Identification of circulating microRNAs
as potential biomarkers for hepatic
necroinflammation in patients with
autoimmune hepatitis
Chisato Saeki ‍ ‍,1,2 Juntaro Matsuzaki,3,4 Masahiko Kuroda,5 Koji Fujita,5Makiko Ichikawa,6 Satoko Takizawa,6 Keiko Takano,1 Tsunekazu Oikawa,1
Yasuni Nakanuma,7 Masayuki Saruta,1 Takahiro Ochiya,3,8 Akihito Tsubota9

To cite: Saeki C, Matsuzaki J,
Kuroda M, et al. Identificationof circulating microRNAs
as potential biomarkers forhepatic necroinflammationin patients with autoimmune
hepatitis. BMJ Open Gastro
2022;9:e000879. doi:10.1136/
bmjgast-2022-000879► Additional supplemental
material is published online
only. To view, please visit thejournal online (http://​dx.​doi.​
org/​10.​1136/​bmjgast-​2022-​
000879).

CS, JM and MK contributed
equally.
Received 17 January 2022
Accepted 16 March 2022

© Author(s) (or theiremployer(s)) 2022. Re-­use
permitted under CC BY-­NC. No
commercial re-­use. See rights
and permissions. Publishedby BMJ.
For numbered affiliations see
end of article.
Correspondence toChisato Saeki;​chisato@​jikei.​ac.​jp
Akihito Tsubota;​atsubo@​jikei.a​ c.​jp

ABSTRACT
Objective MicroRNAs (miRNAs) are implicated in thepathogenesis of autoimmune diseases and could bebiomarkers of disease activity. This study aimed to
identify highly expressed circulating miRNAs in patientswith autoimmune hepatitis (AIH) and to evaluate theirassociation with clinical characteristics.
Methods Microarray analyses were performed, andmiRNA expression profiling for AIH, primary biliary
cholangitis (PBC) and overlap syndrome (OS) using theserum of patients and healthy individuals was done.
Samples were divided into discovery and test sets
to identify candidate miRNAs that could discriminate
AIH from PBC; the former included 21 AIH and 23 PBC
samples, while the latter included five AIH and eight PBC
samples.
Results Among 11 candidate miRNAs extracted in the
discovery set, 4 (miR-­3196, miR-­6125, miR-­4725–3 p
and miR-­4634) were specifically and highly expressedin patients with AIH in the test set. These four miRNAs
discriminated AIH from PBC with high sensitivity (0.80–1.00) and specificity (0.88–1.00). In situ hybridisation
analysis revealed that these miRNAs were expressed in
the cytoplasm of hepatocytes in patients with AIH. Their
expression levels were highest in untreated patients with
AIH, followed by those in untreated patients with OS. They
drastically or moderately decreased after prednisolone
treatment. Histological analysis demonstrated that theexpression levels of miR-­3196, miR-­6125 and miR-­4634
in patients with AIH and OS were correlated with severe
hepatic necroinflammatory activity.
Conclusion These circulating miRNAs are suggested toreflect hepatic necroinflammatory activity and serve as
AIH-­related and treatment-­responsive biomarkers. ThesemiRNAs could be beneficial in developing new therapeuticstrategies for AIH.

INTRODUCTION
Autoimmune hepatitis (AIH) is a progressive, chronic, autoimmune liver disease characterised by elevated serum transaminase,autoantibodies, hyperglobulinaemia andinterface hepatitis on histology.1–3 Although

Saeki C, et al. BMJ Open Gastro 2022;9:e000879. doi:10.1136/bmjgast-2022-000879

Summary box
What is already known about this subject?
► MicroRNAs (miRNAs) are involved in the pathogene-sis of autoimmune diseases and circulating miRNAs
could serve as biomarkers for disease activity.
► Approximately half of patients with autoimmune
hepatitis (AIH) have persistent histological activity
on liver biopsy specimens, even after biochemical
remission. Therefore, non-­invasive biomarkers aredesirable to determine disease activity for formulating therapeutic strategies.

What are the new findings?
► Comprehensive miRNA microarray analysis identi-

fied four circulating miRNAs (miR-­3196, miR-­6125,
miR-­4725–3 p and miR-­4634) that were specificallyand highly expressed in patients with AIH.
► These four miRNAs were strongly or moderately expressed in the cytoplasm of hepatocytes in patientswith AIH.
► The expression levels of these four miRNAs drastically decreased after prednisolone treatment.
► The expression levels of miR-­3196, miR-­6125 andmiR-­
4634 were correlated with severe hepatic
necroinflammatory activity on histology in patientswith AIH.

