MOLECULAR CARCINOGENESIS 50:136142 (2011)

Circulating MicroRNAs, miR-21, miR-122, and

miR-223, in Patients With Hepatocellular
Carcinoma or Chronic Hepatitis
Jian Xu,1 Chen Wu,1 Xu Che,2 Li Wang,3 Dianke Yu,1 Tongwen Zhang,1 Liming Huang,1 Hui Li,3 Wen Tan,1
Chengfeng Wang,2** and Dongxin Lin1*
1
State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing, China
2
Department of Abdominal Surgery, Cancer Institute and Hospital, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing, China
3
Department of Epidemiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing, China

Numerous studies have shown that aberrant microRNA (miRNA) expression is associated with the development and
progression of various types of human cancer and serum miRNAs are potential biomarkers. This study examined
whether some commonly deregulated miRNAs in hepatocellular carcinoma (HCC) are presented in serum of patients
with HCC and can serve as diagnostic markers. Serum miRNAs (miR-21, miR-122, and miR-223) were quantified by
real-time quantitative RT-PCR in 101 patients with HCC and 89 healthy controls. In addition, 48 patients with chronic
type B hepatitis were also analyzed for comparison. We found that the median levels of miR-21, miR-122, and miR-
223 were significantly higher in patients with HCC than those in healthy controls (P 7.48
1013, P 6.93
109,
and P 3.90
1012, respectively). However, these elevated serum miRNAs were also detected in patients with
chronic hepatitis (P 2.05
1012, P 4.52
1016, and P 1.65
1011, respectively). Moreover, serum miR-21
and miR-122 in patients with chronic hepatitis were higher than in patients with HCC (P 3.99
104 and
P 4.97
108), although no such significant difference was found for miR-223. Receiver-operator characteristic
(ROC) curve analyses suggest that these serum miRNAs may be useful markers for discriminating patients with HCC
or chronic hepatitis from healthy controls, but not patients with HCC from patients with chronic hepatitis. Our
results indicate that serum miR-21, miR-122 and miR-223 are elevated in patients with HCC or chronic hepatitis
and these miRNAs have strong potential to serve as novel biomarkers for liver injury but not specifically for HCC.
2010 Wiley-Liss, Inc.

Key words: miRNA; hepatocellular carcinoma; hepatitis; serum

INTRODUCTION great research interest. Numerous studies have

Hepatocellular carcinoma (HCC) is the sixth most
common cancer worldwide in terms of numbers of
cases and the third most common cause of cancer
death because of its poor prognosis [1]. It has been
well known that infection with hepatitis B and C
virus (HBV and HCV) is the major etiological factor
for the development of HCC. Currently, there are no
reliable biomarkers for early detection of primary
HCC and most patients with primary HCC are
diagnosed at advanced stages, which is associated
with poor prognosis and low survival rate of the
disease. a-Fetoprotein (a-AFP) has mainly been used
in clinic for diagnosis of primary HCC; however, its
sensitivity and specificity are not satisfying [2]. Thus,
the identification of more effective and reliable
markers for early detection of primary HCC is
extremely important and numerous efforts have
been made during the past decades.
MicroRNAs (miRNAs), a class of small RNAs that
regulate mRNA translation and function as onco-
genes or tumor suppressor genes, have attracted
shown that aberrant miRNA expression is associated
with the development and progression of various
types of human cancer [37]. The published studies
demonstrated an alteration of miRNAs expression
profiles in primary HCC compared with normal liver
tissue. It has been shown that among the deregulated
miRNAs in HBV-related HCC, miR-10b, miR-18,
Additional Supporting Information may be found in the online
version of this article.
Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B
virus; miRNA, microRNA; Ct, cycle threshold; ROC, receiver-operator
characteristic.
*Correspondence to: Department of Etiology & Carcinogenesis,
Cancer Institute & Hospital, Chinese Academy of Medical Sciences,
Beijing 100021, China.
**Correspondence to: Department of Abdominal Surgery,
Cancer Institute & Hospital, Chinese Academy of Medical Sciences,
Beijing 100021, China
Received 1 August 2010; Revised 18 October 2010; Accepted
27 October 2010
DOI 10.1002/mc.20712
Published online 10 December 2010 in Wiley Online Library
(wileyonlinelibrary.com).

