- Email: [email protected]
Serum microRNA-125b correlates with hepatitis B viral replication and liver necroinflammation
Accepted Manuscript Serum microRNA-125b Correlates with Hepatitis B Viral Replication and Liver Necroinflammation Fahong Li, Pu Zhou, Wanyu Deng, Jinyu Wang, Richeng Mao, Yao Zhang, Jing Li, Yu Jie, Feifei Yang, Yuxian Huang, Mengji Lu, Jiming Zhang PII:
S1198-743X(16)00016-1
DOI:
10.1016/j.cmi.2015.12.024
Reference:
CMI 493
To appear in:
Clinical Microbiology and Infection
Received Date: 17 September 2015 Revised Date:
7 December 2015
Accepted Date: 21 December 2015
Please cite this article as: Li F, Zhou P, Deng W, Wang J, Mao R, Zhang Y, Li J, Jie Y, Yang F, Huang Y, Lu M, Zhang J, Serum microRNA-125b Correlates with Hepatitis B Viral Replication and Liver Necroinflammation, Clinical Microbiology and Infection (2016), doi: 10.1016/j.cmi.2015.12.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Original Article
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Serum microRNA-125b Correlates with Hepatitis B Viral Replication and
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Liver Necroinflammation
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Fahong Li,*,1 Pu Zhou,*,1 Wanyu Deng,‡ Jinyu Wang,* Richeng Mao,* Yao Zhang,*
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Jing Li,* Yu Jie,* Feifei Yang,* Yuxian Huang, * Mengji Lu,‡ Jiming Zhang*,§
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*
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Shanghai, China
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‡
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Essen, Germany
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Department of Infectious Diseases, Huashan Hospital, Fudan University,
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Institute of Virology, University Hospital of Essen, University of Duisburg-Essen,
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§
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Ministry of Health (MOH&MOE), Fudan University, Shanghai, China
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Key Laboratory of Medical Molecular Virology of the Ministry of Education and
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Fahong Li and Pu Zhou contributed equally to the study.
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Running Title: microRNA-125b, HBV replication, and liver injury
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Corresponding authors:
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Jiming Zhang
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Room 510, Building 5, 12 Middle Wulumuqi Road, Shanghai, China
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Phone: +86 21 52888125; Fax: +86 21 52886140
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Email: [email protected]
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Mengji Lu
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Virchowstrasse 179, Institute of Virology, University Hospital of Essen, University
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of Duisburg-Essen, Essen, Germany
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Phone: 0201-723 3530; Fax: 0201-723 5929
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E-mail: [email protected]
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ABSTRACT
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MicroRNAs (miRNAs) were recently reported to play an important role in
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hepatitis B virus (HBV) infection and related diseases. We evaluated the
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correlation
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necroinflammation in Chinese patients with chronic hepatitis B (CHB) infection.
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Serum miRNA-125b levels in samples from 211 CHB patients were determined
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by reverse transcriptase-polymerase chain reaction. Liver biopsies were
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collected from 138 patients. Serum miRNA-125b, miRNA-122, and miRNA-124
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levels were determined. Correlations between serum miRNA-125b, viral
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replication, and liver necroinflammation were analyzed. The receiver operating
11
characteristic (ROC) curve was used to assess the discriminating power of
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serum miRNA-125b to grade liver necroinflammation (G). HepG2.2.15 cells were
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transfected with miRNA-125b mimics. Intracellular viral core DNA was extracted
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and analyzed by Southern blot. We found that serum miRNA-125b was
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positively correlated with the serum HBV DNA level. HBV replication capacity
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increased after transfection with miRNA-125b mimics. CHB patients with
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moderate-to-severe liver necroinflammation (G≥ 2) showed significantly higher
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(P < 0.001) serum miRNA-125b levels than those with G<2. In patients with
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alanine aminotransferase (ALT) levels lower than 2 upper limit of normal, serum
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miRNA-125b combined with miRNA-124 yielded an area under the ROC curve
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of 0.816, with 70.4% sensitivity and 84.9% specificity to discriminate the grade of
miRNA-125b,
viral
replication,
and
liver
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between
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ACCEPTED MANUSCRIPT liver necroinflammation (G ≥ 2).Thus, we concluded that miRNA-125b may
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enhance HBV replication. Serum miRNA-125b correlates with viral load. Serum
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miRNA-125b alone or combined with miRNA-124 has potential to discriminate
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grades of liver necroinflammation, particularly in Chinese CHB patients with
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normal or mildly increased ALT levels.
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Key words: hepatitis B virus; microRNA-125b; viral replication; liver
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necroinflammation prediction
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INTRODUCTION An estimated 248 million patients worldwide are chronically infected with
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hepatitis B virus (HBV) [1]. In China, a high prevalence of HBV infection, with a
4
hepatitis B surface antigen (HBsAg) carrier rate of 7.18% in 2006, has been
5
reported [2]. Chronic HBV infection (CHB) leads to cirrhosis and hepatocellular
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carcinoma, which is associated with a high mortality rate [3]. Recent reports
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have highlighted that microRNAs (miRNAs) may participate in regulating HBV
8
infection and related diseases [4-6].
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miRNAs are endogenous, small, noncoding RNAs that regulate
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expression of a variety of genes at the post-transcriptional level [7]. Recent data
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have shown that host miRNAs may be involved in host–virus interactions and
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play an important role in regulating the viral life cycle [5]. In patients with CHB,
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miRNA-122 inhibits viral replication in HBV-related hepatocellular carcinoma [8].
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Moreover, miRNA-1 promotes HBV replication by increasing HBV core promoter
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transcriptional activity [9]. Other miRNAs associated with viral replication have
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been
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miRNA-141, miRNA-1231, and miRNA-125a-5p [10-13].
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identified
more
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recently,
including
miRNA-199a-3p,
miRNA-210,
Besides a role in HBV replication, several miRNAs have been found to
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correlate with liver damage in CHB patients, including miRNA-210, miRNA-21,
20
miRNA-124, and miRNA-125a [5, 13-15]. For example, liver miRNA-125a may
21
correlate with liver disease progression [13]. An association of miRNA-124 with
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liver necroinflammation was also reported in CHB patients, showing potential as
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a novel biomarker for liver necroinflammation, particularly in patients with normal
3
or mildly increased alanine aminotransferase (ALT) levels [15]. As a homolog of miRNA-125a, serum miRNA-125b was identified to be
5
aberrantly expressed in hepatitis B antigen (HBeAg)-positive and -negative
6
children [16]. Recently, serum miRNA-125b was reported to correlate with serum
7
HBV DNA and HBsAg level in CHB patients [4]. miRNA-125b was also
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upregulated in patients with CHB, HBV-positive cirrhosis, and HBV-positive
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hepatocarcinoma [17]. Liver miRNA-125b was suggested to play an important
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role in regulating the hepatic pro-inflammatory state in quercetin-fed mice [18]. Therefore, we hypothesized that miRNA-125b may correlate with both
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viral replication and liver necroinflammation in CHB patients. In this study, we
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aimed to identify the correlation between miRNA-125b, viral replication, and liver
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necroinflammation in adult CHB patients; In addition, the potential predictive
15
value of miRNA-125b alone or in combination with other miRNAs for liver
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necroinflammation in CHB patients was investigated. The effect of miRNA-125b
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on HBV replicative ability in vitro was examined.
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MATERIALS AND METHODS
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Patients A total of 785 patients with CHB were screened between 2009 and 2014 at
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Huashan Hospital, Fudan University (Shanghai, China). Exclusion criteria
5
included
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cytomegalovirus, hepatitis virus C, A, D, and E, or human immunodeficiency
7
virus; inadequate serum samples; or incomplete clinical data. Finally, a total of
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211 patients with CHB were included in this study and were grouped according
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to the 4 phases of CHB infection, based on the 2009 EASL guidelines [19]:
or
virologically
confirmed
co-infection
with
immune-tolerant
immune-reactive
(IR),
inactive
carrier
(IC),
and
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HBeAg-negative CHB (ENH). All patients enrolled were infected with genotype B
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or C. From the total of 211 CHB patients, 138 underwent a liver biopsy at
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baseline. Of these, 29 patients achieved a virological response with entecavir
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therapy and underwent a second liver biopsy at 48 weeks. Additionally, 35 age-
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and gender-matched healthy volunteers were enrolled as healthy controls (HCs).