How might it impact on clinical practice in theforeseeable future?
► The miRNAs identified in this study could serve asnon-­invasive biomarkers associated with hepatic
necroinflammatory activity in patients with AIH.

environmental, genetic and epigenetic factors
and T cell-­mediated immune responses
are considered the pathogenesis of AIH,
the molecular mechanisms underlying thedisease have not been clearly elucidated.4 5Therefore, diagnostic biomarkers and radical
treatments specific for AIH have not yet beendeveloped.3 5
1

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Hepatology

Repeat liver biopsies are useful in assessing diseaseactivity and treatment response; however, these areinvasive, time-­consuming and burdensome to patients.
Therefore, in real-­world clinical settings, clinical remission is assessed based on biochemical findings, such as
the normalization of transaminase and IgG levels.6 Of
note, approximately half of AIH patients with serum
parameter normalization after immunosuppressive treatment have persistent histological activity on liver biopsy
specimens, which is associated with fibrosis progression
and poor prognosis.7 8 Therefore, to develop a novel
treatment and improve the liver-­related mortality inpatients with AIH, it is important to better understandthe molecular mechanisms underlying the disease andto identify molecules that sensitively reflect histologicaldisease activity.8 9
Primary biliary cholangitis (PBC) is another autoimmune liver disease characterized by the destruction ofsmall intrahepatic bile ducts, progressive cholestasis, andthe presence of highly specific serum antimitochondrial
antibodies (AMAs).10–12 Some patients with PBC manifest the features of AIH and are commonly classified ashaving so-­called ‘overlap syndrome (OS)’13–15; OS hasa frequency of 7%–13% in patients with autoimmune
liver disease.14 Of note, patients with OS are more likely
to have oesophageal varices, gastrointestinal bleeding,
ascites, hepatic failure and/or liver transplantationcompared with those with typical PBC. Therefore, thestandard treatments for both PBC (such as ursodeoxycholic acid (UDCA)) and AIH (such as corticosteroids)
are required to achieve a complete treatment response inmost patients with OS.15 16
MicroRNAs (miRNAs) constitute a class of small
noncoding RNAs that directly bind to the 3’ untranslated
regions of specific target genes, thereby inducing post-­
transcriptional inhibition.17–25 miRNAs play a crucial rolein various biological processes, including tissue development and differentiation, immunity, cell proliferation,
apoptosis and oncogenesis.17 18 Their dysregulation isassociated with liver diseases, such as viral hepatitis, alcoholic liver disease, non-­alcoholic fatty liver disease andhepatocellular carcinoma.20 21 Therefore, elucidating
the mechanisms underlying miRNA dysregulation could
provide new therapeutic strategies for liver diseases.23
Circulating RNAs, which are protected from RNases
and remain stable in blood, are attractive, non-­invasive
biomarkers for disease diagnosis and disease stage/
activity determination.20–25 However, only few studies
have evaluated the expression profiles of circulating
miRNAs in patients with AIH.
The aim of this study was to identify miRNAs that werehighly expressed in patients with AIH by comparing thecirculating miRNA profiles of untreated patients with
AIH, PBC and OS and healthy controls. Furthermore,changes in the expression levels of identified miRNAs
prior to and after treatment were assessed, and their association with treatment response was evaluated.
2

METHODS
Participants and samples
In this study, 57 treatment-­naïve patients with AIH(n=28) or PBC (n=29) and 7 patients with OS at the Jikei
University School of Medicine (Tokyo, Japan) and Fuji
City General Hospital (Shizuoka, Japan) were enrolled.
Twenty-­seven healthy volunteers who underwent routine
medical check-­up at the Jikei University School of Medicine were recruited as controls. They all had normal
alanine aminotransferase (ALT;≤35 U/L) and gamma-­
glutamyltransferase (≤50 U/L) levels with no history ofsignificant diseases. Patients with other liver diseases,
such as alcoholic liver disease, drug-­induced liver disease,viral hepatitis and non-­alcoholic steatohepatitis, were
excluded.
Serum samples were collected from patients at thetime of diagnosis or exacerbation of the disease and fromhealthy volunteers at the time of consent. In addition,paired samples were obtained from 21 of the 28 patientswith AIH at least 3 months after treatment, from 23 of
the 29 patients with PBC at least 12 months after treatment, and from all of the seven patients with OS at least
6 months after additional treatment. All serum samples
were stored at −80 ℃ until analysis. Written informed
consent was obtained from each patient. In cases where
serum samples had already been stored in anotherapproved study, participants were notified of the opportunity to opt out of the study through the website of each
institution.
Diagnosis and treatment
AIH was diagnosed based on the revised International
Autoimmune Hepatitis Group criteria.26 The patientsin this study were diagnosed as having probable or definite AIH and were treated with prednisolone alone(0.6–1.0 mg/kg/day). We defined remission of AIH
as the normalization of ALT and IgG (≤1700 mg/dL)levels.6 Diagnosis of PBC was made if patients met any
one of the following criteria: (1) biochemical evidenceof cholestasis and histological evidence of chronic non-­suppurative destructive cholangitis; (2) the presence of
AMA with histologically compatible PBC features and(3) the presence of AMA with PBC clinical features andcourse characteristics, irrespective of histological findings.12 All patients with PBC were treated with UDCA
(13–15 mg/kg/day) or UDCA plus bezafibrate (400 mg/
day). In this study, biochemical response to these treatments was defined according to the Barcelona criteria,27
that is, an alkaline phosphate (ALP) decrease of >40%of baseline values or normal ALP levels (≤325 U/L) after
12 months of treatment. OS was diagnosed according
to the Paris criteria.15 Six patients with OS were treatedwith a combination of prednisolone and UDCA, and theremaining one was treated with UDCA alone.
Histological assessments
All patients underwent liver biopsy for diagnostic
purposes. All liver specimens were histopathologically
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Open access

