Analytical Biochemistry 578 (2019) 45–50

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Role of identified RNA N6-methyladenosine methylation in liver T

a,1 c,1 c d c b,*
Kechen Xu , Yuchao Sun , Baixiang Sheng , Yiwen Zheng , Xiaoli Wu , Keyang Xu
a
Clinical Laboratory Center, Wuyi First People's Hospital, Wuyi, 321200, Zhejiang, China
b
The Fourth Clinical Medicine School of Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China
c
The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, 322000, Zhejiang, China
d
Hangzhou Medical College, Hangzhou, 310053, Zhejiang, China

A R T I C LE I N FO A B S T R A C T

Keywords: N6-Methyladenosine (m6A) is the most abundant and important internal modification site of RNA methylation in
N6-methyladenosine viruses and eukaryotic. m6A RNA methylation plays key roles in the regulation of post-transcriptional gene
Methylation expression, including messenger RNA (mRNA), microRNA (miRNA) and long noncoding RNA (lncRNA). And
Messenger RNA m6A methylation regulates the various aspects of RNA metabolism, including structure, maturation, stability,
Noncoding RNA
splicing, export, translation and decay. Liver is a vital metabolic and digestive organ in the pathophysiological
Liver
processes. Recent studies suggested that m6A RNA modification highly regulates hepatic function and devel-
opment of liver diseases. Here, we aim to summarize the biological and clinical significance of m6A modification
in hepatic growth and hepatic disease including viral hepatitis, non-alcoholic fatty liver disease (NAFLD), and
liver cancer.

1. Introduction
Methylated RNA nucleotides widely exist in all kingdoms of life,
which play crucial roles in pathophysiological processes. Methylated
nucleosides in messenger RNA (mRNA) were firstly discovered from
Novikoff hepatoma cells in 1974 [1] and then several methylation
modifications in eukaryotic messenger RNA (mRNA) were identified,
including N7-methylguanosine at the cap, N6-methyl-2′-O -methyla-
denosine, 2′-O-methylation within the cap and the internal positions,
and internal N6-methyladenosine (m6A) and 5-methylcytosine [2].
Among them, m6A as a reversible modification site can be methylated
by “writers”, demethylated by “erasers”, and recognized by “readers”
[3]. Over 12,000 m6A sites are identified with a typical consensus in the
transcripts of more than 7000 human genes [4]. The sites of m6A
modification are identified to preferentially concentrate on two distinct
coordinates: around stop codons and within long internal exons [4].
Furthermore, m6A methylation has been widely reported in regulation
of mRNA [5] and non-coding RNA (ncRNA) [6].
m6A methylation can regulate mRNA at various stages, including
structure, maturation, stability, splicing, export, translation and decay
[7]. The dynamic m6A modification is recognized by YTH domain fa-
mily 2 (YTHDF2) to affect the stability of mRNA [8]. Human YTHDF1
recognizes m6A-modified mRNAs and then enhances mRNA translation
efficiency by interacting with initiation factors and ribosomes [9].
Furthermore, some researchers have found that YTHDF2 can selectively
bind m6A-methylated mRNA and control RNA decay in a methylation-
dependent manner [10]. As a splicing modulator, m6A affects mRNA
maturation by recruiting m6A regulators to pre-mRNA [11]. And m6A
methylation of mRNA can inhibit its tRNA accommodation and trans-
lation elongation rate [12]. Furthermore, m6A modification can reg-
ulate the structure and function of noncoding RNA, including tRNA,
rRNA, small nuclear RNA, miRNA and long non-coding RNA [6]. The
expression of m6A demethylase fat mass and obesity-associated protein
(FTO) can affect the steady state level of certain miRNAs, including up-
regulation of hsa-miR-6505-5p, hsa-miR-651-5p and hsa-miR-493-5p,
and down-regulation of hsa-miR-7-5p, hsa-miR-92a-1-5p and hsa-miR-
6769a-3p [13]. In reverse, miRNA145 can also modulate m6A levels by
targeting the 3′-untranslated mRNA region of the m6A binding protein
YTHDF2 [14]. In addition, m6A modification of lncRNA can induce the
proliferation, migration and apoptosis of cancer cells, including pan-
creatic cancer, ovarian cancer and hepatoma [15]. It was also reported
that the m6A methyltransferase-like 16 (METTL16) can methylate di-
verse cellular RNAs in HEK293 cells, including 8 pre-mRNAs, 355
mRNAs, 68 lncRNAs and others [16]. And RNA m6A methylation is
highly involved in hepatic cell differentiation, metabolic processes and
pathologic development [17]. m6A may be a potential therapeutic and
diagnostic target for hepatic diseases.
Here, we aim to summarize the functions and clinical implications

