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ORIGINAL ARTICLE
N
6
-Methyladenosine modification of circDcbld2
in Kupffer cells promotes hepatic fibrosis via
targeting miR-144-3p/Et-1 axis
Sai Zhu
a,b,y
, Xin Chen
a,y
, Lijiao Sun
a,y
, Xiaofeng Li
a,y
, Yu Chen
c
,
Liangyun Li
a
, Xiaoguo Suo
a
, Chuanhui Xu
a
, Minglu Ji
a
,
Jianan Wang
a
, Hua Wang
a
, Lei Zhang
a
, Xiaoming Meng
a
,
Cheng Huang
a
, Jun Li
a,*
a
Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs,
School of Pharmacy, Anhui Medical University, Hefei 230032, China
b
Department of Nephropathy, the First Affiliated Hospital, Anhui Medical University, Hefei 230022, China
c
Department of Pharmacy, the Second Affiliated Hospital of Bengbu Medical College, Bengbu 233000, China
Received 1 April 2024; received in revised form 22 June 2024; accepted 26 July 2024
KEY WORDS
Kupffer cells;
circDcbld2;
miR-144-3p;
Et-1;
N
6
-methyladenosine;
Wtap;
Igf2bp2;
Hepatic fibrosis
Abstract Kupffer cells (KCs), as residents and sentinels of the liver, are involved in the formation of
hepatic fibrosis (HF). However, the biological functions of circular RNAs (circRNAs) in KCs to HF have
not been determined. In this study, the expression levels of circRNAs, microRNAs, and messenger RNAs
(mRNAs) in KCs from a mouse model of HF mice were investigated using microarray and circRNA-Seq
analyses. circDcbld2 was identified as a candidate circRNA in HF, as evidenced by its up-regulation
in KCs. Silver staining and mass spectrometry showed that Wtap and Igf2bp2 bind to cirDcbld2. The
suppression of circDcbld2 expression decreased the KC inflammatory response and oxidative stress
and inhibited hepatic stellate cell (HSCs) activation, attenuating mouse liver fibrogenesis. Mechanisti-
cally, Wtap mediated the N
6
-methyladenosine (m6A) methylation of circDcbld2, and Igf2bp2 recognized
m6A-modified circDcbld2 and increased its stability. circDcbld2 contributes to the occurrence of HF
by binding miR-144-3p/Et-1 to regulate the inflammatory response and oxidative stress. These findings
indicate that circDcbld2 functions via the m6A/circDcbld2/miR-144-3p/Et-1 axis and may act as a
potential biomarker for HF treatment.
*Corresponding author.
E-mail address: lj@ahmu.edu.cn (Jun Li).
y
These authors made equal contributions to this work.
Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.
https://doi.org/10.1016/j.apsb.2024.11.003
2211-3835 ª2025 The Authors. Published by Elsevier B.V. on behalf of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese
Academy of Medical Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Chinese Pharmaceutical Association
Institute of Materia Medica, Chinese Academy of Medical Sciences
Acta Pharmaceutica Sinica B
www.elsevier.com/locate/apsb
www.sciencedirect.com
Acta Pharmaceutica Sinica B 2025;15(1):296e313
ª2025 The Authors. Published by Elsevier B.V. on behalf of Chinese Pharmaceutical Association and
Institute of Materia Medica, Chinese Academy of Medical Sciences. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Hepatic fibrosis (HF), a chronic liver disease, is characterized by
excessive extracellular matrix (ECM) deposition
1,2
. HF can
progress to cirrhosis or even cancer resulting in persistent liver
injury
3-5
, and is caused by various conditions, such as alcohol
abuse and non-alcoholic steatohepatitis (NASH)
6
. Macrophage
infiltration in the liver accompanies persistent liver injury
7-9
.
Interestingly, the uncontrolled secretion of inflammatory factors
and chemokines by macrophages are important driving force for
HF formation
10
. In addition, inflammation-induced changes in the
liver microenvironment lead to dysregulation of liver function,
reducing the capacity for self-repair and accelerating HF
progression
11
.
Kupffer cells (KCs) and recruited macrophages are crucial
regulators of inflammation in the organ and are considered
potential new targets for the treatment and prevention of HF
12
.
Activated macrophages synthesize and release various inflamma-
tory cytokines and chemokines that accelerate inflammatory
infiltration and collectively drive activated hepatic stellate cells
(HSCs) to transform into myofibroblasts
12,13
, while simulta-
neously producing enormous amounts of actin alpha 2, smooth
muscle, aorta (a-SMA)
14
. Therefore, it is essential to explore the
mechanisms by which macrophage activation affects HSC acti-
vation during HF formation. Recent work has shown that liver
macrophages promote HF by enhancing HSC activation in a nu-
clear factor kappa B (NF-kB)-dependent manner
15
. However, the
precise molecular mechanisms by which macrophages are
involved in HF remain largely unclear.
N
6
-methyladenosine (m6A), a common modification of
messenger RNA (mRNA)
16,17
, has recently gained attention owing
to its potential involvement in Circular RNAs (circRNA) function.
circRNAs, are small non-coding RNAs (ncRNAs) with a cova-
lently closed single-stranded loop configuration produced by
exon skipping or direct back-splicing of precursor mRNA (pre-
mRNA)
18
, exhibit different manifestation m6A modification
patterns from those of traditional mRNA
19,20
.
Recent advances in RNA sequencing technology have revealed
that most circRNAs exhibit tissue-specific expression profiles
21
.
circRNAs act as competing endogenous RNAs (ceRNAs) and
specifically adsorb microRNAs (miRNAs) to affect downstream
target gene expression or regulate gene expression at the levels of
transcription and splicing
22-24
. Differential circRNA expression is
associated with various pathological processes, including HF
25
.
circRNAs are also dysregulated in macrophages during disease
development
26,27
. Our group has studied the roles of non-coding
RNAs in the development of HF extensively
28-32
. We have
shown that circFbxw4 (F-box and WD repeat domain containing
4) affects HF progression via the miR-18b-3p/Fbxw7 (F-box and
WD repeat domain containing 7) axis in HSCs
28
. Furthermore,
circUbe2k
33
(ubiquitin conjugating enzyme E2 K), circPsd3
29
(Pleckstrin and Sec7 domain containing 3), and circMcph1
34
(Microcephalin 1) play significant roles in HF. The functions
and mechanisms of circRNAs that are differentially expressed in
macrophages during HF formation have been a focus of our
research
35
.
In this study, the expression patterns of circRNAs, miRNAs, and
mRNAs in KCs extracted from a mouse model of HF were
analyzed to explore the potential biomarkers. We identified a novel
abnormally expressed circRNA, circDcbld2, derived from the
Discoidin, CUB and LCCL domain containing 2 (Dcbld2)gene
locus. circDcbld2 expression levels were significantly increased in
HF formation and were elevated in patients with HF. The inhibition
of circDcbld2 reduced the expression of inflammatory factors in
macrophages, attenuated oxidative stress, and affected HSC acti-
vation to mitigate liver fibrogenesis in mice. Mechanistically, we
discovered that circDcbld2 binds to miR-144-3p to regulate
Endothelin 1 (Et-1) expression. These findings indicate that the
circDcbld2/miR-144-3p/Et-1 axis may have important functions in
macrophages and contribute to the pathogenesis of HF. Further-
more, our investigation revealed an interaction between circDcbld2
and WT1 associated protein (Wtap), a pivotal m6A writer protein,
and the stability of circDcbld2 was increased via insulin-like growth
factor 2 mRNA binding protein 2 (Igf2bp2), a pivotal m6A reader
protein. Therefore, our results support the value of circDcbld2 as a
novel potential therapeutic biomarker for HF.