2010 WILEY-LISS, INC.

 SERUM miRNAs IN LIVER CANCER OR HEPATITIS
miR-20, miR-21, miR-221/222, and miR-224 are up-
regulated but miR-122, miR-145, miR-150, miR-
199a, miR-199b, miR-200b, miR-214, and miR-223
are down-regulated [8,9]. Many of these miRNAs are
thought to play important role in hepatocarcino-
genesis and other types of human cancer. Among the
deregulated miRNAs, miR-21 functions as an onco-
gene and its up-regulation promotes malignant cell
proliferation, invasion and metastasis [10,11],
whereas miR-223 may function as tumor suppressor
gene and is commonly repressed in HCC [12]. MiR-
122 is a liver-specific miRNA and loss of its expression
contributes to the malignant phenotype of HCC cells
[1315].
MiRNAs in serum or plasma have been considered
as promising novel biomarkers for cancer diagnosis
and prognosis [16]. For example, it has been reported
that 3 miRNAs (miR-21, miR-155, and miR-210) are
elevated in serum of patients with diffuse large B-cell
lymphoma compared with healthy individuals and
miR-21 elevation is also associated with relapse-free
survival [17]. Recently, more studies have been
emerging to show that serum miRNAs are potential
diagnostic or prognostic markers for certain types of
cancer including lung [18,19], prostate [20], color-
ectal [21], ovarian [22], and breast [23] cancers.
However, no study has so far been reported on serum
miRNAs and HCC. On the basis of these findings, we
hypothesized that some deregulated miRNAs in HCC
would also present in serum of patients with HCC
and might serve as diagnostic markers.
To examine this hypothesis, we selected three
commonly deregulated miRNAs in HCC, miR-21,
miR-122, and miR-233, for analysis in patients with
primary HCC and healthy controls. Because the
expression levels of some miRNAs in liver tissue and/
or serum are also elevated due to liver injury [2426]
and it is a common nature that primary HCC is often
accompanied with chronic hepatitis [27], in this
study we also analyzed these three serum miRNAs in
patients with chronic type B hepatitis.
MATERIALS AND METHODS
Study Subjects
This study consisted of 101 patients with advanced
primary HCC, 48 patients with chronic type B
hepatitis, and 89 healthy controls. Patients with
HCC and patients with chronic type B hepatitis were
recruited at Cancer Hospital and Peking Union
Hospital, Chinese Academy of Medical Sciences
(Beijing), respectively. Healthy controls were ran-
domly selected from a database consisting of 2500
individuals based on a physical examination. Each
participant donated 5-ml blood sample for serum
miRNA analysis. For HCC patients, blood samples
were taken at the time of initial consultation before
definitive surgical intervention and/or adjuvant
therapy. For patients with chronic type B hepatitis,
Molecular Carcinogenesis
137
blood samples were taken at the time when the
disease was active. This study was approved by the
Institutional Review Board of the Chinese Academy
of Medical Sciences Cancer Institute and Hospital.
RNA Extraction and Reverse Transcription
Blood sample was centrifuged at 3000g for 10 min
at 48C to completely remove cellular components,
and the supernatant (serum) was collected. RNA was
then extracted from 100 ml of serum using the
TRIZOL reagent (Invitrogen, Carlsbad, CA) accord-
ing to the protocol provided by the manufacturer.
Briefly, 1.0-ml TRIZOL reagent and 200-ml chloro-
form were added to the serum sample and the
mixture was vortexed for 15 s and stood at 258C for
3 min. After centrifugation at 12 000g for 15 min at
48C, the supernatant was transferred to a fresh tube
and 500-ml isopropanol was added. After incubation
at 208C for 20 min, the mixture was centrifuged at
12 000g for 10 min at 48C to remove the supernatant
and the RNA pellet was washed with 75% ethanol.
After removal of ethanol by centrifugation at 7500g
for 5 min at 48C, RNA was air-dried for 5 min and
then dissolved in 30-ml RNase-free water. The purity
of isolated RNA was determined by OD260/280 using
a Nanodrop ND-1000 (Thermo Scientific, Worcester,
MA), with the value around 2.0 indicating high
purity. Each sample of 11.5-ml RNA was polyadeny-
lated and reversely transcribed to cDNA in a final
volume of 30 ml using polyadenylation polymerase
(New England Biolabs, Beverly, MA) and First-Strand
cDNA Synthesis Kit (Takara, Dalian, China) with
oligo-d(T) primer. The cDNA product was 1:5 diluted
with water and stored at 808C for analysis.
Validation of Internal Reference for Serum miRNA
Quantification
To detect the interest miRNAs in serum sample
by quantitative real-time PCR, it must have stable
internal references. Because there are no universal
internal references that have been validated to use, 7
miRNAs that are presented in human serum, RNU48,
RNU6B, let-7a, miR-16, miR-142-3p, miR-181a, and
miR-181c [18,20,28], were empirically chosen for
examination in serum samples from seven healthy
individuals, four patients with HCC, and four
patients with chronic type B hepatitis. The stability
of these miRNAs was then tested with geNorm
software [29] and the most reliable miRNAs were
chosen as internal references.
Quantification of Serum miRNAs
Real-time quantitative PCR (qPCR) quantification
of miRNAs was performed with ABI Prism 7900HT
sequence detection system (Applied Biosystems,
Foster City, CA) using SYBR Green PCR Master
Mixture (Takara). The miRNA-specific primers
(Supplementary Table I) were designed on the basis
of the miRNA sequences obtained from the miRBase
138 XU ET AL.