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Patients characteristic are summarized in Table 1.
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(IT),
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serologically
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Informed written consent was obtained from all patients and the study was
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approved by the Institutional Ethics Committee for human studies at Huashan
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Hospital, Fudan University, Shanghai, China. All procedures were in accordance
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with the Declaration of Helsinki.
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Liver Histology
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Liver specimens were collected using a 16-gauge Menghini needle, fixed in
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formalin, embedded in paraffin, and stained with either hematoxylin and eosin or
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Masson’s trichrome stain. The sections were then independently examined by
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two experienced
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Necroinflammatory lesions and fibrosis were evaluated based on the modified
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Scheuer scoring system [20].
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Blood Sampling
were
unaware
of
clinical
status.
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pathologists
Blood samples were collected at the outpatient department in serum tubes.
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Samples were centrifuged at 3000 ×g for 10 min at 4°C, and the supernatant
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serum was aliquoted and recentrifuged at 3000 ×g for an additional 10 min at
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4°C. Serum samples were stored at −80°C until use.
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miRNA
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Transcription–Polymerase Chain Reaction (qRT-PCR)
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and
Quantitative
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Isolation
Real-time
Reverse
The isolation and quantification of serum miRNA was performed using
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miRcute miRNA extraction, first-strand cDNA synthesis, and qPCR detection kits
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according to the manufacturer instructions (TIANGEN, Beijing, China). qRT-PCR
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was performed as previously described [15].
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Cell Culture and Transfection
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Cells of the human hepatoma cell line HepG2.2.15 that harbor integrated
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dimers of the HBV genome (GenBank accession number: U95551) were used
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for transfection. Cells were cultured in Dulbecco’s modified Eagle’s medium with
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ACCEPTED MANUSCRIPT penicillin (100 U/ml), streptomycin (100 mg/ml), and 400 µg/ml G418,
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supplemented with 10% fetal bovine serum. Cells were seeded at a density of
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5 × 105/well in 6-well plates and transfected with miRNA-125b mimics (QIAGEN,
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Germany) at step-increased concentrations of 5, 10, 20, and 40 nM using
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Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer
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instructions.
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5’-UCCCUGAGACCCUAACUUGUGA-3’. Duplicate plates were used for
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samples. miRNA mimic negative control was transfected at indicated
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concentrations
sequence
of
miRNA-125b
mimic
was
just
as
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The
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miRNA-125b
and
the
sequence
is
5'-UCACAACCUCCUAGAAAGAGUAA-3’ (miRIDIAN, GE Healthcare). miRNA-1
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( 5'-UGGAAUGUAAAGAAGUAUGUAU-3’) was used as a positive control for the
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influence on HBV replication (QIAGEN, Germany). Functional effectiveness
13
experiment of miRNA-125b on its known target gene enhancer of zeste homolog
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2 (EZH2) was conducted. On the second day after transfection, cells were
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treated with fresh medium containing 2% fetal bovine serum and 1% dimethyl
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sulfoxide.
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Analysis of HBV Replication In Vitro
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At 96 h post-transfection, the cell culture supernatant was collected for
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HBsAg and HBeAg detection using enzyme-linked immunosorbent assay
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according to the manufacturer instructions (Kehua, China). Cells were harvested
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and intracellular, encapsulated HBV DNA was extracted and analyzed by
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Southern blot analysis as previously described [21]. 72h after transfection, the
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expression of gene EZH2 was detected by western blot [9].
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Statistical Analysis Data were analyzed using SPSS software for Windows (version 18.0; IBM,
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Endicott, NY, USA). Statistical significance was determined by a Kruskal-Wallis
6
test, Mann-Whitney t-test, and Wilcoxon matched-pairs t-test. Spearman’s
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correlation was used to evaluate the correlation coefficient (r). The distribution of
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miRNA-125b in CHB patients was evaluated at different natural stages of
9
infection. Correlations between serum miRNA-125b and viral load as well as
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HBsAg level were analyzed. The relationship between serum miRNA-125b, liver
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necroinflammation, and fibrosis were investigated. Additionally, the predictable
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value of serum miRNA-125b for liver necroinflammation was assessed. The
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interrelations of miRNA-125b, miRNA-122, and miRNA-124 were analyzed.
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The diagnostic value for the differentiation of liver histology in CHB patients was
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assessed by receiver operating characteristic (ROC) and area under the ROC
16
curve (AUC). Binary logistic regression was used to generate a composite
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variable. Logistic regression was also performed to analyze the correlation
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between potential factors and liver necroinflammation or fibrosis. All statistical
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tests were two-tailed, and a p-value < 0.05 was considered statistically
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significant.
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RESULTS
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Positive Correlation of Serum miRNA-125b Levels and Viral Load in
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Patients with CHB In all patients, we observed that an increase in the average level of serum
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miRNA-125b was proportional to the increase in serum HBV DNA and HBsAg
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(Figure 1A and 1B). miRNA-125b levels in CHB patients with HBV DNA load > 7
6
log10 IU/ml were significantly higher than in those with lower viral loads (P <
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0.001). CHB patients with viral load < 5 log10IU/ml showed the lowest average
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serum miRNA-125b level (P < 0.05; Figure 1A). miRNA-122, showed a similar
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positive correlation with HBV DNA level (Figure 1C).
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We also analyzed the relationship between serum miRNA-125b and HBsAg
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levels, a known biomarker for viral replication. Similarly, serum miRNA-125b
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levels increased significantly with those of HBsAg (Figure 1B). The serum
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miRNA-125b level in CHB patients with HBsAg levels < 3 log10 IU/ml were
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significantly lower than in those with HBsAg ≥ 3–4 log10IU/ml (P < 0.001) or >4
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log10IU/ml (P < 0.001) (Figure 1B). miRNA-122 levels showed a similar positive
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correlation with HBsAg (Figure 1D). In contrast, miRNA-124 showed no
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association with serum HBV DNA and HBsAg levels (Figure 1E and 1F).
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Distribution of Serum miRNA-125b Levels at Different Phases of CHB
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Infection
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CHB patients showed significantly higher miRNA-125b levels than HCs
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miRNA-125b levels were observed at the IR, followed by IT and ENH phase, and
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then IC phase (Figure 2B). Nonetheless, patients in all subgroups showed
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significantly higher serum miRNA-125b levels compared to the HCs (P < 0.001).
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Serum miRNA-125b Levels Correlate with Liver Necroinflammation
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Severity in CHB Patients and Decreased after Antiviral Therapy
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Patients with no or mild liver necroinflammation and fibrosis showed a
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significantly lower miRNA-125b level than those with moderate-to-severe liver
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necroinflammation and fibrosis (P<0.001; Figure 2C). Subgroup analysis
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showed that serum miRNA-125 levels were significantly higher in CHB patients
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with moderate-to-severe necroinflammation (G2–4; n = 76) than in those with no
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or mild necroinflammation (G0–1; n = 62) (P < 0.001) (Figure 2D). miRNA-125b
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levels were significantly correlated with ALT (r =0.379, P<0.001) (Figure S1).
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Multivariate analysis showed that miRNA-125b (odds ratio [OR] = 1.932, P
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= 0.022), miRNA-124 (OR = 3.502, P = .002), and ALT (OR = 1.026, P < 0.001)
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are independent predictors for liver necroinflammation (Table S1). However,
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miRNA-125b showed no correlation with fibrosis (P=0.172). Serum miRNA-125b
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levels decreased significantly after therapy in the majority of patients (n = 29)
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compared with those at baseline (P=0.007), which is consistent with the
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histological improvement observed (Figure S2).