2. Materials and methods
2.1. Animals and model establishment
C57BL/6J mice (6e8 weeks of age) were obtained from the
Animal Experiment Center of Anhui Medical University (Anhui,
China). Before the experiment, mice were housed in an environ-
ment with adequate food and water for one week. Euthanasia was
performed 3 days after the last injection for modeling. A set of
mice was used for the perfusion extraction of primary cells and
detection of related indicators, and the remaining mice were used
for histopathological examination of tissue following para-
formaldehyde embedding. The animal studies were approved and
reviewed by the Animal Experimentation Ethics Committee of
Anhui Medical University.
Various methods were used for HF model were establishment.
Firstly, for carbon tetrachloride (CCl
4
)-induced HF model, as
described previously
33
. Carbon tetrachloride (CCl
4
) and olive oil
(1:4, v/v) were administered to mice by intraperitoneal injection
(1 mL/kg), biweekly for 6 weeks to trigger HF. Vehicle mice were
injected with olive oil (same volume).
Secondly, the model of cholestatic HF was established through
bile duct ligation (BDL) as described previously
36
. The cholestatic
liver fibrosis model was housed in a specific pathogen-free (SPF)-
class experimental animal room at the Experimental Animal
Center of Anhui Medical University. Mice in the BDL group were
established by common bile duct ligation, operated by double
ligation using non-resorbable surgical sutures. The sham group
underwent identical procedures without ligation. After 15 days,
mice were sacrificed to observe fibrosis.
N
6
-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 297
Thirdly, another model of HF was established through the
injection of Thioacetamide (TAA) (200 mg/kg, diluted in saline) 3
times weekly for 8 weeks
37
. Mice were sacrificed 24 h after the
last administration, and fractional liver tissues were collected for
histopathological examination.
2.2. Generation of macrophage-specific Wtap knockout mice
Wtap-cKO (Wtap-conditional knockout) (C57BL/6J background)
mice were generated in which Wtap was specifically depleted
from macrophages (global Wtap knockout is embryonically
lethal). Wtap
F/F
(Wtap
Flox/Flox
) mice were purchased from
GemPharmatech Co., Ltd. (Nanjing, China). Wtap
F/F;Lyz-Creþ
(Wtap-cKO) mice were generated by mating Wtap
F/F
mice with
macrophage-specific promoter (Lysozyme 2)-driven Cre (Lyz-Cre)
mice.
2.3. circRNA expression analysis
2.3.1. RNA extraction and quality control
Primary macrophages were extracted from the vehicle and CCl
4
-
treated mice. Each group of samples contained primary macro-
phages from six mice for sequencing. Total RNAs of KCs were
extracted using the MIRNeasy Mini Kit (Qiagen, Hilden, Ger-
many). Then, the RNase-Free DNase Set and RNA Clean XP Kit
(Beckman Coulter, Brea, CA, USA) were used to purify the
extracted RNA. Quantitative detection was performed using the
NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham,
MA, USA) and Agilent Bioanalyzer 2100 (Agilent Technologies,
Santa Clara, CA, USA).
2.3.2. Library preparation and high-throughput sequencing
Following the manufacturer’s instructions, RNA-seq libraries were
constructed using the TruSeqStranded Total RNA Sample
Preparation Kit (Illumina, USA). Subsequently, the Qubit2.0
Fluorometer (Life Technologies, USA) and Agilent 2100 Bio-
analyzer (Agilent Technologies, USA) were used for quantifica-
tion and validation. The library was diluted to 10 pmol/L and
sequenced using the Illumina HiSeq 2500 system (Illumina,
USA). Sequencing and library validation and construction were
performed by Origin Biotech (Shanghai, China).
2.3.3. Data analysis
Quality control for RNA-Seq reads was performed using FastQC1
(v.0.11.3). Ribosomal RNA reads, poor reads and Illumina TruSeq
adapter sequences were trimmed using seqtk. Then, BWA-MEM
(v.2.0.4) was used to map mouse reference genes and trimmed
reads. circRNA sequencing data were analyzed using CIRI, and
matched against the circBase database. The counts were normal-
ized to SRPBM. The thresholds for differential expression were
adjusted P-value <0.05 and |log
2
(fold change)| 1.0. All of the
dysregulated genes were evaluated using a heat map and KEGG
pathway enrichment analyses.
2.4. RNA sequencing and functional enrichment analysis
RNA samples were isolated and purified using TRIzol (Thermo
Fisher Scientific, USA) according to the manufacturer’s protocol.
PolyA mRNA was specifically captured through two rounds of
purification using oligo (dT) magnetic beads (Thermo Fisher,
USA). The fragmented RNA was subjected to reverse transcription
to synthesize cDNA (Invitrogen SuperScript™II Reverse
Transcriptase, Cat. 1896649, CA, USA). These compound DNA
and RNA duplexes were then converted to DNA duplexes by
double-strand synthesis using E. coli DNA polymerase I (NEB,
USA) and RNase H (NEB, USA). The two strands were digested
with UDG enzymes (NEB, cat.m0280, MA, US) to form a library
with a fragment size of 300 bp 50 bp (strand-specific library).
Then, the Illumina NovaSeq™6000 platform (LC Bio Technol-
ogy Co., Ltd., Hangzhou, China) was used for paired-end
sequencing following the standard operation in PE150 mode.
Differentially expressed mRNAs were identified using adjusted
P-value <0.05 and |log
2
(fold change)| 1.0 as thresholds. Gene
functions were evaluated using Kyoto Encyclopedia of Genes and
Genomes (KEGG) and Gene Ontology (GO) functional enrich-
ment analyses.
2.5. Microarray analysis
According to the instructions provided, the Affymetrix
GeneChipmiRNA 4.0 Array (Affymetrix, USA) and FlashTag™
Biotin HSR Labeling Kit (Affymetrix, USA) were used. The
GeneChip Hybridization Wash and Stain Kit (Thermo Fisher
Scientific, USA) was used to dye the miRNA array for imaging.
Differentially expressed miRNAs were identified using |log
2
(fold
change)| 1.0), P-value <0.05, and volcano plot filtering. The
targets of miRNAs were obtained using two databases TargetScan
and miRanda.
2.6. ceRNA network (ceRNET) snalysis
A ceRNET (circRNAsemiRNAsemRNAs) was generated based
on dysregulated circRNAs, miRNAs, and mRNAs. The interaction
ceRNET was established using Cytoscape.
2.7. Human samples
Human liver samples were obtained from the First Affiliated
Hospital of Anhui Medical University (Anhui, China). This study
was approved by the Biomedical Ethics Committee of Anhui
Medical University. The volunteers were undertaken with the
understanding and written consent. All protocols adhered to the
principles outlined in the Declaration of Helsinki. Characteristics
of participants are shown in Supporting Information Table S1.