database (http://microrna.sanger.ac.uk/). Melting Validation of miR-181a and miR-181c as Internal

curve analysis was performed at the end of PCR
cycles in order to validate the specificity of the
expected PCR product. All samples were run in
duplicate, including blank controls without cDNA.
The cycle threshold (Ct) is defined as the number of
cycles required for the fluorescent signal to cross the
threshold in qPCR. The formula 2DCt was used to
calculate the levels of miRNAs in serum, where
DCt mean (Ct of internal references)Ct of target
miRNA.
Statistical Analysis
MannWhitney test was performed to compare
the differences in serum miRNA levels between
patients and healthy controls. All tests were two-
sided test and P < 0.05 was considered statistically
significant. Receiver-operator characteristic (ROC)
curves were established to evaluate the value of
serum miRNA levels for discriminating patients with
HCC or chronic hepatitis from healthy controls, and
patients with HCC from patients with chronic
hepatitis. Statistical analyses were performed with
SPSS 16.0 software (SPSS, Inc., Chicago, IL).
RESULTS
Characteristics of Study Subjects
Select characteristics of study subjects are sum-
marized in Table I. There were no significant differ-
ences in sex distribution among the three groups of
study subjects. However, although there was no
significant difference between patients with HCC
and healthy controls in terms of age distribution,
patients with chronic hepatitis were younger than
healthy controls and patients with HCC (both
P < 0.001). Seventy-six patients (75.2%) with HCC
were HBsAg positive, whereas none of healthy
controls carried HBV.
References for Quantification
In order to select internal references for quantifi-
cation of serum miRNAs, we examined the levels of
RNU6B, RNU48, let-7a, miR-16, miR-142-3p, miR-
181a, and miR-181c by using quantitative RT-PCR in
15 serum samples (seven healthy individuals, four
HCC patients, and four chronic type B hepatitis
patients). Data analysis with geNorm showed that
combined use of both miR-181a and miR-181c as
internal references had an M value of 0.154, indicat-
ing that these two miRNAs are stably expressed in
serum and are optimal references for the PCR
quantification of other serum miRNAs [29].
Increased Levels of Serum miRNAs in Patients With HCC
We first analyzed the profiles of serum miR-21,
miR-122, and miR-223 in a training set consisting of
10 healthy controls and 10 HCC patients. The
median levels of these serum miRNAs were signifi-
cantly higher in patients with HCC than in healthy
controls (0.68 vs. 0.12, P 0.028; 0.67 vs. 0.22,
P 0.007; and 3.13 vs. 0.24, P 0.005; respectively).
Similar trend of the results were obtained in the
validation set of 79 healthy controls and 91 HCC
patients, with the levels of serum miR-21, miR-122,
and miR-223 being significantly elevated in patients
than in controls (3.85 vs. 0.14, P 6.90
1012; 1.40
vs. 0.40, P 9.19
108; and 27.81 vs. 0.46,
P 1.01
1010; respectively). Pooled analysis of
the training and validation sets showed that differ-
ences in the levels of these serum miRNAs between
HCC patients and healthy controls were more
significant (3.36 vs. 0.14, P 7.48
1013; 1.19 vs.
0.34, P 6.93
109; and 17.20 vs. 0.35,
P 3.90
1012; respectively; Figure 1). However,
we noted that although HCC patients had signifi-
cantly higher levels of these three serum miRNAs