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Differentiating Power of miRNA-125b alone or Combined with miRNA-124
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for Liver Necroinflammation Grade in CHB patients ALT was an important biomarker of liver necroinflammation and an indicator
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of prior CHB treatment. In all CHB patients, ALT showed the highest diagnostic
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value for differentiating significant liver necroinflammation (G ≥ 2; AUC = 0.838)
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(Figure 3A). In patients with normal ALT levels, the differentiating power of
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miRNA-125b was comparable to that of miRNA-124 (AUC = 0.757 versus 0.723
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P > 0.05) (Figure 3B). The sensitivity, specificity, and accuracy for miRNA-125b
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were 75.0%, 79.1%, and 78.0%, and those for miRNA-124 were 43.8%, 95.4%,
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and 81.4%, respectively (Table S2). The combination of miRNA-125b and
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miRNA-124 showed higher diagnostic value (AUC = 0.801, accuracy of 83.1%).
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In patients with an ALT < 2 upper limit of normal (ULN), miRNA-125b (AUC =
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0.759) showed higher diagnostic value than miRNA-124 (AUC = 0.743) and ALT
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(AUC = 0.697) (Figure 3C). The sensitivity, specificity, and accuracy values for
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miRNA-125b were 77.8%, 73.6%, and 75.1%, respectively. The AUC of the
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combination of miRNA-125b and miRNA-124 was 0.816, whereas sensitivity,
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specificity, and accuracy values were 70.4%, 84.9%, and 79.6%, respectively.
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These data suggest that serum miRNA-125 alone or combined with miRNA-124
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is superior to ALT in predicting the degree of liver necroinflammation in patients
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with normal-to-mildly elevated ALT.
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miRNA-125b Enhanced HBV Replication Capacity In Vitro
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As shown in Figure 4, HBV replication capacity increased with higher doses of
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miRNA-125b mimic treatment to HepG2.2.15 cells. A similar trend was observed
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for the supernatant HBsAg and HBeAg level. In contrast, miRNA mimic negative
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control had no effect on viral replication, HBsAg or HBeAg level. The negative
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regulating function of miRNA-125b mimic on EZH2 expression was observed as
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reported previously [22]. miRNA-1 mimic was observed to enhance HBV
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replication capacity as ever demonstrated [9] (Figure S3).
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DISCUSSION
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ACCEPTED MANUSCRIPT Previous studies have shown that miRNA-125b plays an important role in
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the pathogenesis of various tumors such as skin cancer, lung cancer, and
3
hepatoma [23]. In the present study, we investigated the association of
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miRNA-125b, viral replication, and liver necroinflammation in adult CHB patients,
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as well as the effect of miRNA-125b on HBV replication in vitro.
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Several miRNAs have been found to correlate with HBV replication, liver
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necroinflammation or fibrosis (Table S3) [5-6, 12, 24-25]. In this study, serum
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miRNA-125b was correlated with viral load in adult CHB patients, which is
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consistent with previous studies [4, 16]; the in vitro results supported these
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clinical findings. Additionally, the miRNA-125b level of patients in the IT phase
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was lower than that in the IR phase, indicating that miRNA-125b may be
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influenced by other factors other than HBV replication, such as liver
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necroinflammation.
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We demonstrated that the serum miRNA-125b level is not only related to
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liver necroinflammation but also capable of differentiating the severity in CHB
16
patients.
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necroinflammation and an indicator of antiviral therapy [26]. According to the
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AASLD guideline for the treatment of CHB, ALT ≥ 2 ULN is used to indicate
19
antiviral treatment [26]. However, ALT alone is inadequate to grade liver
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necroinflammation. Indeed, liver biopsy of a study in the US revealed that 37%
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of patients with normal ALT showed significant fibrosis and inflammation [27].
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been
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as
a
biomarker
of
liver
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showed significant inflammation (G > 2) and 36.4% showed significant fibrosis
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(stage > 2) [28]. Therefore, a part of patients with normal or mildly elevated ALT
4
(1 ULN < ALT < 2 ULN) should receive antiviral therapy. Currently, liver biopsy is
5
the gold standard for evaluating liver necroinflammation and fibrosis [29].
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However, most patients refuse liver biopsy since it is invasive and painful.
7
Therefore, novel noninvasive biomarkers to evaluate liver necroinflammation
8
with higher sensitivity and diagnostic power are urgently needed. Some
9
noninvasive methods such as FibroScan have been used to investigate the
10
extent of liver fibrosis, but not necroinflammation [30]. Recently, miRNA-124 was
11
identified as an independent predictive factor for liver necroinflammation,
12
especially in patients with normal or mildly elevated ALT levels [15]. However, its
13
sensitivity
14
necroinflammation severity is relatively low. In our study, we found that serum
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miRNA-125b
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miRNA-125b was superior to miRNA-124 and ALT in differentiating grades of
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liver necroinflammation in CHB patients with normal or mildly elevated ALT.
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Furthermore, the combination of miRNA-125b with miRNA-124 markedly
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improved the power of differentiating liver necroinflammation, as well as
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sensitivity and accuracy. Though miRNA-125b and miRNA-124 both correlated
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with liver necroinflammation, only miRNA-125b also correlated with viral
and
diagnostic
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correlated
with
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to
differentiate
necroinflammation
and
liver
that
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replication. A very recent study demonstrated consistently that miRNA-125b correlates
3
positively with serum HBV DNA and HBsAg level [4]. Our study further
4
demonstrated miRNA-125b enhanced HBV replication capacity in vitro. On the
5
other hand, the study of Akamatsu et al. did not prove an association of serum
6
miRNA-125b level with liver necroinflammatory activity which is inconsistent with
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our findings [4]. This discrepancy could be explained by the difference in the
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patient
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necroinflammatory activity.
especially
the
proportion
of
patients
with
severe
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It was known that persistent HBV infection can lead to cirrhosis and
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hepatocellular carcinoma (HCC) with high mortality rate [3]. Long term changes
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in both serum HBV DNA and ALT levels independently predict the risk for
13
hepatocellular carcinoma [31]. It was reported that intrahepatic DNA levels
14
correlates strongly with serum HBV DNA levels and with the degree of fibrosis
15
[32]. Advanced fibrosis and severe necroinflammatory activity, in particular
16
severe lobular activity, are independent predisposing risk factors for the
17
development of HCC in patients with CHB [33]. In our study, we found that
18
miRNA-125b correlates both serum HBV DNA and ALT levels. However, the
19
relationship of miRNA-125b and HCC was not investigated yet. It was reported
20
that the expression of miRNA-125b decreases in HCC tumor tissues and cell
21
lines compared to non-malignant controls and overexpression of miRNA-125b
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significantly
suppress
cell
proliferation,
migration,
invasion,
and
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angiogenesis in HCC cell lines [34]. The level of miRNA-125b was also identified
3
as a HCC diagnostic discriminator from both noncancerous and normal liver
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tissues, serving as a HCC survival predictor [34]. Therefore, in the future the
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correlation between serum miRNA-125b and risk of HCC would be further
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investigated in patients with HBV-related chronic hepatitis.
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In our study, we also found a correlation between miRNA-122 and
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miRNA-125b as Akamatsu et al. reported [4]. However, the possible relationship
9
between miRNA-125b and miRNA-122 in CHB patients is obscure. It was
10
reported that miRNA-122 down-regulates its target cyclin G1, and thus interrupts
11
the negatively regulation of cyclin G1 on p53 and then facilitate p53-mediated
12
inhibition of HBV replication [35]. Thus miRNA-122 suppresses HBV replication
13
through cyclin G 1-modulated P53 activity in patients with hepatitis B. However,
14
the mechanism of miRNA-125b regulating HBV replication is still unknown. It
15
was reported that overexpression of miRNA-125b may repress the endogenous
16
level of p53 protein and then suppress apoptosis in human neuroblastoma cells
17
and lung fibroblast cells [36]. Therefore, we hypothesize that miRNA-125b may
18
repress the level of p53 protein and then correspondingly enhance HBV
19
replication. The counteraction of miRNA-122 and miRNA-125b on HBV
20
replication
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histopathological lesion.