2.8. circDcbld2 suppression in mice
pHBAAV-circDcbld2 (circDcbld2-KD) and control were designed
and synthesized by Hanbio Biotechnology (Hanbio Biotech-
nology, China). Mice were injected with circDcbld2-KD and the
vector at 1 10
12
vg/mL into the tail vein. After a week of
observation, the model was established by circDcbld2-KD
administration. The transfection efficiency for circDcbld2 was
measured by qRT-PCR in primary macrophages.
2.9. Flow cytometry analysis
Cells were incubated with anti-Cd11b-FITC (BD Biosciences,
USA) and anti-F4/80-PE (BD Biosciences, USA) antibodies.
Next, the CytoFLEX flow cytometer (Beckman Coulter, USA)
was used to detect compounds. Cd11b
þ
and F4/80
þ
cells were
considered macrophages for subsequent experiments, and
CytExpert software (Beckman Coulter, USA) was used to analyze
data.
298 Sai Zhu et al.
2.10. Histology and immunohistochemistry
Paraffin-embedded and 4% paraformaldehyde-fixed liver tissues
(4 mm) were used for immunofluorescence (IF) and immunohis-
tochemical (IHC) staining of Et-1 (Abcam, USA), F4/80 (Bioss,
bsm-34028M), and a-SMA (Abcam, USA). Liver pathology was
evaluated by Sirius red and H&E staining. Sections were scanned
using a digital slide scanner (Pannoramic MIDI, 3DHISTECH,
Hungary).
2.11. Examination of oxidative stress markers
Commercial assay kits for the detection of Tatalase (CAT; #A007-
1-1), glutathione (GSH; #A006-2-1), malondialdehyde (MDA;
#A003-1-3), and superoxide dismutase (SOD; #A001-3-2) were
obtained from Nanjing Jiancheng Bioengineering Institute
(Nanjing, China). Oxidative stress damage of ROS in liver tissue
for the HF model was examined by ROS assay kit (Bestbio, #BB-
470534) following the manufacturer’s protocols.
2.12. DNA sequencing
RNA was reverse-transcribed into cDNA by using
PrimeScript
TM
RT Master Mix (Takara, Japan). Polymerase chain
reaction (PCR) was performed using 2 Taq Master Mix (Takara,
Japan) following the manufacturer’s instructions. The PCR prod-
ucts were identified by using DNA sequencing (ABI3730XL,
USA).
2.13. Pull-down assay
A biotinylated circDcbld2 probe was designed for binding to the
junction site of circDcbld2. Bone marrow-derived macrophages
(BMDMs) were washed with PBS, and the cell lysate was incu-
bated with 3 mg of biotinylated probe for 4 h. Lysates were
incubated with RNase-free BSA (10 mg/mL) and Pierce™
Streptavidin Magnetic Beads (Thermo Fisher Scientific, USA) at
4C (3 h) to decreasing nonspecific binding. RNA complexes
were rotated with probe-bead complexes at 4 C overnight. After
washing the beads three times, relative protein levels were
analyzed through Western blotting.
2.14. Fluorescence in situ hybridization (FISH)
In situ hybridization probes, i.e., FAM-labelled circDcbld2 and
FAM-labelled miR-144-3p probes, were established by
GenePharma (Shanghai, China). Briefly, cells were washed three
times with sterile PBS and fixed with 4% paraformaldehyde at
room temperature. A FISH Kit (GenePharma, China) was
employed for protein detection according to the manufacturer’s
instructions, with hybridization at 37 C overnight in a dark moist
chamber. DAPI was used to stain nuclei. Images were collected by
an inverted fluorescence microscope (Leica, Japan).
2.15. Isolation of bone marrow-derived macrophages, Kupffer
cells, hepatocytes, and hepatic stellate cells
Previously described methods were used to isolate KCs
38
, hepa-
tocytes, HSCs
28
, and BMDM
39
.In situ perfusion was performed
using collagenase followed by differential centrifugation accord-
ing to a density gradient. A catheter was inserted in the liver portal
vein of the mouse and dissect the inferior vena cava and perfusion.
The digested liver was passed through a sieve (200-mesh). Then,
25% and 50% Percoll were used to separate macrophages. The
isolated cells were resuspended in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% fetal bovine serum (FBS). Non-
adherent cells and culture medium were discarded after the liver
macrophages adhered to the flask surface for 40 min. Macro-
phages were identified by using flow cytometry
40
. Hepatocytes
were isolated from the mouse liver by in situ collagenase perfu-
sion. The liver was perfused with HBSS (2%FBS) via the portal
vein, followed by 0.27% collagenase IV. Perfused livers were
dissected and teased through 70 mm nylon mesh cell strainers.
Hepatocytes were collected following centrifugation at 50 g
(2 min three times). Primary HSCs were isolated through two-step
collagenase (Sigma)epronase (Sigma) perfusion of mouse livers.
Then, OptiPrep (Axis Shield, Norway) was used to for density
gradient centrifugation (11.5% and 20%)
41
. Furthermore, tibias
and femurs were isolated from mice via cutting at the knee joint.
Bone marrows were collected and cultured for 7 days and termi-
nally differentiated into macrophages.
2.16. siRNA-circDcbld2 and over-expression circDcbld2
plasmid transfection
Small interfering RNAs (siRNAs) and an over-expression plasmid
for circDcbld2 were obtained from Hanheng Biotechnology
(Shanghai, China). BMDMs were transfected with siRNA-
circDcbld2 with Lipofectamine 2000 (Invitrogen, USA). The
culture medium was replaced, after 6 h, followed by incubation for
an additional 24 h. The transfection efficiency of circDcbld2 was
detected using qRT-PCR. Sequences are shown in Supporting
Information Table S2.
2.17. miR-144-3p mimics and inhibitor transfection
miR-144-3p mimics and the inhibitor were structured in Hanheng
Biotechnology (Shanghai, China). The transfection methods were
consistent with those for circDcbld2. A qRT-PCR analysis was
used to measure the transfection efficiency of the miR-144-3p
mimics and inhibitor. Related sequences are shown in Supporting
Information Table S3.
2.18. RNA immunoprecipitation (RIP)
The Magna RIP RNA-Binding Protein Immunoprecipitation Kit
(Millipore Sigma, 17-701) was used for an RIP analysis following
the manufacturer’s protocols. Magnetic beads precoated with
Igf2bp2 or IgG (Millipore) were incubated with the cell suspen-
sion at 4 C overnight. Proteinase K was used to treat
RNAeprotein complexes to remove protein impurities. Finally,
RNA was purified using TRIzol and detected using qRT-PCR.
2.19. m6A MeRIP-qRT-PCR
RNA samples were collected in Wtap-cKO or Wtap
F/F
BMDMs
under lipopolysaccharide (LPS) stimulation. The RNA was soni-
cated to obtain 100e150 nt fragments, which were incubated with
m6A antibody to analyze m6A enrichment using qRT-PCR.
Briefly, fragmented RNA was combined with the m6A antibody
in RIP immunoprecipitation buffer and incubated at 4 C over-
night, followed by incubation in proteinase K buffer at 55 C for
30 min. TRIzol was used to extract RNA, and m6A immunopur-
ification of mRNA was detected using qRT-PCR.