Table I. Distribution of Select Characteristics of Healthy Controls, Patients With Primary Hepatocellular Carcinoma, and
Patients With Chronic Type B Hepatitis

Healthy controls (n 89) Patients with HCC (n 101) Patients with chronic hepatitisa (n 48)

No. % No. % No. %

Sex
Male 68 76.4 78 77.2 31 64.6
Female 21 23.6 23 22.8 17 35.4
Age (yr)
50 36 40.5 39 38.6 38 79.2
5160 26 29.2 30 29.7 7 14.6
6170 18 20.2 21 20.8 2 4.1
>70 9 10.1 11 10.9 1 2.1
HBV status
HBsAg 0 0.0 76 75.2 48 100.0
HBsAg 100 100.0 25 24.8 0 0.0
a
P < 0.001, compared with healthy controls or patients with HCC in terms of age.

Molecular Carcinogenesis
 SERUM miRNAs IN LIVER CANCER OR HEPATITIS
Figure 1. Serum levels of miR-21 (a), miR-122 (b), and miR-233 (c)
in healthy controls (n 89), patients with hepatocellular carcinoma
(HCC, n 101), and patients with chronic type B hepatitis (n 48).
Values shown (log10 scale at Y-axis) are normalized to miR-181a and
miR-181c. The median levels of the three serum miRNAs in patients
with HCC or chronic hepatitis are significantly higher than that in
healthy controls (all P < 0.0001). The median levels of serum miR-21
and miR-122 in patients with chronic hepatitis are significantly higher
than that in patients with HCC (both P < 0.0001), whereas the
median levels of serum miR-223 are not significantly different
between these two groups of subjects (P 0.957).
than healthy controls, overlap to a certain extent
occurred between these two groups of study subjects.
Increased Levels of Serum miRNAs in Patients With
Type B Hepatitis
We examined 48 serum samples of patients with
chronic type B hepatitis and found that the median
Molecular Carcinogenesis
139
levels of miR-21 (22.86 vs. 0.14, P 2.05
1012),
miR-122 (11.02 vs. 0.34, P 4.52
1016), and miR-
223 (11.44 vs. 0.35, P 1.65
1011) were also
elevated in these patients than in healthy controls
(Figure 1). Interestingly, the levels of miR-21 and
miR-122 in serum of patients with chronic hepatitis
were significantly higher than those in patients with
HCC (22.86 vs. 3.36, P 3.99
104 and 11.02 vs.
1.19, P 4.97
108; Figure 1a and b), whereas no
significant difference was observed for miR-223
between the two groups of study subjects (11.44 vs.
17.20, P 0.955; Figure 1c).
Evaluation of miRNAs as Potential Diagnostic Markers
To evaluate whether these serum miRNAs can be
used as potential diagnostic markers for HCC or
hepatitis, ROC curve analyses were performed. It was
revealed that the levels of serum miR-21, miR-122,
and miR-223 were potential markers for discriminat-
ing HCC patients from healthy controls, with ROC
curve areas of 0.87 (95% CI: 0.810.93), 0.79 (95%
CI: 0.710.86), and 0.86 (95% CI: 0.800.92),
respectively (Figure 2a). At the cut-off values of 0.46
(for miR-21), 0.70 (for miR-122), and 1.91 (for miR-
223), the sensitivity and specificity for these markers
were 84% and 73.5%, 70.7% and 69.1%, and 80.0%
and 76.5%, respectively. Similarly, ROC curve anal-
yses revealed that these three miRNAs were useful
markers for discriminating patients with chronic
hepatitis from healthy controls. ROC curve areas for
miR-21, miR-122, and miR-223 were 0.91 (95% CI:
0.840.97), 0.93 (95% CI: 0.880.98), and 0.88 (95%
CI: 0.810.942), respectively (Figure 2b). At the cut-
off values of 3.48 for miR-21, 1.50 for miR-122, and
1.73 for miR-223, sensitivity and specificity were
80.0% and 95.6%, 80.0% and 91.2%, and 80.0% and
75.0%, respectively. However, ROC curve analyses
revealed that these three miRNAs were unable to
differentiate patients with HCC from patients with
chronic hepatitis (data not shown).
DISCUSSION
In this study, we found that serum miR-21, miR-
122, and miR-223 were significantly elevated in
patients with HCC or patients with chronic type B
hepatitis compared with healthy controls. In addi-
tion, the levels of serum miR-21 and miR-122 were
significantly higher in patients with chronic type B
hepatitis than in patients with HCC. All these three
miRNAs showed potential diagnostic values for HCC
or chronic hepatitis. However, since the significantly