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may
possibly
contribute
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to
viral
persistence
and
liver
ACCEPTED MANUSCRIPT There are several limitations to this study. First, the mechanisms by which
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miRNA-125b enhance HBV replication and predict liver necroinflammation
3
remained to be evaluated in the future study. Second, the larger number of
4
patients with normal or mildly elevated ALT should be included for verification in
5
the future, confirming the diagnostic values in clinics. Third, as our study was
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carried out in Chinese patients who were characterized by Genotype B and C,
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further studies are required in CHB patients from different geographical areas or
8
with different genotypes to confirm these data.
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In conclusion, we discovered that serum miRNA-125b correlated with viral
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replication and enhanced HBV replication capacity in vitro. Serum miRNA-125b
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alone or combined with miRNA-124 may have excellent differentiating power for
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liver necroinflammation severity in Chinese CHB patients with normal or mildly
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elevated ALT.
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ACKNOWLEDGEMENTS
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We thank Dr. Haiyan Lü (Hua shan Hospital, Fudan University) for her help
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during the study.
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CONFLICT OF INTEREST
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The authors have no conflicts of interest to declare.
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FUNDING
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ACCEPTED MANUSCRIPT This study was funded by the National Natural Science Foundation of China
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(81471933,81271833), the Major Science and Technology Special Project of
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China (2012ZX10002007-001-002, 2013ZX10002001), the “973” Project
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(2012CB519001), and the Special Research Fund of Ministry of Health for
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Non-Profit Sector (201302010).
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Abbreviations:
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ALT, alanine aminotransferase
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AUC, area under receiver operating characteristic curve
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CHB, chronic hepatitis B
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ENH, HBeAg-negative CHB
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EZH2, enhancer of zeste homolog 2
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G, grading of necroinflammation
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HBeAg, hepatitis B e antigen
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HBsAg, hepatitis B surface antigen
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HBV, hepatitis B virus
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HCs, healthy controls
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HCC, hepatocellular carcinoma
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IC, inactive carrier
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IT, immune tolerant
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IR, immune-reactive
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miRNA, microRNA; qRT-PCR, quantitative reverse transcription polymerase
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ROC, receiver operating characteristic
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S, staging of fibrosis
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ULN, upper limit of normal
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REFERENCES
SC
6
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[31]. Chen CF, Lee WC, Yang HI et al. Changes in serum levels of HBV-DNA and
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[32]. Wong DK, Yuen MF, Tse E et al. Detection of intrahepatic HBV DNA and
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Microbiology 2004; 42: 3920-24
[33]. Lee SH, Chung YH, Kim JA et al. Histological characteristics
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predisposing to development of hepatocellular carcinoma in patients with
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chronic hepatitis B. J Clin Pathol 2011; 64: 599-604 [34]. Li W, Xie L, He X et al. Diagnostic and prognostic implications of
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[35]. Wang S, Qiu L, Yan X et al. Loss of microRNA 122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G(1) -modulated P53 activity. Hepatology. 2012; 55(3):730-41. [36]. Le MT, Teh C, Shyh-Chang N et al. MicroRNA-125b is a novel negative regulator of p53. Genes Dev. 2009;23(7):862-76
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Figure 1. Correlations of serum miRNA-125b, miRNA-122, and miRNA-124
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with HBV DNA and HBsAg. (A) Serum miRNA-125b and HBV DNA; (B) serum
4
miRNA-125b and HBsAg; (C) serum miRNA-122 and HBV DNA; (D) serum
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miRNA-122 and HBsAg; (E) serum miRNA-124 and HBV DNA; (F) serum
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miRNA-124 and HBsAg;
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Figure 2. Correlations of serum miRNA-125b with liver necroinflammation
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and fibrosis. (A) Serum miRNA-125b levels in patients with HBV infection and
9
healthy controls. (B) Distribution of serum miRNA-125b levels in patients at
10
different phases of the course of CHB infection. (C, D) Patients were divided into
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groups by grading of necroinflammation or staging of fibrosis or both.
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Abbreviations: miRNA, microRNA; HBV, hepatitis B virus; HC, healthy control; IT,
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immune-tolerant; IR, immune-reactive; IC, inactive carrier; ENH, hepatitis B e
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antigen negative chronic hepatitis B; G, grading of necroinflammation; S, staging
15
of fibrosis; n, number.
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Figure 3. Differentiating power of serum ALT, miRNA-125b, miRNA-124,
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and combined miRNA-125b and miRNA-124 for liver necroinflammation
18
severity in CHB patients.
19
miRNA-125b, miRNA-124, and the combination of miRNA-125b and miRNA-124
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for differentiating the severity of liver necroinflammation in patients with CHB (n
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= 138) from HCs (n = 35). (B) Moderate-to-severe necroinflammation (≥G2) in
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(A) ROC curves and AUC are presented for ALT,
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U/L (n = 80).
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Figure 4. miRNA-125b enhanced HBV replication capacity in vitro.
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(A) HepG2.2.15 cells were transfected with step-increased levels of
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miRNA-125b mimic. Cells were harvested and intracellular, encapsulated HBV
6
DNA was extracted and analyzed by Southern blot analysis. (B) Supernatant
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HBsAg and HBeAg levels of HepG2.2.15 cells were determined by specific
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ELISAs after transfection with indicated doses of miRNA-125b mimic. (C)
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Functional effectiveness experiment of miRNA-125b on gene enhancer of zeste
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homolog 2. Abbreviations: miRNA, microRNA; HBV, hepatitis B virus; HBeAg,
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hepatitis B e antigen; HBsAg, hepatitis B surface antigen; EZH2, enhancer of
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zeste homolog 2;
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before and after antiviral therapy (n = 29). (A) Changes in the proportion of necroinflammation (G0–4) and fibrosis (S0–4). (B) Serum miRNA-125 levels of
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CHB patients before and after antiviral therapy.
Figure S3. HepG2.2.15 cells were transfected with miRNA control (10nM),
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miRNA-1 (5 nM) and miRNA-125b (5 nM). Cells were harvested and the levels of HBV replicative intermediates were determined by southern blot as previous
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Table 1. Baseline characteristics of enrolled individuals CHB patients (n = 211)
HCs (n = 35)
P value
Characteristic
IT (n = 37)
IR (n = 109)
IC (n = 29)
ENH (n = 36)
Gender (male, %)
23 (62.2 %)
77 (70.6 %)
20 (69.0 %)
28 (77.8 %)
27 (10)
32 (17)
36 (14)
42 (21)
34 (14)
.49
60.5 (86.3)
21.0 (17.0)
< .01
4.00 (2.16)
N/A
3.31 (0.83)
N/A
ALT (U/L)
29.5 (11.5)
HBV DNA (log10 IU/ml)
7.37 (0.94)
7.00 (2.54)
2.10 (1.00)
HBsAg (log10 IU/ml)
4.72 (0.52)
3.97 (0.91)
3.09 (1.58)
20/17
56/53
13/16
HBV Genotype (n, B/C)
124.0 (154.6)
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(18.5)
IT (n = 11)
IR (n = 74)
IC (n = 20)
9 (81.8 %)
53 (71.6 %)
17 (85.0 %)
Age (years)
30 (8)
33 (17)
37 (20)
ALT (U/L)
36 (18)
120 (128)
HBV DNA (log10IU/ml)
7.89 (1.01)
HBsAg (log10 IU/ml) HBV Genotype (n, B/C)
27 (81.8 %)
.44 .83
28 (20)
59 (87)
< .01
7.01 (2.13)
2.67 (1.86)
4.00 (2.50)
4.55 (1.15)
4.00 (0.89)
3.01 (0.58)
3.25 (0.92)
5/6
34/40
9/11
15/18
2/9/0/0/0
6/9/27/29/3
11/9/0/0/0
2/14/10/7/0
16/4/0/0/0
2/7/11/5/8
Necroinflammation
9/2/0/0/0
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Stage of fibrosis S0/S1/S2/S3/S4 (n)
P value
42 (21)
Grading of
G0/G1/G2/G3/G4 (n)
ENH (n = 33)
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Gender (male, %)
.37
17/16
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CHB patients with liver biopsy (n = 138)
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Age (years)
29 (82.8%)
11/14/28/11/10
For age, ALT, HBsAg, and HBV DNA titers, data are presented as median (interquartile range). P-values < .05 are considered as significant. N/A, not available. Abbreviations: ALT, alanine aminotransferase ; CHB, chronic hepatitis B; HBV, hepatitis B virus; HBsAg,
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hepatitis B surface antigen; IT, immune-tolerant; IR, immune-reactive; IC, inactive carrier; ENH, HBeAg
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negative hepatitis. G, grading of necroinflammation; S, staging of fibrosis.