N
6
-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 299
2.20. Dot blot
Extracted RNA was boiled in a metal bath at 90 C for 5 min and
cooled immediately. The RNA sample point on the nylon mem-
brane (UV lamp for 2 h) was blocked with 5% skim milk (2 h) and
incubated with the m6A antibody overnight. The nylon membrane
was washed with TBST (Tris-buffered saline containing 0.1%
Tween 20) three times on the next day. The secondary antibody
was applied, followed by detection using the Amersham Imager
600 System. Methylene blue was used to stain the membranes.
2.21. RNA extraction and qRT-PCR analysis
Total RNA was extracted with TRIzol from cells or tissues. The
NanoDrop 2000 Spectrophotometer was employed to detect the
purity and concentration of extracted RNA. PrimeScrip™RT
Master Mix was used to synthesize cDNA. Gapdh and b-actin
expression were used as internal controls. Primer sequences are
listed in Supporting Information Tables S4eS6.
2.22. Nuclear and cytoplasmic fractionation analysis
The Cytoplasmic & Nuclear RNA Purification Kit (#BB-36021,
BestBio) was used for a nuclear and cytoplasmic fractionation
analysis. The nuclear and cytoplasmic components were extracted
through centrifugation (1200 gfor 5 min). Then, each fraction
was collected separately.
2.23. Actinomycin D (Act-D) and RNase R treatment
Linear RNA was removed with RNase R (Epicentre Technologies,
USA). The samples (RNase Rþand RNase Re) were supple-
mented with reaction buffer (10 , 0.6 mL) and DEPC-water
(0.2 mL) for 20 min at 37 C. circRNA and mRNA exposed to
RNase R were detected with CFX96 qRT-PCR (Bio-Rad, CA).
BMDM transcription was blocked with Actinomycin D (4, 8, 16,
and 24 h) (2 mg/mL, Sigma, USA). qRT-PCR was used to evaluate
the stability of circDcbld2 and the mRNA level of linear Dcbld2.
2.24. Luciferase reporter assay
circDcbld2 sequences with miR-144-3p target sites were synthe-
sized and cloned into the pSI-Check2 reporter vector downstream
of firefly luciferase (circDcbld2-wild type and circDcbld2-mutant)
by Hanheng Biotechnology (Shanghai, China), respectively. The
reporter vector, miR-144-3p mimics, or negative control were
transfected in HEK-293T cells using Lipofectamine 3000 (Invi-
trogen, CA). Activity levels of firefly and Renilla luciferase were
measured using a Dual-Luciferase system (Promega, USA) ac-
cording to the manufacturer’s protocol and detected by GloMax
Multi Jr (Promega, USA).
2.25. Western blotting
Whole-protein samples were transferred to a PVDF membrane
(Millipore, USA). The antibodies used in the analysis were as
follows: b-actin (1:500, Sino Biological lnc., 100166-MM10),
Wtap (1:500; Cell Signaling Technology, #56501), Igf2bp2
(1:500; Abcam, ab124930). a-SMA (1:500; Abcam, ab7817),
Col1a1(1:500; Bioss, bs-0578R). Then, membranes were
incubated with secondary antibodies (HRP-coupled) (1:1000,
ZSGB-Bio, China) for 60 min at room temperature. Finally, pro-
tein signals were analyzed using an enhanced chemiluminescence
(ECL) system (Bio-Rad, USA).
2.26. Statistical analysis
Data are shown as the mean standard error of mean (SEM)
and were analyzed by one-way analysis of variance (ANOVA),
followed by NewmaneKeuls post hoc tests implemented in Prism
8.0 (GraphPad Software, USA). Correlations in expression
levels were evaluated using Pearson’s correlation coefficients.
Pvalue <0.05 were considered statistically significant.
3. Results
3.1. circDcbld2 up-regulated in mouse KCs by high-throughput
sequencing
The secretion of inflammatory factors and chemokines from KCs
is critical during HF
42
. To identify candidate circRNAs involved
in HF, primary KCs (isolated from vehicle or HF mice) were
examined by circRNA high-throughput sequencing. The progres-
sion of HF formation is shown in Supporting Information
Fig. S1A. We first successfully established BDL, CCl
4
,
and TAA-induced HF mouse models and confirmed injury and
pathological characteristics (Fig. 1A and Fig. S1BeS1D). In-
flammatory factors were increased in KCs (Fig. S1E) and tissues
(Fig. S1F) from HF mice than from control mice. The serological
indicator alanine/aspartate-transaminase (ALT/AST) in
CCl
4
-induced HF mice is summarized in Fig. S1G. These results
indicate successful model construction.
We detected differential expression of 3138 circRNAs between
KCs extracted from HF mice and control mice, of which 457
circRNAs were recorded in circBase. The circRNAs with
log
2
FC 1.0 were selected for subsequent analyses. Further
analysis indicated 99 circRNAs were expressed differently in
HF (Supporting Information Files S1 and S2), including 39
up-regulated and 60 down-regulated circRNAs (Supporting
Information File S3). These dysregulated circRNAs, after valida-
tion, may be related to the development of HF. The relative
expression levels of circRNAs are shown in Supporting
Information Fig. S2A and the circRNA gene distribution is
shown in Fig. S2B. Back-splicing from host genes is considered
the source of most circRNAs
18
. The biological characteristics
(including length, localization, host genes, and exons/introns) of
the circRNAs were evaluated. We selected 13 circRNAs for model
validation (Fig. S2C). Based on validation and expression
intensity results, we selected circDcbld2 for further analysis.
Notably, we found that circDcbld2 was up-regulated in HF mice
induced by CCl
4
, TAA, and BDL (Fig. 1B). We also detected
circDcbld2 expression in BMDMs (Fig. 1C), KCs (Fig. 1D),
HSCs, and hepatocytes, extracted from CCl
4
-induced HF mice.
Levels of circDcbld2 were significantly elevated in KCs
and BMDMs in HF, and did not differ significantly in HSCs and
hepatocytes (Fig. 1E). To explore the expression changes of
circDcbld2 (hsa_circ_0066631) in human liver fibrosis, we
extracted RNA from human liver tissues for detection, revealing
that circDcbld2 as up-regulated (Fig. 1FeG and Fig. S2D). These
findings indicate that the expression pattern of circDcbld2 corre-
sponds with the pathology of HF; accordingly, circDcbld2 is a
potential biomarker during HF formation.
300 Sai Zhu et al.
Figure 1 circDcbld2 up-regulated in mouse KCs by high-throughput sequencing. (A) Pathology observation stained with Sirius red staining
were performed in CCl
4
-induced, BDL-induced, TAA-induced and vehicle mouse liver tissues sections. Scale bar Z100 mm. (B) The up-
regulated of circDcbld2 in HF mice induced by CCl
4
, TAA and BDL (nZ6). (C) The process of BMDM extraction. (D) KCs were identi-
fied by flow cytometry. (E) The expression of circDcbld2 in KCs, BMDM, HSCs and Hepatocytes (nZ6). (F) Pathology observation of human
fibrotic liver tissues sections stained with H&E and IHC of a-SMA. (G) circDcbld2 expressed in human fibrotic liver tissues. The bar shows the
mean SEM. *P<0.05, ***P<0.001 vs vehicle group.