higher levels of serum miR-21 and miR-223 have
been reported in patients with other types of human
cancer compared with healthy controls [17,22], they
may not be specific markers for HCC or chronic
hepatitis. Nevertheless, because miR-122 has been
shown to be liver-specific [30] and our ROC analyses
yielded a curve area of 0.93 for chronic hepatitis and
0.79 for HCC, this miRNA could serve as a potential
140
Figure 2. Receiver-operator characteristic (ROC) curves of the miR-21, miR-122, and miR-233 for discriminating
hepatocellular carcinoma (a) or chronic type B hepatitis (b).
marker for chronic hepatitis and perhaps HCC if liver
injury caused by other factors can be excluded.
It has been shown that miR-122 and miR-223 are
frequently down-regulated in HCC compared with
adjacent benign liver [12,14,15,3133]. Thus, it
appears contrary and unexpected that the levels of
miR-122 and miR-223 are elevated in serum of HCC
patients. Our results showed that the elevated serum
miR-122 and miR-223 are presented not only in
patients with HCC but also in patients with chronic
hepatitis, suggesting that the elevated miR-122 and
miR-223 in the serum of patients may reflect liver
injury but not tumor itself. Hepatocytes contain
abundant miR-122 and miR-223 and damage of
hepatocytes caused by inflammation due to virus
infection or cancer would be expected to release
significant amount of these miRNAs into the circu-
lation. Because serum miRNAs have been shown to
be very stable [30], miRNAs leaked from damaged
hepatocytes would accumulate in blood to a high
level. This might explain why miR-122 and miR-223
are down-regulated in HCC tissues but elevated in
serum of HCC patients. These findings suggest that
the two miRNAs might be markers of liver damage
rather than HCC. Similar results have been obtained
in the previous studies using animal model, showing
that drug-induced liver injury elevated plasma miR-
122 levels [24,26].
MiR-21 is expressed in various human tissues
including the liver and over-expression of this
miRNA has been observed in many types of cancer
such as breast cancer [34], lung cancer [35], colon
cancer [36], and HCC [10,11,37,38]. Recent studies
have shown that serum miR-21 levels are signifi-
cantly increased in patients with defused large B-cell
lymphoma or ovarian cancer [17,22]. In the present
study, we found higher levels of serum miR-21 in
Molecular Carcinogenesis
XU ET AL.
patients with HCC than in healthy controls, which is
consistent with the previous studies and suggests
that miR-21 may be a universal serum marker for
several cancers. However, we also detected a signifi-
cantly higher miR-21 levels in serum of patients with
chronic type B hepatitis; furthermore, the median
level of serum miR-21 in patients with chronic
hepatitis was much higher than that in patients with
HCC (22.86 vs. 3.36, P < 0.001). This finding points
out that elevated serum miR-21 could also come
from tissue injury such as hepatitis. Since patients
with chronic hepatitis may have more serious
damage of hepatocytes than patients with HCC, it
is reasonable to see much higher level of serum miR-
21 and miR-122 in patients with chronic hepatitis
than in patients with HCC. Elevation of certain
miRNAs in serum or plasma has been proven to come
from corresponding tissue damage. For example,
elevated plasma miR-208 and miR-1 have been
shown to come from damaged cells due to myocar-
dial injury [39] and acute myocardial infarction [40].
An important issue in detection of serum miRNAs
is that there are no widely accepted universal internal
references for quantification. RNU6B, RNU48, let-7a,
miR-16, miR-142-3p, miR-181a, and miR-181c have
been used as internal references in tissue samples but
not all in serum samples [18,20,28]. In the present
study, these candidate miRNAs were therefore
initially tested as internal references in a set of serum
samples. Analysis of the resultant Ct values with
geNorm software usually used for selection of most
reliable reference genes [29] showed that miR-181a
and miR-181c are the most stable and optimal as