evaluated by a single liver-­specialised pathologist (YN).
The severity of inflammation and fibrosis in AIH was
graded according to the Ishak score.28 In brief, thisgrading system comprises four necroinflammatory categories: (1) periportal or periseptal interface hepatitis,(2) confluent necrosis, (3) focal lytic necrosis, apoptosis
and focal inflammation and (4) portal inflammation;
histological grading scores, which ranged from 0 to 18,
were calculated by summing the scores for each category,
and were graded in this study as follows; total score 0–3,
A0; score 4–8, A1; score 9–12, A2 and score 13–18, A3.
Fibrosis stages ranged from 0 to 6, with stage 6 defined
as liver cirrhosis.28 The histological staging and gradingof PBC were assessed based on the Nakanuma’s classification.29 30 In brief, scores for fibrosis, bile duct loss andchronic cholestasis were summed for staging as follows:
total score 0, stage 1 (no progression); score 1–3, stage 2(mild progression); score 4–6, stage 3 (moderate progression); and score 7–9, stage 4 (advanced progression).
Cholangitis activity (CA) and hepatitis activity (HA) were
graded as CA0–3 and HA0–3, respectively. The histological staging and grading of OS were assessed based on
both the Ishak score and Nakanuma’s classification.

Analysis of miRNA expression by microarray
Our standard operating procedures for miRNA
microarray analysis have been previously published.31–34
In Brief, total RNA was extracted from 300 µL serum
samples obtained from patients with AIH, PBC andOS, and from healthy individuals using a 3D-­Gene
RNA extraction reagent (Toray Industries, Tokyo,
Japan). Comprehensive miRNA expression analysis was
performed using the 3D-­Gene miRNA labelling kit andthe 3D-­Gene Human miRNA Oligo Chip (Toray Industries), designed to detect 2588 miRNAs registered in
the miRBase release 21 database (http://www.mirbase.​org/). To normalise microarray signals, three preselected internal control miRNAs (miR-­149–3 p, miR-­2861,
and miR-­4463) were used. All microarray data from thisstudy are publicly available in the GEO database (accession #GSE- 140249) (https://www.ncbi.nlm.nih.gov/​geo/).