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-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 301
3.2. Characterization of circDcbld2
circDcbld2 (mmu_circ_0000693) is derived from the host gene
Dcbld2 located on chromosome 16 (58424673e58433576)
(366 nt). Genomic structure and back-splicing point analyses
revealed that circDcbld2 consists of two exons from the Dcbld2
(exons 2e3) (Fig. 2A). Furthermore, the qRT-PCR product of
circDcbld2 verified that the circRNA matched the circDcbld2
sequence from circBase and showed head-to-tail splicing. Using
convergent and divergent primers designed for circDcbld2, and
genomic DNA (gDNA) and complementary DNA (cDNA) as
templates, 1% agarose gel electrophoresis revealed a distinct,
single product of circDcbld2 using divergent primers from cDNA
only, while no product was obtained from gDNA (Fig. 2B). To
verify the circRNA characteristics of circDcbld2, we confirmed
resistance to RNase R digestion (Fig. 2C). Furthermore, an Acti-
nomycin D (Act D) analysis showed that circDcbld2 had greater
stability than that of the linear Dcbld2 transcript in KCs (Fig. 2D).
We also treated circDcbld2 and linear Dcbld2 with RNase R,
revealing that circDcbld2 was resistant to RNase R digestion
(Fig. 2E). FISH and cytoplasmic and nuclear fractionation assays,
with U6 and Gapdh as controls, revealed that circDcbld2 localized
to both the nucleus and cytoplasm (Fig. 2F and G).
3.3. Wtap mediates circDcbld2 m6A modification and increases
the stability via Igf2bp2
Cytoplasmic circRNAs act as miRNA sponges or as RNA-binding
proteins (RBPs), while nuclear circRNAs mainly interact with
RBPs
43
. Given the location of circDcbld2, RNA pull-down and
mass spectrometry (MS) were employed to explore its functions
(Fig. 3A). We detected m6A-related proteins with protein
molecular weights of 40e70 kDa and found two proteins, WTAP
and IGF2BP2, were consistently pulled down by the biotin-labeled
circDcbld2 antisense probe (Fig. 3B and C). The pull-down con-
centration and silver staining of the polypeptide sites of Wtap and
Igf2bp2 are shown in Fig. 3D and E, and Supporting Information
Fig. S3A and S3B. We then constructed Wtap
F/F;Lyz-Creþ
(Wtap-
cKO) mice and confirmed that the knockout efficiency through
Western blotting (Fig. 3F and Fig. S3C). Colorimetric and dot blot
assays indicated the m6A methylation level was lower in Wtap-
cKO mice than in Wtap
F/F
(Fig. S3D and S3E). We predicted a
credible methylation site of circDcbld2 using SRAMP (http://
www.cuilab.cn/sramp)(Fig. S3F). Luciferase reporter gene con-
structs with wild-type or mutant circDcbld2 further verified the
m6A modification of circDcbld2 by Wtap (Fig. S3G). The lucif-
erase reporter assay indicated that the level of wild-type
circDcbld2 decreased significantly after Wtap-cKO administra-
tion but not in response to mutant circDcbld2 (Fig. S3H).
Furthermore, circDcbld2 levels were significantly decreased in
Wtap-cKO than in Wtap
F/F
(Fig. 3G). MeRIP-qPCR results proved
that circDcbld2 was enriched for m6A modifications in the LPS-
stimulated Wtap
F/F
group, while this enrichment was decreased
in the LPS-stimulated Wtap-cKO group (Fig. 3H). These results
demonstrated that the Wtap-related m6A modification regulated
circDcbld2 expression. Recently, Igf2bp2 has been identified as an
m6A-related “reader” protein involved in the stability of down-
stream genes by recognizing m6A modifications
44
. Silver staining
and mass spectrometry revealed enrichment for the stability-
related protein Igf2bp2; therefore, we further evaluated whether
m6A modification affects circDcbld2 stability. The results showed
that Wtap-cKO significantly reduced the stability of circDcbld2, as
determined using Act D treatment (Fig. 3I). An RIP analysis
further proved that circDcbld2 and Igf2bp2 interact in LPS-
stimulated BMDMs (Fig. 3J). RNA stability analysis indicated
that Igf2bp2 inhibition reduced circDcbld2 stability following Act
D treatment (Fig. 3K), and shortened the RNA half-life of
circDcbld2 (Fig. S3I), indicating that Igf2bp2 participated in the
maintenance of circDcbld2 stability. Furthermore, RIP-qPCR
analysis showed that an Igf2bp2-specific antibody resulted in
significantly greater circDcbld2 enrichment in the Wtap
F/F
group
than in the IgG control, and this increase was obviously reduced in
the Wtap-cKO group (Fig. S3J). Therefore, the increase in
circDcbld2 expression in the model group may be due to the
modified and identified of circDcbld2 m6A site by Wtap and
Igf2bp2 to enhance the stability of circDcbld2, this clarifying the
mechanism underlying circDcbld2 up-regulation in liver fibrosis.
3.4. circDcbld2 increases inflammation and oxidative stress of
BMDMs
To analyze the function of circDcbld2 in BMDMs, we constructed
siRNA of circDcbld2 (Fig. 4A). After analyzing the efficiency of
siRNA-circDcbld2 transfection in BMDMs (Fig. 4B), we per-
formed RNA-seq (Supporting Information Fig. S4A). Genes
associated with KEGG pathways (TNF, HIF-1, and FoxO
signaling pathways), and GO terms (inflammatory response,
oxidoreductase activity) were affected by circDcbld2 silencing
(Fig. 4C and D). In addition, circDcbld2 suppression decreased
the secretion of inflammatory factors and chemokines such as
IL-1b,Tnf-a, and Mcp-1, as determined by ELISA (Fig. 4E). We
further found that circDcbld2 silencing reduced the mRNA levels
of oxidative stress markers, such as Nox1,Nox2,Nox4, and
p22
phox
(Fig. 4F). Moreover, while SOD and GSH were decreased
in LPS-stimulated BMDMs compared with controls, these de-
creases were attenuated by circDcbld2 knockdown (Fig. S4B and
S4C). In addition, the over-expression of circDcbld2 had the
opposite effects compared with those for circDcbld2 silencing
(Fig. 4G and H, Fig. S4D and S4E). Taken together, these results
show that circDcbld2 silencing effectively reduces LPS-induced
inflammation and oxidative stress in BMDM.
3.5. Pro-fibrogenic activities and pro-oxidative activities effects
of circDcbld2 in HF mice
To further explore the influence of circDcbld2 on HF, circDcbld2-
KD was injected into HF mice through the tail vein. We verified
the efficiency of circDcbld2 inhibiting in KCs (Supporting
Information Fig. S5A). Functionally, the degrees of liver
collagen deposition and parenchymal distortion were reduced,
vascular architecture was altered, and F4/80
þ
macrophagocytes
and a-SMA
þ
myofibroblasts were reduced in HF following
circDcbld2-KD treatment (Fig. 5A and Fig. S5B). We further
evaluated HF-related damage indicators, Timp-1,a-SMA, Col1a1,
and Tgf-b1, in liver tissues and the inflammation markers IL-1b,
Tnf-a, and Mcp-1 in KCs (Fig. S5C). The ALT/AST indicator in
CCl
4
-induced HF mice decreased following circDcbld2-KD
administration (Fig. S5D). circDcbld2-KD administration
decreased the levels of inflammatory factors and chemokines
(IL-1b,Tnf-a,and Mcp-1), as analyzed by ELISA (Fig. S5E).