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S1198-743X(16)00016-1
DOI:
10.1016/j.cmi.2015.12.024
Reference:
CMI 493
To appear in:
Clinical Microbiology and Infection
Received Date: 17 September 2015 Revised Date:
7 December 2015
Accepted Date: 21 December 2015
Please cite this article as: Li F, Zhou P, Deng W, Wang J, Mao R, Zhang Y, Li J, Jie Y, Yang F, Huang Y, Lu M, Zhang J, Serum microRNA-125b Correlates with Hepatitis B Viral Replication and Liver Necroinflammation, Clinical Microbiology and Infection (2016), doi: 10.1016/j.cmi.2015.12.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Original Article
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Serum microRNA-125b Correlates with Hepatitis B Viral Replication and
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Liver Necroinflammation
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Fahong Li,*,1 Pu Zhou,*,1 Wanyu Deng,‡ Jinyu Wang,* Richeng Mao,* Yao Zhang,*
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Jing Li,* Yu Jie,* Feifei Yang,* Yuxian Huang, * Mengji Lu,‡ Jiming Zhang*,§
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*
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Shanghai, China
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‡
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Essen, Germany
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Department of Infectious Diseases, Huashan Hospital, Fudan University,
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Institute of Virology, University Hospital of Essen, University of Duisburg-Essen,
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§
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Ministry of Health (MOH&MOE), Fudan University, Shanghai, China
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1
Key Laboratory of Medical Molecular Virology of the Ministry of Education and
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Fahong Li and Pu Zhou contributed equally to the study.
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Running Title: microRNA-125b, HBV replication, and liver injury
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Corresponding authors:
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Jiming Zhang
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Room 510, Building 5, 12 Middle Wulumuqi Road, Shanghai, China
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Phone: +86 21 52888125; Fax: +86 21 52886140
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Email: [email protected]
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Mengji Lu
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Virchowstrasse 179, Institute of Virology, University Hospital of Essen, University
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of Duisburg-Essen, Essen, Germany
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Phone: 0201-723 3530; Fax: 0201-723 5929
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E-mail: [email protected]
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ABSTRACT
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MicroRNAs (miRNAs) were recently reported to play an important role in
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hepatitis B virus (HBV) infection and related diseases. We evaluated the
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correlation
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necroinflammation in Chinese patients with chronic hepatitis B (CHB) infection.
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Serum miRNA-125b levels in samples from 211 CHB patients were determined
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by reverse transcriptase-polymerase chain reaction. Liver biopsies were
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collected from 138 patients. Serum miRNA-125b, miRNA-122, and miRNA-124
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levels were determined. Correlations between serum miRNA-125b, viral
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replication, and liver necroinflammation were analyzed. The receiver operating
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characteristic (ROC) curve was used to assess the discriminating power of
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serum miRNA-125b to grade liver necroinflammation (G). HepG2.2.15 cells were
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transfected with miRNA-125b mimics. Intracellular viral core DNA was extracted
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and analyzed by Southern blot. We found that serum miRNA-125b was
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positively correlated with the serum HBV DNA level. HBV replication capacity
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increased after transfection with miRNA-125b mimics. CHB patients with
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moderate-to-severe liver necroinflammation (G≥ 2) showed significantly higher
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(P < 0.001) serum miRNA-125b levels than those with G<2. In patients with
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alanine aminotransferase (ALT) levels lower than 2 upper limit of normal, serum
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miRNA-125b combined with miRNA-124 yielded an area under the ROC curve
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of 0.816, with 70.4% sensitivity and 84.9% specificity to discriminate the grade of
miRNA-125b,
viral
replication,
and
liver
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serum
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between
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ACCEPTED MANUSCRIPT liver necroinflammation (G ≥ 2).Thus, we concluded that miRNA-125b may
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enhance HBV replication. Serum miRNA-125b correlates with viral load. Serum
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miRNA-125b alone or combined with miRNA-124 has potential to discriminate
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grades of liver necroinflammation, particularly in Chinese CHB patients with
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normal or mildly increased ALT levels.
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Key words: hepatitis B virus; microRNA-125b; viral replication; liver
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necroinflammation prediction
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INTRODUCTION An estimated 248 million patients worldwide are chronically infected with
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hepatitis B virus (HBV) [1]. In China, a high prevalence of HBV infection, with a
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hepatitis B surface antigen (HBsAg) carrier rate of 7.18% in 2006, has been
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reported [2]. Chronic HBV infection (CHB) leads to cirrhosis and hepatocellular
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carcinoma, which is associated with a high mortality rate [3]. Recent reports
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have highlighted that microRNAs (miRNAs) may participate in regulating HBV
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infection and related diseases [4-6].
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miRNAs are endogenous, small, noncoding RNAs that regulate
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expression of a variety of genes at the post-transcriptional level [7]. Recent data
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have shown that host miRNAs may be involved in host–virus interactions and
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play an important role in regulating the viral life cycle [5]. In patients with CHB,
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miRNA-122 inhibits viral replication in HBV-related hepatocellular carcinoma [8].
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Moreover, miRNA-1 promotes HBV replication by increasing HBV core promoter
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transcriptional activity [9]. Other miRNAs associated with viral replication have
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been
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miRNA-141, miRNA-1231, and miRNA-125a-5p [10-13].
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identified
more
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recently,
including
miRNA-199a-3p,
miRNA-210,
Besides a role in HBV replication, several miRNAs have been found to
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correlate with liver damage in CHB patients, including miRNA-210, miRNA-21,
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miRNA-124, and miRNA-125a [5, 13-15]. For example, liver miRNA-125a may
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correlate with liver disease progression [13]. An association of miRNA-124 with
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liver necroinflammation was also reported in CHB patients, showing potential as
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a novel biomarker for liver necroinflammation, particularly in patients with normal
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or mildly increased alanine aminotransferase (ALT) levels [15]. As a homolog of miRNA-125a, serum miRNA-125b was identified to be
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aberrantly expressed in hepatitis B antigen (HBeAg)-positive and -negative
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children [16]. Recently, serum miRNA-125b was reported to correlate with serum
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HBV DNA and HBsAg level in CHB patients [4]. miRNA-125b was also
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upregulated in patients with CHB, HBV-positive cirrhosis, and HBV-positive
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hepatocarcinoma [17]. Liver miRNA-125b was suggested to play an important
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role in regulating the hepatic pro-inflammatory state in quercetin-fed mice [18]. Therefore, we hypothesized that miRNA-125b may correlate with both
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viral replication and liver necroinflammation in CHB patients. In this study, we
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aimed to identify the correlation between miRNA-125b, viral replication, and liver
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necroinflammation in adult CHB patients; In addition, the potential predictive
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value of miRNA-125b alone or in combination with other miRNAs for liver
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necroinflammation in CHB patients was investigated. The effect of miRNA-125b
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on HBV replicative ability in vitro was examined.
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MATERIALS AND METHODS
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Patients A total of 785 patients with CHB were screened between 2009 and 2014 at
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Huashan Hospital, Fudan University (Shanghai, China). Exclusion criteria
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included
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cytomegalovirus, hepatitis virus C, A, D, and E, or human immunodeficiency
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virus; inadequate serum samples; or incomplete clinical data. Finally, a total of
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211 patients with CHB were included in this study and were grouped according
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to the 4 phases of CHB infection, based on the 2009 EASL guidelines [19]:
or
virologically
confirmed
co-infection
with
immune-tolerant
immune-reactive
(IR),
inactive
carrier
(IC),
and
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HBeAg-negative CHB (ENH). All patients enrolled were infected with genotype B
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or C. From the total of 211 CHB patients, 138 underwent a liver biopsy at
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baseline. Of these, 29 patients achieved a virological response with entecavir
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therapy and underwent a second liver biopsy at 48 weeks. Additionally, 35 age-
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and gender-matched healthy volunteers were enrolled as healthy controls (HCs).