Statistical analysis
Serum samples from patients with AIH and PBC weredivided into the discovery and test cohorts. The discovery
cohort was used to select candidate miRNAs that could
discriminate between AIH and PBC, and the test cohort
Detection of miRNAs in liver tissues by in situ hybridisation
was used to confirm the results obtained from theTo evaluate the expression of miRNAs in the liver, in situ
discovery cohort. In this study, miRNAs with medianhybridisation was performed using a RiboMap in situ
signal values ≥26 were defined as abundant miRNAs
hybridisation kit (Ventana Medical Systems, Tucson, AZ)
in serum and were included in subsequent analyses.
on a Ventana Discovery automated in situ hybridisation
To identify robust biomarkers that could discriminate
instrument (Ventana Medical Systems). Digoxigenin-­between AIH and PBC, a cross-­validation score was calculabelled, LNA-­
modified DNA probes against miR-­lated for each miRNA in the discovery set. This score was
3196, miR-­6125, miR-­4725–3 p, miR-­4634 and scramble
based on leave-­one-­out cross-­validation and obtained bysiRNA were obtained from Exiqon (Exiqon, Vedbaek,
summing the results (hit or not) of each leave-­out sample
Denmark). In situ hybridisation steps after the deparafand dividing it by the number of repeats (the same asfinisation step were performed based on the standard
total sample number), as previously described.34 miRNAs
protocol provided in the manufacturer’s RiboMap appliwith cross-­validation scores >0.8 were selected for further
cation note (http://www.ventanamed.com). The initial
analyses. The diagnostic sensitivity, specificity and area
fixation step was performed by incubating the sections
under the curve (AUC) were calculated for the selected
in a formalin-­based RiboPrep reagent (Ventana Medical
miRNAs. In the test set, we determined the performances
Systems) for 32 min at 37°C. After acid treatment usingof the selected miRNAs in discriminating between AIH
a hydrochloride-­
based RiboClear reagent (Ventana
and PBC, and miRNAs with AUC >0.8 were considered to
Medical Systems) for 12 min at 37°C, the slides werebe AIH-­specific.
treated with a ready-­to-­use protease 2 reagent after an
Statistical analyses were performed using R V.3.1.2 (Rinitial cell conditioning CC2 reagent (Ventana Medical
Foundationfor Statistical Computing, http://www.R-​
Systems) for 8 min at 90°C. The initial denaturing prehyproject.org),​compute.​es package V.0.2–4, hash package
bridisation step was done on the sections for 8 min at
V.2.2.6,
MASS
package V.7.3–45, mutoss package V.0.1–
91°C after being hybridised with an anti-­sense LNA ribo10,
pROC
packageV.1.8 and IBM SPSS Statistics V.22
probe (5 pmol/slide) using a RiboHybe hybridisation(IBM
Japan,
Tokyo,
Japan). Principal component analbuffer (Ventana Medical Systems) for 10 hours at 55°C.
ysis
(PCA)
was
performed
using the Partek Genomics
After three stringency washes with 2×RiboWash (Ventana
Suiteversion
6.6.
Continuous
variables were presented
Medical Systems) for 4 min at 51°C, postprobe fixationasmean±SD.
Unpairedandpairedt-­tests were used to
was performed using a RiboFix reagent for 20 min atevaluatethedifferencesinthedistributionof continuous
37°C, followed by incubation of antidigoxigenin alkaline
variables
betweenthetwogroups.
The
Fisher’s
exact
phosphatase Fab fragments 1:800 (Roche) for 30 min at
test
was
used
to
evaluatethesignificanceofdifferences
37°C. A signal was detected using a BlueMap NBT/BCIPin the distribution of categorical variables between thesubstrate kit for 4 hours at 37°C. Finally, the sections were
two groups. Correlations between variables were analysedcounterstained with Kernechtrot (nuclear fast red) as ausing the Pearson’s correlation test.
marker stain and covered with a glass coverslip.
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3

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Open accessFigure 1 The strategy used to identify highly expressed microRNAs (miRNAs) in autoimmune hepatitis (AIH). (A) Serumsamples were obtained from 28 untreated patients with AIH and 29 untreated patients with primary biliary cholangitis (PBC).
these samples were divided into two sets: the discovery set and the test set. paired serum samples were obtained from 21
treated patients with AIH, 23 treated patients with PBC, 7 patients with overlap syndrome, and 27 healthy individuals (controls).
(B) Flow diagram for selecting candidate miRNAs. Of the total 2588 miRNAs, 2259 were excluded due to their low expression
levels. Eleven candidate miRNAs with cross-­validation scores >0.8 were selected in the discovery set; of these, four miRNAs,
with area under the curve (AUC)>0.8, were selected in the test set. (C) Principal component analysis (PCA) for the 23 untreated
AIH and 21 untreated PBC samples in discovery set using 329 highly expressed miRNAs. The axes show the first threeprincipal components, with their respective fractions of explained variance. The first three principal components explain 53.7%
of variance. miRNA profiles of AIH and pBC were well segregated.