Immunofluorescence staining showed that fibrogenic factors
(Col1a1), and macrophage factors (iNos and F4/80) were
302 Sai Zhu et al.
Figure 2 Characterization of circDcbld2. (A) The genomic structure and backsplicing point of circDcbld2. (B) Divergent primers amplified
circDcbld2 from cDNA by PCR and an agarose gel electrophoresis, rather than from gDNA, Gapdh was used as a linear control. (C) circDcbld2
from cDNA was analyzed by divergent primers even exposed to Rnase R digestion, the opposite result showed from gDNA. (D) Actinomycin D
was added to detected the circDcbld2 and linear Dcbld2 expression in KCs at the indicated time points. (E) circDcbld2 resisted to Rnase R
digestion (nZ6). (F, G) Fluorescence in situ hybridization and Cytoplasmic and nuclear fractionation assay revealed that circDcbld2 localized to
both the nucleus and cytoplasm. Scale bar Z20 mm. The bar shows the mean SEM. ns, no significance; **P<0.01, ***P<0.001 vs Linear
Dcbld2 group (D); Rnasegroup (E) and Nucleus group (F).
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-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 303
Figure 3 Wtap mediates circDcbld2 m6A modification and increases the stability via Igf2bp2. (A, B) The process of RNA pull-down and MS.
(C) Western blot analysis of WTAP and IGF2BP2. (D, E) The polypeptide sites of Wtap and Igf2bp2 in pull-down concentration and silver
staining. (F) The Western blot analysis of knockout efficiency of WTAP. (G) Relative expression of circDcbld2 decreased in Wtap-cKO group. (H)
Wtap-mediated circDcbld2 m6A modifications was detected with MeRIP-qPCR analysis. The m6A modification of circDcbld2 was decreased
following Wtap-cKO (nZ4). (I) The stability of circDcbld2 was weaken by Wtap-cKO. (J) qRT-PCR analysis of RIP in LPS-stimulated BMDMs
indicated the binding of Igf2bp2 protein and circDcbld2 (nZ4). (K) The stability of circDcbld2 was decreased following Igf2bp2 administration.
The data represent the mean SEM. *P<0.05, **P<0.01, vs Wtap
F/F
group (G), m6A group in Wtap
F/F
(H), LPS þWtap
F/F
group (I), Control
group (J) and LPS þsiRNA-NC-Igf2bp2 group (K).
304 Sai Zhu et al.
Figure 4 circDcbld2 increases inflammation and oxidative stress of BMDMs. (A) A siRNA target site of circDcbld2 was constructed. (B)
Silencing efficiency of siRNA-circDcbld2 in BMDMs following transfection. (C, D) GO and KEGG enrichment analysis in BMDMs following
circDcbld2 knock-down. (E) circDcbld2 suppression decreased the release of IL-1b,Tnf-a, and Mcp-1 by ELISA (nZ3). (F) mRNA expression
of Nox1,Nox2,Nox4, and p22
phox
were reduced by circDcbld2 administration (nZ3). (G) circDcbld2 over-expression enhanced the release of
IL-1b,Tnf-a, and Mcp-1by ELISA (nZ3). (H) mRNA expression of Nox1,Nox2,Nox4, and p22
phox
were increased following circDcbld2 over-
expression (nZ3). The data represent the mean SEM. *P<0.05, ***P<0.001 vs siRNA-NC group (B) and control group (E, F, G, H);
#
P<0.05,
##
P<0.01,
###
P<0.001 vs LPS þsiRNA-NC group (E, F) and LPS þOE-NC group (G, H).
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-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 305
increased in the HF model and decreased following circDcbld2-
KD administration (Supporting Information Fig. S6A). NAD(P)
H oxidase subunits (Nox1,Nox2,Nox4, and p22
phox
) increased in
primary KCs from HF mice, and these increases were mitigated by
circDcbld2-KD treatment (Fig. S6B). These results suggest that
circDcbld2-KD could effectively reduce inflammatory injury and
fibrosis in HF mice. Moreover, while CCl
4
induction increased
oxidative stress hallmarks significantly (MDA), CCl
4
-induced in-
creases in expression were attenuated by circDcbld2-KD treat-
ment. Levels of antioxidants, such as CAT, GSH, and SOD, were
Figure 5 Pro-fibrogenic activities and pro-oxidative activities effects of circDcbld2 in HF mice. (A) Pathology observation of H&E staining
and Sirius red staining in HF with circDcbld2-KD, IHC stain of a-SMA and F4/80. Representative images were presented, scale bar, 100 mm.
Quantification of Sirius red and a-SMA (nZ6). (B) IF staining confirmed that the level of oxidative stress activation (ROS) was highly in HF
mice and decreased following circDcbld2-KD (nZ6). Representative images were presented, scale bar Z50 mm. The data represent the
mean SEM. ***P<0.001 vs vehicle group;
###
P<0.001 vs CCl
4
-induced liver fibrosis group.
306 Sai Zhu et al.
restored in CCl
4
-induced HF mice after circDcbld2-KD adminis-
tration (Fig. S6C). Immunofluorescence staining confirmed that
ROS levels were high in CCl
4
-induced HF mice and were reduced
in circDcbld2-KD mice (Fig. 5B). These results show that
circDcbld2 affects hepatic oxidative stress and inflammatory
infiltration in HF model mice.
3.6. Silencing circDcbld2 reduces HSC activation in HF mice
HSCs, which are the primary source of mature myofibroblasts,
play a crucial role in HF formation. To explore the influence of
inflammatory factors and chemokines secreted by activated mac-
rophages on HSCs, we co-cultured primary HSCs with the
BMDM supernatant, transfected with circDcbld2-siRNA
(Fig. 6A). We detected a-SMA, Col1a1,Timp-1, and Tgf-b1
mRNA expression (Fig. 6B) and analyzed the Col1a1 and a-SMA
protein levels in HSCs (Fig. 6C). Silencing circDcbld2 in BMDMs
could effectively reduce HSC activation. Furthermore, Immuno-
fluorescence staining showed that macrophage factors (F4/80) and
HSC activation factors (a-SMA) were elevated in the HF model
but reduced following circDcbld2-KD administration in vivo
(Fig. 6D). Finally, we extracted primary HSCs from HF mice and
explored the expression levels of the HSC activation factor
a-SMA and fibrogenic factor Col1a1following circDcbld2-KD
administration. Silencing circDcbld2 through circDcbld2-KD
administration effectively reduced the expression of a-SMA and
Col1a1 in primary HSCs (Fig. 6E). Furthermore, the a-SMA,
Col1a1,Timp-1, and Tgf-b1mRNA levels were decreased
following circDcbld2-KD treatment (Fig. 6F). Taken together,
silencing circDcbld2 alleviated the secretion of inflammatory
factors and chemokines in macrophages, reduced HSC activation,
inhibited the differentiation of activated HSCs into myofibro-
blasts, decreased collagen deposition, and attenuated CCl
4
-
induced HF injury.