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Patients characteristic are summarized in Table 1.
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(IT),
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serologically
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Informed written consent was obtained from all patients and the study was
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approved by the Institutional Ethics Committee for human studies at Huashan
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Hospital, Fudan University, Shanghai, China. All procedures were in accordance
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with the Declaration of Helsinki.
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Liver Histology
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Liver specimens were collected using a 16-gauge Menghini needle, fixed in
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formalin, embedded in paraffin, and stained with either hematoxylin and eosin or
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Masson’s trichrome stain. The sections were then independently examined by
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two experienced
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Necroinflammatory lesions and fibrosis were evaluated based on the modified
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Scheuer scoring system [20].
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Blood Sampling
were
unaware
of
clinical
status.
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pathologists
Blood samples were collected at the outpatient department in serum tubes.
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Samples were centrifuged at 3000 ×g for 10 min at 4°C, and the supernatant
10
serum was aliquoted and recentrifuged at 3000 ×g for an additional 10 min at
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4°C. Serum samples were stored at −80°C until use.
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miRNA
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Transcription–Polymerase Chain Reaction (qRT-PCR)
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and
Quantitative
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Isolation
Real-time
Reverse
The isolation and quantification of serum miRNA was performed using
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miRcute miRNA extraction, first-strand cDNA synthesis, and qPCR detection kits
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according to the manufacturer instructions (TIANGEN, Beijing, China). qRT-PCR
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was performed as previously described [15].
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Cell Culture and Transfection
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Cells of the human hepatoma cell line HepG2.2.15 that harbor integrated
20
dimers of the HBV genome (GenBank accession number: U95551) were used
21
for transfection. Cells were cultured in Dulbecco’s modified Eagle’s medium with
8
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2
supplemented with 10% fetal bovine serum. Cells were seeded at a density of
3
5 × 105/well in 6-well plates and transfected with miRNA-125b mimics (QIAGEN,
4
Germany) at step-increased concentrations of 5, 10, 20, and 40 nM using
5
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer
6
instructions.
7
5’-UCCCUGAGACCCUAACUUGUGA-3’. Duplicate plates were used for
8
samples. miRNA mimic negative control was transfected at indicated
9
concentrations
sequence
of
miRNA-125b
mimic
was
just
as
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miRNA-125b
and
the
sequence
is
5'-UCACAACCUCCUAGAAAGAGUAA-3’ (miRIDIAN, GE Healthcare). miRNA-1
11
( 5'-UGGAAUGUAAAGAAGUAUGUAU-3’) was used as a positive control for the
12
influence on HBV replication (QIAGEN, Germany). Functional effectiveness
13
experiment of miRNA-125b on its known target gene enhancer of zeste homolog
14
2 (EZH2) was conducted. On the second day after transfection, cells were
15
treated with fresh medium containing 2% fetal bovine serum and 1% dimethyl
16
sulfoxide.
17
Analysis of HBV Replication In Vitro
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At 96 h post-transfection, the cell culture supernatant was collected for
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HBsAg and HBeAg detection using enzyme-linked immunosorbent assay
20
according to the manufacturer instructions (Kehua, China). Cells were harvested
21
and intracellular, encapsulated HBV DNA was extracted and analyzed by
9
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Southern blot analysis as previously described [21]. 72h after transfection, the
2
expression of gene EZH2 was detected by western blot [9].
3
Statistical Analysis Data were analyzed using SPSS software for Windows (version 18.0; IBM,
5
Endicott, NY, USA). Statistical significance was determined by a Kruskal-Wallis
6
test, Mann-Whitney t-test, and Wilcoxon matched-pairs t-test. Spearman’s
7
correlation was used to evaluate the correlation coefficient (r). The distribution of
8
miRNA-125b in CHB patients was evaluated at different natural stages of
9
infection. Correlations between serum miRNA-125b and viral load as well as
10
HBsAg level were analyzed. The relationship between serum miRNA-125b, liver
11
necroinflammation, and fibrosis were investigated. Additionally, the predictable
12
value of serum miRNA-125b for liver necroinflammation was assessed. The
13
interrelations of miRNA-125b, miRNA-122, and miRNA-124 were analyzed.
14
The diagnostic value for the differentiation of liver histology in CHB patients was
15
assessed by receiver operating characteristic (ROC) and area under the ROC
16
curve (AUC). Binary logistic regression was used to generate a composite
17
variable. Logistic regression was also performed to analyze the correlation
18
between potential factors and liver necroinflammation or fibrosis. All statistical
19
tests were two-tailed, and a p-value < 0.05 was considered statistically
20
significant.
21
RESULTS
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Positive Correlation of Serum miRNA-125b Levels and Viral Load in
2
Patients with CHB In all patients, we observed that an increase in the average level of serum
4
miRNA-125b was proportional to the increase in serum HBV DNA and HBsAg
5
(Figure 1A and 1B). miRNA-125b levels in CHB patients with HBV DNA load > 7
6
log10 IU/ml were significantly higher than in those with lower viral loads (P <
7
0.001). CHB patients with viral load < 5 log10IU/ml showed the lowest average
8
serum miRNA-125b level (P < 0.05; Figure 1A). miRNA-122, showed a similar
9
positive correlation with HBV DNA level (Figure 1C).
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We also analyzed the relationship between serum miRNA-125b and HBsAg
11
levels, a known biomarker for viral replication. Similarly, serum miRNA-125b
12
levels increased significantly with those of HBsAg (Figure 1B). The serum
13
miRNA-125b level in CHB patients with HBsAg levels < 3 log10 IU/ml were
14
significantly lower than in those with HBsAg ≥ 3–4 log10IU/ml (P < 0.001) or >4
15
log10IU/ml (P < 0.001) (Figure 1B). miRNA-122 levels showed a similar positive
16
correlation with HBsAg (Figure 1D). In contrast, miRNA-124 showed no
17
association with serum HBV DNA and HBsAg levels (Figure 1E and 1F).
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Distribution of Serum miRNA-125b Levels at Different Phases of CHB
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Infection
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CHB patients showed significantly higher miRNA-125b levels than HCs
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ACCEPTED MANUSCRIPT (Figure 2A). Among the subgroups of CHB patients, the highest serum
2
miRNA-125b levels were observed at the IR, followed by IT and ENH phase, and
3
then IC phase (Figure 2B). Nonetheless, patients in all subgroups showed
4
significantly higher serum miRNA-125b levels compared to the HCs (P < 0.001).
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Serum miRNA-125b Levels Correlate with Liver Necroinflammation
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Severity in CHB Patients and Decreased after Antiviral Therapy
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Patients with no or mild liver necroinflammation and fibrosis showed a
9
significantly lower miRNA-125b level than those with moderate-to-severe liver
10
necroinflammation and fibrosis (P<0.001; Figure 2C). Subgroup analysis
11
showed that serum miRNA-125 levels were significantly higher in CHB patients
12
with moderate-to-severe necroinflammation (G2–4; n = 76) than in those with no
13
or mild necroinflammation (G0–1; n = 62) (P < 0.001) (Figure 2D). miRNA-125b
14
levels were significantly correlated with ALT (r =0.379, P<0.001) (Figure S1).
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Multivariate analysis showed that miRNA-125b (odds ratio [OR] = 1.932, P
16
= 0.022), miRNA-124 (OR = 3.502, P = .002), and ALT (OR = 1.026, P < 0.001)
17
are independent predictors for liver necroinflammation (Table S1). However,
18
miRNA-125b showed no correlation with fibrosis (P=0.172). Serum miRNA-125b
19
levels decreased significantly after therapy in the majority of patients (n = 29)
20
compared with those at baseline (P=0.007), which is consistent with the
21
histological improvement observed (Figure S2).