RESULTS
Characteristics of the study participants
A total of 142 samples consisting of 49 samples from
28 patients with AIH (including 21 paired samples), 52
samples from 29 patients with PBC (including 23 paired
samples), 14 samples from 7 patients with OS (including
7 paired samples), and 27 samples from 27 healthy
controls (online supplemental table S1) were subjected
to miRNA microarray analysis. First, untreated AIH andPBC samples were classified into the discovery and test
cohorts to identify circulating miRNAs that could discriminate AIH from PBC (figure 1A, online supplemental
table S2). The discovery set included 23 untreated AIH
and 21 untreated PBC samples. The test set included
five untreated AIH and eight untreated PBC samples.
Regarding treatment response, all patients with AIH(n=21) met the remission criteria, and all patients withPBC (n=23) were biochemical responders. Among theseven patients with OS, four had already been receiving
4

treatment for either PBC or OS and developed acuteexacerbation of liver disease when the first samples werecollected. For the management of disease exacerbation,
prednisolone was added to the preceding UDCA monotherapy in three patients, and its dose was increased in
one patient who was on UDCA and prednisolone combination therapy. The remaining three patients were newly
diagnosed with OS and were treatment-­naïve. All patientswith OS had normalised ALT and ALP levels after thetreatment, and only one had elevated IgG levels despite
the treatment.
Identification of highly expressed miRNAs in AIH
The expression levels of 2588 miRNAs were comprehensively evaluated in the miRNA microarray analysis.
First, 2259 miRNAs with a median signal value <26 were
excluded. The remaining 329 miRNAs were retainedfor further analyses (figure 1B). PCA mapping was
performed to assess the miRNA expression patterns in
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Table 1 Selected serum miRNAs for discrimination between AIH and PBC
Discovery set

Test set

CV score

AUC

95% CI

AUC

95% CI

Elevated in AIH miR-­3665

0.91

0.94

0.88 to 1.00

0.38

0.02 to 0.73

 miR-­8072

0.86

0.95

0.89 to 1.00

0.53

0.18 to 0.87

 miR-­3196

0.84

0.95

0.89 to 1.00

0.90

0.70 to 1.00

 miR-­6125

0.84

0.91

0.82 to 1.00

0.90

0.73 to 1.00

 miR-­6836–3 p

0.82

0.90

0.80 to 0.99

0.38

0.03 to 0.73

 miR-­1908–5 p

0.82

0.86

0.73 to 0.98

0.70

0.35 to 1.00

 miR-­6729–5 p

0.82

0.90

0.81 to 0.99

0.80

0.52 to 1.00

 miR-­4725–3 p

0.82

0.91

0.83 to 1.00

0.88

0.65 to 1.00

 miR-­6789–5 p

0.82

0.92

0.84 to 1.00

0.78

0.51 to 1.00

 miR-­4634

0.82

0.91

0.82 to 1.00

0.98

0.90 to 1.00

Elevated in PBC miR-­4655–5 p

0.82

0.82

0.68 to 0.95

0.80

0.45 to 1.00

Bold indicates the AUC values of miRNAs extracted in the test set.
AIH, autoimmune hepatitis; AUC, area under the curve; CV, cross-­validation; PBC, primary biliary cholangitis.; .

the discovery set (figure 1C), indicating that there weredistinct differences in the miRNA expression profile
between AIH and PBC.
Subsequently, miRNAs that could discriminate AIH
from PBC were investigated based on leave-­
one-­
out
cross-­validation, and 11 miRNAs (10 elevated in AIH
and one elevated in PBC) with cross-­validation scores>0.8 in the discovery set were selected (figure 1B,
table 1). Finally, the diagnostic performance of these
11 miRNAs was assessed in the test set, and 4 miRNAs(miR-­3196, miR-­6125, miR-­4725–3 p and miR-­4634)
were highly accurate in discriminating AIH from PBC,
with AUCs >0.8 (figure 1B, table 1). The AUC, sensitivity and specificity values for the four miRNAs were
determined in the test set: miR-­3196 had 0.90, 0.80 and
1.00, respectively; miR-­6125 had 0.90, 0.80 and 0.88,
respectively; miR-­
4725–3 p had 0.88, 1.00 and 0.88,
respectively; and miR-­
4624 had 0.98, 1.00 and 0.88,
respectively (figure 2A).
Changes in circulating miRNA expression levels after
treatment
Changes in the expression levels of the four miRNAs
prior to and after treatment in patients with AIH, PBC
and OS and healthy controls were evaluated using
microarray data (figure 2B). The median expression
levels of the four miRNAs were highest in pretreatment
AIH samples, followed by those in OS samples collected at
disease diagnosis or exacerbation. More importantly, theexpression levels of these miRNAs (except miR-­4725–3 p
in OS patients) significantly decreased in posttreatment
AIH and OS samples. These findings suggest that thesemiRNAs reflect the disease activity of AIH and that OS
may dominantly involve AIH features.
Saeki C, et al. BMJ Open Gastro 2022;9:e000879. doi:10.1136/bmjgast-2022-000879