internal references under our experimental condi-
tions. With this couple of serum miRNAs as internal
references for quantification, our results in the
present study should be reliable. However, to
 SERUM miRNAs IN LIVER CANCER OR HEPATITIS
validate whether these elevated serum miRNAs can
serve as biomarkers for hepatitis or HCC, further
studies with larger sample size are needed. In
addition, further studies should also include patients
with type C hepatitis and patients with liver injury
induced by other factors such as chemicals and drugs
for comparison.
In conclusion, our study demonstrated that three
miRNAs commonly deregulated in primary HCC,
miR-21, miR-122, and miR-223, are presented at
higher levels in serum of patients with HCC
compared with healthy individuals, suggesting that
these serum miRNAs, especially miR-122 known to
be liver specific, might serve as potential markers of
the cancer. However, because these miRNAs are also
presented at significantly higher levels in serum of
patients with chronic hepatitis, they are more likely
to reflect liver injury caused by inflammation.
Therefore, caution should be taken when one
intends to use these miRNAs as markers of HCC or
other types of human cancer.
ACKNOWLEDGMENTS
This study was supported by the State Key Basic
Research Program (2004CB518701).
REFERENCES
1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,
2002. CA Cancer J Clin 2005;55:74108.
2. Zinkin NT, Grall F, Bhaskar K, et al. Serum proteomics and
biomarkers in hepatocellular carcinoma and chronic liver
disease. Clin Cancer Res 2008;14:470477.
3. Zhang HH, Wang XJ, Li GX, Yang E, Yang NM. Detection of
let-7a microRNA by real-time PCR in gastric carcinoma.
World J Gastroenterol 2007;13:28832888.
4. Wu W, Sun M, Zou GM, Chen J. MicroRNA and cancer:
Current status and prospective. Int J Cancer 2007;120:953
960.
5. Visone R, Petrocca F, Croce CM. Micro-RNAs in gastro-
intestinal and liver disease. Gastroenterology 2008;135:
18661869.
6. Iorio MV, Casalini P, Piovan C, et al. MicroRNA-205 regulates
HER3 in human breast cancer. Cancer Res 2009;69:2195
2200.
7. Garzon R, Heaphy CE, Havelange V, et al. MicroRNA 29b
functions in acute myeloid leukemia. Blood 2009;114:
53315341.
8. Varnholt H, Drebber U, Schulze F, et al. MicroRNA gene
expression profile of hepatitis C virus-associated hepatocel-
lular carcinoma. Hepatology 2008;47:12231232.
9. Ladeiro Y, Couchy G, Balabaud C, et al. MicroRNA profiling in
hepatocellular tumors is associated with clinical features and
oncogene/tumor suppressor gene mutations. Hepatology
2008;47:19551963.
10. Connolly E, Melegari M, Landgraf P, et al. Elevated
expression of the miR-17-92 polycistron and miR-21 in
hepadnavirus-associated hepatocellular carcinoma contrib-
utes to the malignant phenotype. Am J Pathol 2008;173:
856864.
11. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST,
Patel T. MicroRNA-21 regulates expression of the PTEN
tumor suppressor gene in human hepatocellular cancer.
Gastroenterology 2007;133:647658.
12. Wong QW, Lung RW, Law PT, et al. MicroRNA-223
is commonly repressed in hepatocellular carcinoma and
Molecular Carcinogenesis
141
potentiates expression of Stathmin1. Gastroenterology
2008;135:257269.
13. Bai S, Nasser MW, Wang B, et al. MicroRNA-122 inhibits
tumorigenic properties of hepatocellular carcinoma cells and
sensitizes these cells to sorafenib. J Biol Chem 2009;284:
3201532027.
14. Tsai WC, Hsu PW, Lai TC, et al. MicroRNA-122, a tumor
suppressor microRNA that regulates intrahepatic metastasis
of hepatocellular carcinoma. Hepatology 2009;49:1571
1582.
15. Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thor-
geirsson SS. Loss of miR-122 expression in liver cancer
correlates with suppression of the hepatic phenotype and
gain of metastatic properties. Oncogene 2009;28:35263536.
16. Cortez MA, Calin GA. MicroRNA identification in plasma and
serum: A new tool to diagnose and monitor diseases. Expert
Opin Biol Ther 2009;9:703711.
17. Lawrie CH, Gal S, Dunlop HM, et al. Detection of elevated
levels of tumour-associated microRNAs in serum of patients
