-410,
miR-499) were commonly down-regulated, but were not affected
in the acute liver injury models (Fig. 4), suggesting that these
miRNAs might serve as biomarkers of steatosis. In addition, we
found miRNAs whose expressions were specifically modulated in
each model of chronic liver injury (Table 2), which might
represent markers of each pathology. Previously, Jin et al.
determined the miRNA expression profiles in liver from an
HFD-induced steatosis model rat and found that miR-132 and
miR-30d were up-regulated in liver [27]. In our corresponding
model, the miR-132 was up-regulated, whereas the miR-30d was
down-regulated in plasma. The miRNA expression profle in liver
from MCDD-induced steatohepatitis mice model has been
determined by two research groups. Dolganiuc et al. reported
that 10 and 2 miRNAs were up- and down-regulated, respectively
[28]. Pogribny et al. reported that each of 4 miRNAs were up- and
down-regulated [29]. The only common miRNA in these studies
was miR-200b, which showed up-regulation. We looked at the
expression changes in plasma for these miRNAs in our rat model
of steatohepatitis by MCDD. Among 13 up-regulated miRNAs in
liver, 7 miRNAs including miR-200b were up-regulated, but 4
miRNAs were not changed in plasma (Two miRNAs were absent
in the array platform we used). Among the 6 down-regulated
miRNAs in liver, in plasma 4 miRNAs were up-regulated, but 2
miRNAs were not changed. Recently, Li et al. reported that 16
miRNAs including miR-34, miR-199a-5p, miR-221, miR-146b,
and miR-214 showed progressive up-regulation in rat with hepatic
fibrosis caused by dimethylnitrosamine [30]. Murakami et al.
reported that 11 miRNAs including miR-34, miR-199a-5p, miR-
199, miR-200, and let-7e were up-regulated in a CCl4-induced
fibrosis model mouse [31]. Among them, miR-34 and miR-199a-
5p were common in the two models. In our CCl4-induced fibrosis
model, the miR-34a in plasma was increased, whereas the miR-
199a-5p in plasma was not changed. Taken together, it seems that
Type of liver injury
Up-regulated
miRNAs Fold change (log2)Down-regulated miRNAs Fold change (log2)
miR-30c-2*
212.8
miR-302c*
28.7
miR-153
27.5
miR-9*
26.5
miR-503*
25.7
miR-376c*
24.0
miR-215
21.4
miR-30b*
21.3
miR-29c*
21.2
doi:10.1371/journal.pone.0030250.t003
Table 3. Cont.
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there is no settled rule for the relationship between the changes of
miRNA in liver and in plasma. In addition to the hypothesis from
the acute liver injury model that miRNAs would be released from
liver to plasma, other mechanisms may also be involved in the
chronic liver injury. That might explain why the change of
miRNA expression in liver is not necessarily associated with that in
plasma. It has been recognized that circulating miRNAs are
released from cells in membrane-bound vesicles such as exosomes
or microvesicles. However, recent studies reported that a significant
fraction of the extracellular miRNAs is not within the vesicles, being
Figure 5. Time-dependent changes of plasma ALT and miR-122 levels in individual rat orally administered 1000 mg/kg of APAP
(n=6) with fasting (A) or 300 mg/kg MP (n=5) (B). Graphs with magnified abscissa are also shown. The miR-122 levels represent relative value
to control.
doi:10.1371/journal.pone.0030250.g005
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associated with proteins [32]. It seems that there are three types of
miRNAs: miRNAs which are dominantly vesicle-associated, those
which are dominantly associated with protein, and those which are
equally distributed. In addition, the mechanisms by which miRNAs
are taken up by cells are not fully understood. To understand the
relationship between the miRNA expression profiles in plasma and
those in liver, the complex export and import systems of the
miRNAs in various organs should be clarified.
By the comparison of the miRNA expression profiles in rat
models of various types of liver injury, we could identify miRNAs
that could be specific and sensitive biomarkers of hepatocellular
injury, cholestasis, steatosis, steatohepatitis, and fibrosis. It is
conceivable that the plasma miRNAs would be a superior
noninvasive biomarker in human that could distinguish the
different types of liver injury to conventional biomarkers such as
ALT and ALP, although the analysis to compare the plasma
miRNA expression profiles in patients suffering from various type
liver injury remains to be performed. The plasma miRNAs have a
potential to be used to know the types of liver injury to decide
appropriate therapy, or to know the progress or restoration of liver
injury in clinics. In addition, the plasma miRNAs would be useful
in drug development, since they could detect liver injury caused by
treatment with drug candidates at the early stage, resulting in
saving time and resources in nonclinical study.
In conclusion, the present study demonstrated that the
expression profiles of plasma miRNAs differed according to the
type of liver injury. Although earlier studies reported the changes
of some miRNAs in plasma or tissues with disease using a single
model, implying the possibility of associations with the develop-
ment of disease, comparison of the miRNA expression profiles
across models would be important for understanding the
physiological implications of the miRNAs changes. We could
identify miRNAs which could be specific and sensitive biomarkers
of each type of liver injury (e.g. acute/chronic liver injury or
hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis)
using rat models. Further studies are warranted to elucidate
whether the miRNAs could be used as biomarkers in patients with
various types of liver injury.
Acknowledgments
We acknowledge Mr. Brent Bell for reviewing the manuscript.
Author Contributions
Conceived and designed the experiments: YY MN ST TY. Performed the
experiments: YY ST. Analyzed the data: YY MN ST. Contributed
reagents/materials/analysis tools: YY MN ST KT. Wrote the paper: YY
MN TF TY.
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