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Differentiating Power of miRNA-125b alone or Combined with miRNA-124
3
for Liver Necroinflammation Grade in CHB patients ALT was an important biomarker of liver necroinflammation and an indicator
5
of prior CHB treatment. In all CHB patients, ALT showed the highest diagnostic
6
value for differentiating significant liver necroinflammation (G ≥ 2; AUC = 0.838)
7
(Figure 3A). In patients with normal ALT levels, the differentiating power of
8
miRNA-125b was comparable to that of miRNA-124 (AUC = 0.757 versus 0.723
9
P > 0.05) (Figure 3B). The sensitivity, specificity, and accuracy for miRNA-125b
10
were 75.0%, 79.1%, and 78.0%, and those for miRNA-124 were 43.8%, 95.4%,
11
and 81.4%, respectively (Table S2). The combination of miRNA-125b and
12
miRNA-124 showed higher diagnostic value (AUC = 0.801, accuracy of 83.1%).
13
In patients with an ALT < 2 upper limit of normal (ULN), miRNA-125b (AUC =
14
0.759) showed higher diagnostic value than miRNA-124 (AUC = 0.743) and ALT
15
(AUC = 0.697) (Figure 3C). The sensitivity, specificity, and accuracy values for
16
miRNA-125b were 77.8%, 73.6%, and 75.1%, respectively. The AUC of the
17
combination of miRNA-125b and miRNA-124 was 0.816, whereas sensitivity,
18
specificity, and accuracy values were 70.4%, 84.9%, and 79.6%, respectively.
19
These data suggest that serum miRNA-125 alone or combined with miRNA-124
20
is superior to ALT in predicting the degree of liver necroinflammation in patients
21
with normal-to-mildly elevated ALT.
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miRNA-125b Enhanced HBV Replication Capacity In Vitro
3
As shown in Figure 4, HBV replication capacity increased with higher doses of
4
miRNA-125b mimic treatment to HepG2.2.15 cells. A similar trend was observed
5
for the supernatant HBsAg and HBeAg level. In contrast, miRNA mimic negative
6
control had no effect on viral replication, HBsAg or HBeAg level. The negative
7
regulating function of miRNA-125b mimic on EZH2 expression was observed as
8
reported previously [22]. miRNA-1 mimic was observed to enhance HBV
9
replication capacity as ever demonstrated [9] (Figure S3).
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DISCUSSION
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ACCEPTED MANUSCRIPT Previous studies have shown that miRNA-125b plays an important role in
2
the pathogenesis of various tumors such as skin cancer, lung cancer, and
3
hepatoma [23]. In the present study, we investigated the association of
4
miRNA-125b, viral replication, and liver necroinflammation in adult CHB patients,
5
as well as the effect of miRNA-125b on HBV replication in vitro.
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Several miRNAs have been found to correlate with HBV replication, liver
7
necroinflammation or fibrosis (Table S3) [5-6, 12, 24-25]. In this study, serum
8
miRNA-125b was correlated with viral load in adult CHB patients, which is
9
consistent with previous studies [4, 16]; the in vitro results supported these
10
clinical findings. Additionally, the miRNA-125b level of patients in the IT phase
11
was lower than that in the IR phase, indicating that miRNA-125b may be
12
influenced by other factors other than HBV replication, such as liver
13
necroinflammation.
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We demonstrated that the serum miRNA-125b level is not only related to
15
liver necroinflammation but also capable of differentiating the severity in CHB
16
patients.
17
necroinflammation and an indicator of antiviral therapy [26]. According to the
18
AASLD guideline for the treatment of CHB, ALT ≥ 2 ULN is used to indicate
19
antiviral treatment [26]. However, ALT alone is inadequate to grade liver
20
necroinflammation. Indeed, liver biopsy of a study in the US revealed that 37%
21
of patients with normal ALT showed significant fibrosis and inflammation [27].
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long
been
considered
15
as
a
biomarker
of
liver
ACCEPTED MANUSCRIPT Another study in China reported that 49.2% of CHB patients with ALT < 2 ULN
2
showed significant inflammation (G > 2) and 36.4% showed significant fibrosis
3
(stage > 2) [28]. Therefore, a part of patients with normal or mildly elevated ALT
4
(1 ULN < ALT < 2 ULN) should receive antiviral therapy. Currently, liver biopsy is
5
the gold standard for evaluating liver necroinflammation and fibrosis [29].
6
However, most patients refuse liver biopsy since it is invasive and painful.
7
Therefore, novel noninvasive biomarkers to evaluate liver necroinflammation
8
with higher sensitivity and diagnostic power are urgently needed. Some
9
noninvasive methods such as FibroScan have been used to investigate the
10
extent of liver fibrosis, but not necroinflammation [30]. Recently, miRNA-124 was
11
identified as an independent predictive factor for liver necroinflammation,
12
especially in patients with normal or mildly elevated ALT levels [15]. However, its
13
sensitivity
14
necroinflammation severity is relatively low. In our study, we found that serum
15
miRNA-125b
16
miRNA-125b was superior to miRNA-124 and ALT in differentiating grades of
17
liver necroinflammation in CHB patients with normal or mildly elevated ALT.
18
Furthermore, the combination of miRNA-125b with miRNA-124 markedly
19
improved the power of differentiating liver necroinflammation, as well as
20
sensitivity and accuracy. Though miRNA-125b and miRNA-124 both correlated
21
with liver necroinflammation, only miRNA-125b also correlated with viral
and
diagnostic
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correlated
with
liver
(63.3%)
to
differentiate
necroinflammation
and
liver
that
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power
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replication. A very recent study demonstrated consistently that miRNA-125b correlates
3
positively with serum HBV DNA and HBsAg level [4]. Our study further
4
demonstrated miRNA-125b enhanced HBV replication capacity in vitro. On the
5
other hand, the study of Akamatsu et al. did not prove an association of serum
6
miRNA-125b level with liver necroinflammatory activity which is inconsistent with
7
our findings [4]. This discrepancy could be explained by the difference in the
8
patient
9
necroinflammatory activity.
especially
the
proportion
of
patients
with
severe
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It was known that persistent HBV infection can lead to cirrhosis and
11
hepatocellular carcinoma (HCC) with high mortality rate [3]. Long term changes
12
in both serum HBV DNA and ALT levels independently predict the risk for
13
hepatocellular carcinoma [31]. It was reported that intrahepatic DNA levels
14
correlates strongly with serum HBV DNA levels and with the degree of fibrosis
15
[32]. Advanced fibrosis and severe necroinflammatory activity, in particular
16
severe lobular activity, are independent predisposing risk factors for the
17
development of HCC in patients with CHB [33]. In our study, we found that
18
miRNA-125b correlates both serum HBV DNA and ALT levels. However, the
19
relationship of miRNA-125b and HCC was not investigated yet. It was reported
20
that the expression of miRNA-125b decreases in HCC tumor tissues and cell
21
lines compared to non-malignant controls and overexpression of miRNA-125b
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ACCEPTED MANUSCRIPT can
significantly
suppress
cell
proliferation,
migration,
invasion,
and
2
angiogenesis in HCC cell lines [34]. The level of miRNA-125b was also identified
3
as a HCC diagnostic discriminator from both noncancerous and normal liver
4
tissues, serving as a HCC survival predictor [34]. Therefore, in the future the
5
correlation between serum miRNA-125b and risk of HCC would be further
6
investigated in patients with HBV-related chronic hepatitis.
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In our study, we also found a correlation between miRNA-122 and
8
miRNA-125b as Akamatsu et al. reported [4]. However, the possible relationship
9
between miRNA-125b and miRNA-122 in CHB patients is obscure. It was
10
reported that miRNA-122 down-regulates its target cyclin G1, and thus interrupts
11
the negatively regulation of cyclin G1 on p53 and then facilitate p53-mediated
12
inhibition of HBV replication [35]. Thus miRNA-122 suppresses HBV replication
13
through cyclin G 1-modulated P53 activity in patients with hepatitis B. However,
14
the mechanism of miRNA-125b regulating HBV replication is still unknown. It
15
was reported that overexpression of miRNA-125b may repress the endogenous
16
level of p53 protein and then suppress apoptosis in human neuroblastoma cells
17
and lung fibroblast cells [36]. Therefore, we hypothesize that miRNA-125b may
18
repress the level of p53 protein and then correspondingly enhance HBV
19
replication. The counteraction of miRNA-122 and miRNA-125b on HBV
20
replication
21
histopathological lesion.