Correlation between circulating miRNA expression and liverfunction parameter levels
The correlation between the expression levels of thefour miRNAs and liver function parameters was investigated (figure 2C). Their expression levels had significant
positive correlations with the levels of aspartate aminotransferase (p<0.001 for all; r=0.59, 0.52, 0.48, and 0.60
for miR-­
3196, miR-­
6125, miR-­
4725–3 p and miR-­
4634,
respectively) and ALT (p<0.001 for all; r=0.56, 0.51, 0.54,
and 0.61 for miR-­3196, miR-­6125, miR-­4725–3 p and miR-­
4634, respectively). The expression levels of miR-­4634
and miR-­3196 had significant negative correlations with
albumin levels (p<0.001 and r = −0.43 for both).
Localisation of the identified miRNAs in liver tissues
In situ hybridisation was performed to determine
the localisation of the four miRNAs in liver tissues.
Diffused expressions of the four miRNAs were observedin the cytoplasm of hepatocytes in patients with AIH(figure 3B). miR-­3196 and miR-­6125 were very strongly
stained, while miR-­4725–3 p and miR-­4634 were moderately stained. In patients with PBC and OS, these miRNAs
were also expressed in the cytoplasm of hepatocytes,
but their expression levels were considerably lower thanthose in patients with AIH (figure 3A,C). miR-­3196 wasstrongly stained, miR-­6125 was moderately stained, while
miR-­4725–3 p and miR-­4634 were very weakly stained.
In contrast, the expression of these miRNAs was almost
undetectable in control liver tissues (figure 3D).
Relationship between circulating miRNA expression levels
and histological findings
The correlation between the expression levels of thefour miRNAs and hepatic necroinflammation/fibrosis
5

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Open accessFigure 2 (A) Receiver operating characteristic (ROC) curve analysis of miR-­3196, miR-­6125, miR-­4725–3 p and miR-­4634 in
the discovery and test sets. The area under the curve (AUC), sensitivity and specificity values are shown for each curve. Sky-­
blue areas indicate 95% CIs for ROC curves. (B) The expression of the four microRNAs (miRNAs) in patients with autoimmune
hepatitis (AIH), primary biliary cholangitis (PBC) and overlap syndrome prior to and after treatment, and in healthy individuals.
Box-­and-­whisker plots show the full range of variation, IQR and median values. Outliers are points that fell more than 1.5
times out of the IQR above the third quartile or below the first quartile. P values versus healthy individuals were calculated byunpaired t-­test. P values between prior to and after treatment were calculated by paired t-­test. (C) Correlation between theexpression levels of the four identified miRNAs and liver function parameters in patients prior to treatment (n=64). Numbers
indicate Pearson’s correlation coefficients. Yellow boxes represent statistical significance (p<0.000455), considering the
Bonferroni correction for multiple testing. ALB, albumin, ALP, alkaline phosphate; ALT, alanine aminotransferase; AST, aspartate
aminotransferase; γ-GTP, gamma-­glutamyltransferase; PLT, platelet; TB, total bilirubin.

in patients with AIH and OS was assessed (figure 4A).
The median expression levels of miR-­
3196, miR-­
6125
and miR-­4634 in the necroinflammatory activity grade
A3 group were distinctly higher than those in the grade
6

A0–A2 groups. In contrast, no correlation was foundbetween miRNA expression levels and fibrosis stages.
It was also evaluated whether the expression levelsof the four miRNAs were correlated with histological
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Figure 3 In situ hybridisation detection of miR-­3196, miR-­6125, miR-­4725–3 p and miR-­4634 in liver tissues from patientswith autoimmune liver disease and healthy subjects. The blue-­violet colour represents positive staining. miRNAs expression inpatients with (A)primary biliary cholangitis (PBC), (B)autoimmune hepatitis (AIH), (C)overlap syndrome and (D)in healthy control(magnification, ×400).

severity scores in patients with PBC and OS (figure 4B).
The median expression levels of miR-­3196, miR-­6125, andmiR-­4634 significantly increased in a stepwise mannerwith an increase in hepatitis activity scores. In contrast,
the median expression levels of miR-­6125 significantly
decreased in a stepwise manner with an increase in cholangitis activity. Taken together, these findings suggestthat the expression levels of miR-­3196, miR-­6125, andmiR-­4634 were positively associated with hepatocellularinflammatory activity.
DISCUSSION
miRNAs regulate the activities of immune cells, such as B,
T and dendritic cells. Dysregulated miRNAs are involvedin the pathogenesis of autoimmune diseases.35 Circulating miRNAs could serve as biomarkers for the diagnosis and staging/grading of various diseases, including
autoimmune disease.31–36 However, an extensive analysis of circulating miRNAs in AIH has not sufficiently
proceeded because AIH is relatively rare, so samples
are difficult to obtain. In this study, the miRNA expression profiles between untreated patients with AIH and
PBC were compared using a highly sensitive miRNA
microarray technology; the four circulating miRNAs(miR-­3196, miR-­6125, miR-­4725–3 p and miR-­4634) wereidentified as highly expressed in untreated patients with
AIH, and these could discriminate AIH from PBC withhigh sensitivity and specificity. Of note, their circulatinglevels in patients with AIH drastically decreased after
Saeki C, et al. BMJ Open Gastro 2022;9:e000879. doi:10.1136/bmjgast-2022-000879