3.7. Microarray analysis and identification of circDcbld2/miR-
144-3p interaction
circRNAs commonly function as miRNA sponges. To identify
potential miRNAs that bind circDcbld2, we performed a miRNA
microarray analysis using primary KCs. The results revealed 138
dysregulated miRNAs in HF samples; the abnormal miRNAs were
shown in the heatmap (Supporting Information Fig. S7A,
Supporting Information Files S4 and S5). A circRNA-miRNA
network was constructed based on the dysregulated miRNAs
and circRNAs (Fig. S7B). These results were combined with
target prediction tools (TargetScan) and a microarray analysis to
evaluate potential miRNAs targets of circDcbld2 and to identify
potential miRNAs related to HF (Fig. S7C). We detected eight
miRNAs that may interact with circDcbld2 (Fig. S7D). Sequence
pairing and expression analysis further revealed that circDcbld2
potentially interacts with miR-144-3p (Fig. 7A). Subsequently,
circDcbld2 fragments with the wild-type (WT) sequence or mu-
tations in putative binding sites as well as negative control and
miRNA mimics were used to explore circDcbld2 and miR-144-3p
binding (Fig. 7A). circDcbld2-WT luciferase reporter activity was
suppressed in the presence of miR-144-3p (Fig. 7B). Furthermore,
miR-144-3p was predominantly localized in the cytoplasm in a
FISH analysis (Fig. 7C). miR-144-3p expression, as determined
by qRT-PCR, was lower in LPS-stimulated BMDMs than in
controls, consistent with the results of the microarray analysis
(Fig. 7D). Interestingly, miR-144-3p was increased in BMDMs
following siRNA-circDcbld2 administration compared with the
LPS þsiRNA-NC group (Fig. 7E). Furthermore, transfection with
miR-144-3p mimics effectively reduced the expression levels of
inflammatory factors (IL-1b,Tnf-a, and Mcp-1) and oxidative
stress markers (Nox1,Nox2,Nox4, and p22
phox
)(Supporting
Information Fig. S8A). miR-144-3p mimics could effectively
increase levels of indicators of oxidative stress (SOD and GSH)
(Fig. S8B). miR-144-3p inhibition had opposite effects on markers
of inflammation and oxidative stress compared with those of miR-
144-3p mimics (Fig. S8C and S8D). Cotransfected with siRNA-
circDcbld2 and miR-144-3p mimics resulted in further re-
ductions in the expression levels of inflammatory markers (IL-1b)
(Fig. 7F) and oxidative stress-related indicators (Nox1,Nox2, and
Nox4) than those for siRNA-circDcbld2 single transfection
(Supporting Information Fig. S9A). In addition, SOD and GSH
contents were further increased (Fig. S9B). These results indicate
that circDcbld2 may bind to and regulate the expression of miR-
144-3p in BMDMs.
3.8. circDcbld2 up-regulates Et-1 expression by sponging miR-
144-3p
miRNAs regulate gene expression by binding the 50or 30
untranslated regions of mRNAs to suppress translation. Whole-
transcriptome-seqencing was employed to explore candidate
genes regulated by circDcbld2/miR-144-3p in KCs in HF
(Supporting Information File S6). Differentially expressed
mRNAs in KCs extracted from vehicle and HF mice are shown in
scatter plots in Supporting Information Fig. S10A. These differ-
entially expressed genes were evaluated through GO classification
(Fig. S10B) and KEGG pathway enrichment analyses (Fig. S10C).
Following miRNA microarray and RNA-seq analyses (Fig. S10D),
Et-1 was identified as a candidate target mRNA of miR-144-3p.
The whole-transcriptome-seq analysis revealed that Et-1 expres-
sion is elevated in KCs (Supporting Information File S7). Et-1
binding sites in the miR-144-3p sequence were identified
(Fig. 8A). We found that Et-1 was increased in LPS-stimulated
BMDMs compared with controls (Fig. 8B). Additionally, the
expression of Et-1 decreased with the inhibition of circDcbld2 and
further decreased after the administration of miR-144-3p mimics
(Fig. 8C). In both immunohistochemical and immunofluorescent
analyses of liver tissue, Et-1 expression was decreased in the
CCl
4
-induced HF model following circDcbld2-KD suppression
(Fig. 8D). These results indicate that the circDcbld2/mir-144-3p/
Et-1 axis plays a role in HF; in particular, circDcbld2 sponges
miR-144-3p, thereby affecting Et-1 expression.
4. Discussion
4.1. Stability of circDcbld2 is regulated by m6A modification
Advances in RNA sequencing technology provide a basis for
elucidating the mechanisms underlying the effects of m6A
methylation
45
. In cancer, m6A modifications play significant roles
in RNA stability, interactions, and production
46
. Altering m6A
levels in Mettl3 and Alkbh5 affects circRNA biosynthesis
47,48
.
However, m6A modifications on circRNAs have not been
explored in KCs during HF formation. In this study, we showed
that circDcbld2 is an important promoter of HF and the presence
of methylation sites in the circDcbld2 sequence was predicted. In
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-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 307
Figure 6 Silencing circDcbld2 reduces HSC activation in HF mice. (A) Schematic representation of the co-culture of primary HSCs and
BMDMs. (B) Timp-1,a-SMA, Col1a1, and Tgf-b1mRNA level for primary HSCs, which affected by BMDM with siRNA-circDcbld2 (nZ6).
(C) The protein expression of a-SMA and Col1a1in HSCs, which affected by BMDMs with siRNA-circDcbld2 administration. (D) Immuno-
fluorescent staining indicated a-SMA and F4/80 were enhanced in the CCl
4
-induced HF model and decreased following circDcbld2-KD
administration. Representative images were presented, scale bar Z50 mm. (E) The protein expression level of a-SMA and Col1a1in pri-
mary HSCs by circDcbld2-KD administration. (F) The mRNA expression level of a-SMA, Col1a1,Timp-1 and Tgf-b1in primary HSCs by
circDcbld2-KD administration (nZ6). The data represent the mean SEM. **P<0.01, ***P<0.001 vs LPS þsiRNA-NC-circDcbld2 group
(B) and Vehicle group (F);
###
P<0.001 vs CCl
4
-induced liver fibrosis group (F).
308 Sai Zhu et al.
exploring the upstream regulation mechanism of circDcbld2, we
found a m6A methylation site in circDcbld2 and further confirmed
that circDcbld2 could bind to Wtap and Igf2bp2. The m6A
sequence motif ‘RRm6ACH’ (R ZGorA;HZA, C, or U) is the
consensus recognition sequence for Igf2bp2. We speculated that
Igf2bp2 binds circDcbld2 via the m6A motif. Furthermore, the
increase in circDcbld2 expression in the model group may be due
to the modified and identified circDcbld2 m6A site by Wtap and
Figure 7 Microarray analysis and identification of circDcbld2/miR-144-3p interaction. (A) Schematic of miR-144-3p binding site in
circDcbld2. (B) Renilla luciferase activity analysis of wild-type or mutant circDcbld2 and miRNAs mimics, respectively. (C) miR-144-3p (FAM)
predominantly localized in the cytoplasm with FISH assay. Representative images were presented, scale bar Z20 mm. (D) miR-144-3p sup-
pression was confirmed by qRT-PCR in LPS-stimulated BMDMs (nZ3). (E) Lower level of miR-144-3p in BMDMs was increased following
siRNA-circDcbld2 administration (nZ3). (F) IL-1bmRNA expression was decreased in BMDMs following siRNA-circDcbld2 administration.