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may
possibly
contribute
18
to
viral
persistence
and
liver
ACCEPTED MANUSCRIPT There are several limitations to this study. First, the mechanisms by which
2
miRNA-125b enhance HBV replication and predict liver necroinflammation
3
remained to be evaluated in the future study. Second, the larger number of
4
patients with normal or mildly elevated ALT should be included for verification in
5
the future, confirming the diagnostic values in clinics. Third, as our study was
6
carried out in Chinese patients who were characterized by Genotype B and C,
7
further studies are required in CHB patients from different geographical areas or
8
with different genotypes to confirm these data.
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In conclusion, we discovered that serum miRNA-125b correlated with viral
10
replication and enhanced HBV replication capacity in vitro. Serum miRNA-125b
11
alone or combined with miRNA-124 may have excellent differentiating power for
12
liver necroinflammation severity in Chinese CHB patients with normal or mildly
13
elevated ALT.
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ACKNOWLEDGEMENTS
16
We thank Dr. Haiyan Lü (Hua shan Hospital, Fudan University) for her help
17
during the study.
18
CONFLICT OF INTEREST
19
The authors have no conflicts of interest to declare.
20
FUNDING
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ACCEPTED MANUSCRIPT This study was funded by the National Natural Science Foundation of China
2
(81471933,81271833), the Major Science and Technology Special Project of
3
China (2012ZX10002007-001-002, 2013ZX10002001), the “973” Project
4
(2012CB519001), and the Special Research Fund of Ministry of Health for
5
Non-Profit Sector (201302010).
6
Abbreviations:
7
ALT, alanine aminotransferase
8
AUC, area under receiver operating characteristic curve
9
CHB, chronic hepatitis B
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ENH, HBeAg-negative CHB
11
EZH2, enhancer of zeste homolog 2
12
G, grading of necroinflammation
13
HBeAg, hepatitis B e antigen
14
HBsAg, hepatitis B surface antigen
15
HBV, hepatitis B virus
16
HCs, healthy controls
17
HCC, hepatocellular carcinoma
18
IC, inactive carrier
19
IT, immune tolerant
20
IR, immune-reactive
21
miRNA, microRNA; qRT-PCR, quantitative reverse transcription polymerase
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ROC, receiver operating characteristic
3
S, staging of fibrosis
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ULN, upper limit of normal
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6
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[33]. Lee SH, Chung YH, Kim JA et al. Histological characteristics
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ACCEPTED MANUSCRIPT Figure legends
1
Figure 1. Correlations of serum miRNA-125b, miRNA-122, and miRNA-124
3
with HBV DNA and HBsAg. (A) Serum miRNA-125b and HBV DNA; (B) serum
4
miRNA-125b and HBsAg; (C) serum miRNA-122 and HBV DNA; (D) serum
5
miRNA-122 and HBsAg; (E) serum miRNA-124 and HBV DNA; (F) serum
6
miRNA-124 and HBsAg;
7
Figure 2. Correlations of serum miRNA-125b with liver necroinflammation
8
and fibrosis. (A) Serum miRNA-125b levels in patients with HBV infection and
9
healthy controls. (B) Distribution of serum miRNA-125b levels in patients at
10
different phases of the course of CHB infection. (C, D) Patients were divided into
11
groups by grading of necroinflammation or staging of fibrosis or both.
12
Abbreviations: miRNA, microRNA; HBV, hepatitis B virus; HC, healthy control; IT,
13
immune-tolerant; IR, immune-reactive; IC, inactive carrier; ENH, hepatitis B e
14
antigen negative chronic hepatitis B; G, grading of necroinflammation; S, staging
15
of fibrosis; n, number.
16
Figure 3. Differentiating power of serum ALT, miRNA-125b, miRNA-124,
17
and combined miRNA-125b and miRNA-124 for liver necroinflammation
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severity in CHB patients.
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miRNA-125b, miRNA-124, and the combination of miRNA-125b and miRNA-124
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for differentiating the severity of liver necroinflammation in patients with CHB (n
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= 138) from HCs (n = 35). (B) Moderate-to-severe necroinflammation (≥G2) in
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(A) ROC curves and AUC are presented for ALT,
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U/L (n = 80).
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Figure 4. miRNA-125b enhanced HBV replication capacity in vitro.
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(A) HepG2.2.15 cells were transfected with step-increased levels of
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miRNA-125b mimic. Cells were harvested and intracellular, encapsulated HBV
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DNA was extracted and analyzed by Southern blot analysis. (B) Supernatant
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HBsAg and HBeAg levels of HepG2.2.15 cells were determined by specific
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ELISAs after transfection with indicated doses of miRNA-125b mimic. (C)
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Functional effectiveness experiment of miRNA-125b on gene enhancer of zeste
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homolog 2. Abbreviations: miRNA, microRNA; HBV, hepatitis B virus; HBeAg,
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hepatitis B e antigen; HBsAg, hepatitis B surface antigen; EZH2, enhancer of
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zeste homolog 2;
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before and after antiviral therapy (n = 29). (A) Changes in the proportion of necroinflammation (G0–4) and fibrosis (S0–4). (B) Serum miRNA-125 levels of
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CHB patients before and after antiviral therapy.
Figure S3. HepG2.2.15 cells were transfected with miRNA control (10nM),
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miRNA-1 (5 nM) and miRNA-125b (5 nM). Cells were harvested and the levels of HBV replicative intermediates were determined by southern blot as previous
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Table 1. Baseline characteristics of enrolled individuals CHB patients (n = 211)
HCs (n = 35)
P value
Characteristic
IT (n = 37)
IR (n = 109)
IC (n = 29)
ENH (n = 36)
Gender (male, %)
23 (62.2 %)
77 (70.6 %)
20 (69.0 %)
28 (77.8 %)
27 (10)
32 (17)
36 (14)
42 (21)
34 (14)
.49
60.5 (86.3)
21.0 (17.0)
< .01
4.00 (2.16)
N/A
3.31 (0.83)
N/A
ALT (U/L)
29.5 (11.5)
HBV DNA (log10 IU/ml)
7.37 (0.94)
7.00 (2.54)
2.10 (1.00)
HBsAg (log10 IU/ml)
4.72 (0.52)
3.97 (0.91)
3.09 (1.58)
20/17
56/53
13/16
HBV Genotype (n, B/C)
124.0 (154.6)
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(18.5)
IT (n = 11)
IR (n = 74)
IC (n = 20)
9 (81.8 %)
53 (71.6 %)
17 (85.0 %)
Age (years)
30 (8)
33 (17)
37 (20)
ALT (U/L)
36 (18)
120 (128)
HBV DNA (log10IU/ml)
7.89 (1.01)
HBsAg (log10 IU/ml) HBV Genotype (n, B/C)
27 (81.8 %)
.44 .83
28 (20)
59 (87)
< .01
7.01 (2.13)
2.67 (1.86)
4.00 (2.50)
4.55 (1.15)
4.00 (0.89)
3.01 (0.58)
3.25 (0.92)
5/6
34/40
9/11
15/18
2/9/0/0/0
6/9/27/29/3
11/9/0/0/0
2/14/10/7/0
16/4/0/0/0
2/7/11/5/8
Necroinflammation
9/2/0/0/0
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P value
42 (21)
Grading of
G0/G1/G2/G3/G4 (n)
ENH (n = 33)
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.37
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CHB patients with liver biopsy (n = 138)
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Age (years)
29 (82.8%)
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For age, ALT, HBsAg, and HBV DNA titers, data are presented as median (interquartile range). P-values < .05 are considered as significant. N/A, not available. Abbreviations: ALT, alanine aminotransferase ; CHB, chronic hepatitis B; HBV, hepatitis B virus; HBsAg,
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hepatitis B surface antigen; IT, immune-tolerant; IR, immune-reactive; IC, inactive carrier; ENH, HBeAg
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negative hepatitis. G, grading of necroinflammation; S, staging of fibrosis.
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