prednisolone treatment. Furthermore, in situ hybridisation analysis revealed that the four miRNAs were verystrongly or moderately expressed in the cytoplasm ofhepatocytes in untreated patients with AIH. Given that
it is generally challenging to determine cells that releasecirculating miRNAs, this new finding that the fourmiRNAs are released by hepatocytes will be meaningfulwhen they are identified as other disease biomarkers.
These results suggest that the four miRNAs are closely
associated with the pathogenesis of AIH and may bepotential biomarkers for disease activity.
A previous study reported that 11 circulating miRNAs
were up-­regulated in untreated patients with AIH
compared with healthy controls. Among the 11 miRNAs,miR-­
21–5 p and miR-­
122–5 p levels decreased in theremission phase after treatment.22 However, anotherstudy reported that these two miRNAs were also upregulated in patients with PBC, suggesting that both might not
be specific for AIH.23 In this study, we failed to find theupregulated expression of the two miRNAs. Conversely,
none of the 10 miRNAs up-­regulated in our discovery
set were increased in the previous study.22 There may
be several possible explanations for these differences.
First, the previous study only included a small numberof patients with acute-­onset AIH alone (n=5), and AIH-­
specific miRNAs were identified by comparing patientswith AIH to healthy controls (not including patients with
PBC). Second, our study included 28 patients with AIH(23 discovery and five test) with various disease stages and7

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Open accessFigure 4 (A) Correlation between the expression levels of the four microRNAs (miRNAs) and histological findings in patientswith autoimmune hepatitis (AIH) and overlap syndrome (OS). The scores of A0–A3 represent the necroinflammatory activity
grades, and the scores of F1–F5 represent the fibrosis stages. (B) Correlation between the expression levels of the four miRNAs
and histological findings in patients with primary biliary cholangitis (PBC) and OS. P values for trend were calculated using thePearson’s correlation test. CA, cholangitis activity; HA, hepatitis activity.

activities and compared them to patients with PBC andOS, as well as healthy controls. Third, despite the same
microarray system, we normalized the miRNA signals
using three unique internal controls (miR-­149–3 p, miR-­
2861 and miR-­4463), which are stably expressed in any
samples. Lastly, we excluded miRNAs with signal values<26 because candidate miRNAs should have a certain
expression level as a disease-­specific biomarker.
8

We also focused on the expression patterns of the fourmiRNAs in patients with OS with both AIH and PBC
features. Their median levels were highest in untreatedpatients with AIH, followed by those in untreated patientswith OS. Additionally, their expression levels drasticallydecreased after prednisolone treatment in patients with
AIH and moderately declined in patients with OS after
the initial or additional treatment or increased dose
Saeki C, et al. BMJ Open Gastro 2022;9:e000879. doi:10.1136/bmjgast-2022-000879