*
Corresponding author.
E-mail address: keyangxu@qq.com (K. Xu).
1
These authors contributed equally to this paper.

https://doi.org/10.1016/j.ab.2019.05.005
Received 26 December 2018; Received in revised form 1 May 2019; Accepted 6 May 2019
Available online 07 May 2019
0003-2697/ © 2019 Elsevier Inc. All rights reserved.
K. Xu, et al.
of RNA m6A modification in liver diseases.
2. m6A writers, erasers, readers
The m6A “writers”, a multicomponent protein complex, are con-
sisted of methyltransferase-like 3 (METTL3) [18], methyltransferase-
like 14 (METTL14) [19], Wilms’ tumor 1 associating protein (WTAP)
[20], KIAA1429 21 and RNA binding motif protein 15/15B (RBM15/
15B) [22]. METTL3, a S-adenosyl methionine (SAM)-binding subunit, is
associated with mRNA methylation [18], and the knockdown of
METTL3 can lead to the near-complete loss of m6A [23]. Interaction
between the METTL3 enzymatic subunit of the MTC and retarded
RNAPII has suggested that m6A modification is at least in part co-
transcriptional [24]. METTL3 and METTL14 can form a stable hetero-
dimer as the core of the methylase complex, and the knockdown of
METTL3 or METTL14 both reduces the m6A level due to existing me-
thyltransferase domains [25]. However, only METTL3 has catalytic
activity, and METTL14 just stabilizes METTL3 and its interaction with
the RNA molecule [26]. WTAP as a regulatory subunit is required for
the formation METTL3-METTL14 m6A methyltransferase complex,
which plays a crucial role in the regulation of gene expression and al-
ternative splicing [27]. And KIAA1429 is also required for the intact
catalytic activity of the methyltransferase complex [21]. Furthermore,
RMB15 and RMB15B, two other WTAP interactors, can recruit me-
thyltransferase complex to DRACH (the consensus sequence sites for
m6A methylation) [28].
The demethylated process of m6A “erasers” is mainly achieved
through two enzymes, including FTO [29] and alkB homolog 5
(ALKBH5) [30]. FTO can catalyze the demethylation of m6A residues
both in vitro and in vivo [29]. FTO-mediated demethylation is involved
in m6A modification in different cell contexts such as ultraviolet-in-
duced DNA damage response [31], adipogenesis [32], acute myeloid
leukemia cell transformation and leukemogenesis [33]. However, some
researchers believe that FTO exhibits weak demethylase activities to-
ward m6A in controlling mRNA stability and snRNA biogenesis, and N6,
2′-O-dimethyladenosine (m6Am) as a related nucleotide of m6A might
be real substrate for FTO [34,35]. Therefore, the effects of FTO on m6A
and m6Am are further analyzed, and FTO is indeed the demethylase of
m6Am. But the abundance of m6A is much higher than that of m6Am,
and the change of m6A is more obvious in FTO knockout cell lines [33].
Recent study have founded that FTO can demethylate internal m6A and
cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am
in snRNAs, and N1-methyladenosine (m1A) in tRNA [36]. In addition,
the overexpression of ALKBH5 can reduce the modification of m6A in
mRNAs [30] and small nucleolar RNAs [37]. Moreover, ALKBH5 has
been shown to promote the growth of breast cancer stem cells and
glioblastoma stem cells [6] though increasing the demethylation of 3′
untranslated region of m6A in NANOG [38] and FOXM1 mRNAs [39],
respectively.
m6A “readers” contain YTH domain family proteins, heterogeneous
nuclear ribonucleoproteins and on forth [40]. As the most important
members of m6A “readers”, YTH domain family proteins including
YTHDF1, YTHDF2, YTHDF3, YTHDC1 and YTHDC2, regulate various
gene expression through recognizing m6A modification [41]. The
translation of m6A-modified mRNA is promoted by YTHDF1 and
YTHDF3, the decay of m6A-modified mRNA is accelerated by YTHDF2
and YTHDF3, and the metabolism of m6A-modified mRNA in the cy-