The influence of siRNA-circDcbld2 was further decreased following miR-144-3p mimics (nZ3). The data represent the mean SEM. ns, no
significance; *P<0.05, **P<0.01, ***P<0.001 vs control group (D, E) and LPS group (F);
###
P<0.001 vs LPS þsiRNA-NC-circDcbld2
group (E) and LPS þsiRNA-circDcbld2 group (F).
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-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 309
Igf2bp2, which enhances the stability of circDcbld2, in turn
affecting inflammation and oxidative stress. This explained the
mechanism of circDcbld2 up-regulation in HF. These findings
show for the first time that circDcbld2 binds key m6A writer and
reader proteins, suggesting the potential for m6A modification on
circRNAs.
4.2. circDcbld2 contributes to inflammation, oxidative stress
activation, and HSC activation, and differentiation in HF
Imbalances in inflammation and oxidation play pivotal roles in HF
formation
49
. In this study, circDcbld2 levels were significantly
higher in the HF model than in controls and were elevated in
patients with HF. The differential expression of circDcbld2
prompted us to further investigate its function and mechanism of
action. We discovered that circDcbld2 suppression significantly
reduces the secretion of inflammatory cytokines in macrophages,
alleviates liver fibrogenesis injury, and inhibits chemokine
expression. The down-regulation of circDcbld2 protects against
oxidative stress in macrophages, suggesting that it contributes to
HF by regulating oxidative stress within macrophages. Addition-
ally, HSC trans-differentiation into myofibroblasts is a central
event during HF formation
13
. Therefore, investigating the impact
of inflammatory factors and chemokines secreted by macrophages
on HSC production represents an important direction for our
future research. In this study, silencing circDcbld2 in macrophages
effectively reduced HSC activation and trans-differentiation,
alleviated collagen deposition, and reduced liver injury. Collec-
tively, these results indicate that circDcbld2 exerts pro-fibrotic
effects through increased inflammatory factor and chemokine
secretion, while influencing the oxidative stress balance; further-
more, it facilitates HSC trans-differentiation into myofibroblasts,
thereby accelerating HF progression.
4.3. Identification of the circDcbld2/miR-144-3p/Et-1 axis in
KCs
Despite increasing research on circRNAs in HF, the functions
and mechanisms of circRNAs in KCs remain elusive. HF is
characterized by progressive inflammation and extracellular ma-
trix (ECM) deposition. Liver macrophages play a central role in
driving inflammation and HSC activation to trigger HF
12
. Our
previous studies have demonstrated that the aberrant expression of
circFbxw4 may inhibit HF progression by modulating HSC acti-
vation
28
. Although numerous circRNAs have been investigated in
HSCs, their functions and expression profiles within liver
Figure 8 circDcbld2 up-regulates Et-1 expression by sponging miR-144-3p. (A) Binding sites of miR-144-3p and 30UTR of Et-1.(B)Et-1 was
increased in LPS-stimulated BMDMs (nZ3). (C) Et-1 decreased with the siRNA-circDcbld2 administration and further reduced with miR-144-3p
mimics (nZ3). (D) Immunohistochemistry and immunofluorescence analysis of liver tissue, Et-1 expression was reduced in HF model following
circDcbld2-KD administration. Quantification of fibrosis based on immunohistochemistry analysis of Et-1. Representative images were presented,
scale bar = 100 mm/50 mm. The data represent the mean SEM. **P<0.01, ***P<0.001 vs Control group (B) and LPS group (C) and Vehicle
group (D);
#
P<0.05,
###
P<0.001 vs LPS þsiRNA-circDcbld2 group (C) and CCl
4
-induced liver fibrosis group (D).
310 Sai Zhu et al.
macrophages remain largely unexplored. Furthermore, circRNAs
function as miRNA sponges, transcriptional regulators, RBP-
binding molecules, and protein translation templates in cellular
physiology
50
. Mature miRNAs bind specifically to target mRNAs,
leading to translational repression or mRNA cleavage. Thus,
circRNAs can sequester miRNAs and counteract the miRNA-
mediated suppression of mRNA expression
51
. Our results
revealed that circDcbld2 functions by sponging miR-144-3p.
Notably, circDcbld2 suppression increased miR-144-3p expres-
sion in macrophages. Furthermore, miR-144-3p bound to the 30
untranslated region of Et-1 and down-regulated Et-1 expression
during liver fibrogenesis following decreased levels of circDcbld2.
The impact of circDcbld2 on Et-1 was partially reversed upon
miR-144-3p mimics administration.
5. Conclusions
In this study, we illuminated the first evidence for the pro-
fibrogenic function of circDcbld2 in KCs during the development
of HF and clarified its mechanism of action. We proved that Wtap
interacts with circDcbld2, and Igf2bp2 identifies m6A-modified
circDcbld2 and positively mediates the expression of circDcbld2
by enhancing its stability. In HF, circDcbld2 affected KC in-
flammatory factor production and HSC activation via miR-144-3p/
Et-1. Moreover, circDcbld2 increased the inflammatory response
and oxidative stress during the development of HF and facilitated
HSC trans-differentiation into myofibroblasts, thereby acceler-
ating disease progression (Fig. 9). However, several limitations of
this study need to be addressed. For example, the patient sample
size was small. Although our preliminary findings show that
circDcbld2 is up-regulated in patients with HF, the value of
circDcbld2 as a potential biomarker needs to be validated using
more samples. Moreover, the intercellular communication
between macrophages and HSCs during the experiment was of
interest; however, we only briefly explored the effect of inflam-
matory factors and chemokines secreted by macrophages on HSC
production and the underlying mechanism; these unresolved is-
sues will be a focus of our future research. Our study indicated
that circDcbld2 is a potential biomarker for HF, providing a basis
for the development of novel treatment options.
Figure 9 Wtap mediates the N
6
-methyladenosine (m6A) methylation of circDcbld2, Igf2bp2 recognized m6A-modified circDcbld2 and
increased its stability. circDcbld2 participated in the occurrence of HF by binding miR-144-3p/Et-1 to regulate inflammatory response and
oxidative stress.
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-Methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis 311
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (U19A2001, 82370630, and 82300722); the Anhui
Provincial Natural Science Foundation (No. 2308085QH248,
China); the Research Fund of Anhui Institute of Translational
Medicine (2021zhyx-B06 and 2022zhyx-B07, China); the China
Postdoctoral Science Foundation (No. 2022M710178); the Fund
of Traditional Chinese Medicine Institute of Anhui Dabie
Mountain (No. TCMADM-2024-02, China).
Author contributions
Sai Zhu: Writing eoriginal draft, Resources, Project adminis-
tration, Data curation. Xin Chen: Funding acquisition, Formal
analysis, Data curation. Lijiao Sun: Methodology, Data curation.
Xiaofeng Li: Writing eoriginal draft, Methodology, Formal
analysis. Yu Chen: Software. Liangyun Li: Methodology, Data
curation. Xiaoguo Suo: Methodology. Chuanhui Xu: Software,
Methodology. Minglu Ji: Methodology. Jianan Wang: Data cura-
tion. Hua Wang: Project administration. Lei Zhang: Project
administration. Xiaoming Meng: Methodology, Formal
analysis. Cheng Huang: Software, Methodology. Jun Li:
Writing ereview & editing, Funding acquisition.
Conflicts of interest
The authors declare no conflicts of interest.
Appendix A. Supporting information
Supporting information to this article can be found online at
https://doi.org/10.1016/j.apsb.2024.11.003.
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