with diffuse large B-cell lymphoma. Br J Haematol 2008;
141:672675.
18. Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in
serum: A novel class of biomarkers for diagnosis of cancer
and other diseases. Cell Res 2008;18:9971006.
19. Hu Z, Chen X, Zhao Y, et al. Serum microRNA signatures
identified in a genome-wide serum microRNA expression
profiling predict survival of non-small-cell lung cancer. J Clin
Oncol 2010;28:17211726.
20. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs
as stable blood-based markers for cancer detection. Proc Natl
Acad Sci USA 2008;105:1051310518.
21. Huang Z, Huang D, Ni S, Peng Z, Sheng W, Du X. Plasma
microRNAs are promising novel biomarkers for early
detection of colorectal cancer. Int J Cancer 2010;127:118
126.
22. Resnick KE, Alder H, Hagan JP, Richardson DL, Croce CM,
Cohn DE. The detection of differentially expressed micro-
RNAs from the serum of ovarian cancer patients using a novel
real-time PCR platform. Gynecol Oncol 2009;112:5559.
23. Heneghan HM, Miller N, Lowery AJ, Sweeney KJ, Newell J,
Kerin MJ. Circulating microRNAs as novel minimally invasive
biomarkers for breast cancer. Ann Surg 2010;251:499505.
24. Wang K, Zhang S, Marzolf B, et al. Circulating microRNAs,
potential biomarkers for drug-induced liver injury. Proc Natl
Acad Sci USA 2009;106:44024407.
25. Yu CH, Xu CF, Li YM. Association of MicroRNA-223
expression with hepatic ischemia/reperfusion injury in mice.
Dig Dis Sci 2009;54:23622366.
26. Laterza OF, Lim L, Garrett-Engele PW, et al. Plasma micro-
RNA as sensitive and specific biomarkers of tissue injury. Clin
Chem 2009;55:19771983.
27. Nguyen VT, Law MG, Dore GJ. Hepatitis B-related hepato-
cellular carcinoma: Epidemiological characteristics and dis-
ease burden. J Viral Hepat 2009;16:453463.
28. Ng EK, Chong WW, Jin H, et al. Differential expression of
microRNAs in plasma of patients with colorectal cancer:
A potential marker for colorectal cancer screening. Gut
2009;58:13751381.
29. Ahn K, Huh JW, Park SJ, et al. Selection of internal reference
genes for SYBR green qRT-PCR studies of rhesus monkey
(Macaca mulatta) tissues. BMC Mol Biol 2008;9:78.
30. Chang J, Nicolas E, Marks D, et al. MiR-122, a mammalian
liver-specific microRNA, is processed from hcr mRNA and
may downregulate the high affinity cationic amino acid
transporter CAT-1. RNA Biol 2004;1:106113.
31. Kutay H, Bai S, Datta J, et al. Downregulation of miR-122 in
the rodent and human hepatocellular carcinomas. J Cell
Biochem 2006;99:671678.
32. Girard M, Jacquemin E, Munnich A, Lyonnet S, Henrion-
Caude A. MiR-122, a paradigm for the role of microRNAs in
the liver. J Hepatol 2008;48:648656.
142
33. Murakami Y, Yasuda T, Saigo K, et al. Comprehensive
analysis of microRNA expression patterns in hepatocellular
carcinoma and non-tumorous tissues. Oncogene 2006;25:
25372545.
34. Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene
expression deregulation in human breast cancer. Cancer
Res 2005;65:70657070.
35. Markou A, Tsaroucha EG, Kaklamanis L, Fotinou M,
Georgoulias V, Lianidou ES. Prognostic value of mature
microRNA-21 and microRNA-205 overexpression in non-
small cell lung cancer by quantitative real-time RT-PCR. Clin
Chem 2008;54:16961704.
36. Schetter AJ, Leung SY, Sohn JJ, et al. MicroRNA
expression profiles associated with prognosis and thera-
Molecular Carcinogenesis
XU ET AL.
peutic outcome in colon adenocarcinoma. JAMA 2008;
299:425436.
37. Jiang J, Gusev Y, Aderca I, et al. Association of MicroRNA
expression in hepatocellular carcinomas with hepatitis infection,
cirrhosis, and patient survival. Clin Cancer Res 2008;14:419427.
38. Pineau P, Volinia S, McJunkin K, et al. miR-221 over-
expression contributes to liver tumorigenesis. Proc Natl Acad
Sci USA 2010;107:264269.
39. Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y, Iwai N.
Plasma miR-208 as a biomarker of myocardial injury. Clin
Chem 2009;55:19441949.
40. Ai J, Zhang R, Li Y, et al. Circulating microRNA-1 as a
potential novel biomarker for acute myocardial infarction.
Biochem Biophys Res Commun 2010;391:7377.