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of prednisolone. In contrast, their expression levels in
untreated patients with PBC did not significantly change
despite the treatment. These findings suggest that theexpression levels of the four miRNAs are indicative of
AIH-­related and treatment-­responsive disease activity.
This study demonstrated that the median levels of thefour miRNAs were highest in untreated patients with
AIH, especially in those with necroinflammatory activity
grade A3, and these increased in a stepwise manner withan increase in hepatitis activity in untreated patientswith PBC and OS. Their expression levels significantly
decreased after prednisolone treatment; however, thereduced levels were still higher compared with those inhealthy controls, suggesting that hepatic necroinflammation may still continue even in the remission phaseof AIH. Previous studies reported that approximately
half of AIH patients with biochemical remission after
immunosuppressive treatment showed persistent histological activity on second liver biopsy specimens, which
was associated with fibrosis progression and poor prognosis.7 8 Therefore, they suggested that follow-­up liverbiopsy should be considered even in AIH patients withclinical remission.8 However, liver biopsy is invasive and aburden to patients, making it difficult to obtain a consent
for repeat liver biopsy, especially in patients with clinicalremission. The circulating miRNAs may serve as non-­invasive surrogate biomarkers to estimate the persistenceor disappearance of histological activity and to formulate
therapeutic strategies, such as dose reduction or withdrawal of immunosuppressive agents.
This study had several limitations. First, little is
known about the biological and physiological properties of the four miRNAs identified in this study. Most
studies on these miRNAs have focused on their role andbehaviour exclusively in malignant tumours,37–39 andno studies have investigated their involvement in thepathogenesis and clinical significance in autoimmune
liver diseases Accordingly, the four miRNAs may represent novel biomarkers of hepatic inflammatory activity
in AIH. Second, this study included a small number ofpatients (especially in the test set) and no patients withother liver diseases, such as viral hepatitis, alcoholic
liver disease and non-­alcoholic fatty liver disease. Therefore, it is unclear whether the four miRNAs reflect AIH-­
specific or non-­
specific inflammatory activity. Third,
although the in situ hybridisation revealed that thefour miRNAs were highly expressed in the cytoplasm ofhepatocytes in patients with AIH, it is unclear whether
the upregulation of these miRNAs is involved in thedevelopment of AIH or is a result of hepatic necroinflammation. A large-­scale validation study that includes
other liver diseases is required to determine whether
the four miRNAs are specific and useful biomarkers toestimate the disease activity of AIH. In addition, functional analyses, including the identification of target
genes and mechanisms underlying autoimmune liverdisease, are required to clarify the biological and physiological properties of these miRNAs.

In conclusion, we identified the four circulating
miRNAs that could serve as non-­invasive biomarkers associated with hepatic necroinflammatory activity in patientswith AIH, using a comprehensive and highly sensitive
miRNA microarray technology. These findings may be
helpful in elucidating the mechanisms involved in thepathogenesis of AIH and in developing therapeutic strategies for it.

Saeki C, et al. BMJ Open Gastro 2022;9:e000879. doi:10.1136/bmjgast-2022-000879

9

Author affiliations
1
Division of Gastroenterology and Hepatology, Department of Internal Medicine, The
Jikei University School of Medicine, Tokyo, Japan
2
Division of Gastroenterology, Department of Internal Medicine, Fuji City General
Hospital, Shizuoka, Japan
3
Division of Molecular and Cellular Medicine, National Cancer Center ResearchInstitute, Tokyo, Japan
4
Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Tokyo,
Japan
5
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
6
Toray Industries, Kanagawa, Japan
7
Department of Diagnostic Pathology, Fukui Prefecture Saiseikai Hospital, Fukui,Japan
8
Department of Molecular and Cellular Medicine, Tokyo Medical University, Tokyo,
Japan
9Core Research Facilities, Research Center for Medical Science, The Jikei University
School of Medicine, Tokyo, Japan
Contributors CS, JM and TOc participated in the study conception and design.
CS, KT, TOi and MS collected, analysed and interpreted the data. JM, MI and ST
performed the experiments and analysed the data. YN evaluated the histologicalfindings. MK and KF performed the in situ hybridisation. CS, JM and AT drafted
the manuscript. AT interpreted the data and revised the manuscript extensively.
AT substantively revised and completed the manuscript. CS is responsible for theoverall content as guarantor.
Funding This study was supported by a Development of Diagnostic Technology
for Detection of miRNA in Body Fluids grant from the Japan Agency for Medical
Research and Development and Grants-­in-­Aid for Scientific Research from the
Ministry of Education, Culture, Science and Technology in Japan.
Competing interests MI and ST are employees of Toray Industries, the provider ofthe 3D-­Gene system. The other authors declare no conflicts of interest.
Patient consent for publication Consent obtained directly from patient(s)Ethics approval This study was conducted in accordance with the Declaration of
Helsinki and was approved by the ethics committee of the Jikei University School of
Medicine (approval No. 28-­318, No. 30-­051) and Fuji City General Hospital (approvalNo. 159).
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request.
Supplemental material This content has been supplied by the author(s). It
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not havebeen peer-­reviewed. Any opinions or recommendations discussed are solely
those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability
and responsibility arising from any reliance placed on the content. Where thecontent includes any translated material, BMJ does not warrant the accuracy andreliability of the translations (including but not limited to local regulations, clinicalguidelines, terminology, drug names and drug dosages), and is not responsible
for any error and/or omissions arising from translation and adaptation orotherwise.
Open access This is an open access article distributed in accordance with theCreative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non-­commercially,and license their derivative works on different terms, provided the original work is
properly cited, appropriate credit is given, any changes made indicated, and theuse is non-­commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
ORCID iD
Chisato Saeki http://orcid.org/0000-0002-7407-6142

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