toplasm is enhanced by YTHDF1, YTHDF2 and YTHDF3 [41]. The nu-
clear export and splicing of m6A-modified mRNA are mediated by
YTHDC1 [42,43]. YTHDC2 enhances the translation efficiency of its
targets and also decreases their mRNA abundance by selectively
binding m6A at its consensus motif [44].
In summary, the functions of m6A writers, erasers, readers were
presented in Fig. 1.
46
Analytical Biochemistry 578 (2019) 45–50
Fig. 1. The dynamic and reversible processes and consequences of m6A me-
thylation. RNA, the transcript of DNA, can be modified by m6A methylation.
The processes of m6A methylation are dynamic and reversible, methylated by
“writers”-METTL3, METTL14, WTAP, RBM, and demethylated by “erasers”-FTO
and ALKBH5, as well as recognized by “readers”-YTHDF1, YTHDF2, YTHDF3,
YTHDC1 and YTHDC2. m6A methylation plays critical roles on the regulation of
RNA metabolism, including splicing, export, translation, and stability.
3. m6A methylation and hepatic growth
The dynamic m6A modification affects tremendous genes expres-
sion, alternative splicing patterns and their related signaling pathways,
which may highly involve the functions of cells and tissues [34,45]. The
correlation between m6A and hepatic growth has been shown by pro-
filing transcriptome-wide m6A in porcine liver at three developmental
stages: newborn (0 day), suckling (21 days) and adult (2 years) [17]. In
liver tissue, about 33% post-transcripts of genes can occur as m6A
modification, with 1.33–1.42 m6A peaks per modified gene [17]. Me-
thylated post-transcripts of genes [5] can regulate several hepatic vital
functions such as growth, development, metabolism and protein cata-
bolic [17]. Furthermore, the various levels of m6A methylation are
required for liver biological processes at any particular stage [17]
(Table 1). Therefore, the methylation of RNA m6A has profound im-
pacts on hepatic growth.
4. m6A methylation and viral hepatitis
m6A modification plays a pro-viral or anti-viral role in the genomes
of RNA viruses and transcripts of a DNA virus [46]. For example, m6A
methylation can strongly enhance HIV-1 protein and mRNA expression,
and virus replication by recruiting the cellular YTHDF m6A “reader”
proteins [47,48]. HIV replication and release can be suppressed by
depletion of the METTL3 or METTL14 [48]. However, other study has
found that YTHDF1–3 proteins inhibit HIV-1 infection by blocking viral
reverse transcription and promoting degradation of viral RNA. There-
fore, the functions of m6A in promoting/suppressing HIV infections are
still controversial.
Hepatitis C virus (HCV) replication is negatively regulated though
m6A methylation mediated viral packaging inhibition [46]. YTHDF
proteins can inhibit HCV replication by re-localizing to lipid droplets
and sites of viral assembly, and its depletion significantly enhances the
production of HCV infectious viral particles [49]. Furthermore, the
infectious HCV particle production can also be suppressed by methyl-
transferases METTL3 or METTL14, and enhanced by de-methyl-
transferase FTO [49]. Interestingly, the presence of m6A modifications
in HCV RNA can be recognized to silence innate immune signaling
caused by retinoic acid-inducible gene I (RIG-I-like) receptor [50]. And
previous studies have suggested that invading pathogen nucleic acids
can be recognized and bound by cytoplasmic RIG-I-like receptors to
activate innate immune signaling [51].
HBV, a DNA virus, completes its life cycle by an RNA intermediate
K. Xu, et al.
Table 1
The functions of RNA m6A methylation in liver.
m6A Regulators Functions
Hepatic growth Unclear Methylation of post-transcripts of specific genes, including HOGA1, DPYS, FOLR1, GATM, CYP1A2, ALDH4A1, CYB5R3, SARDH,
GSTZ1, BAAT, GNMT, EDEM2, MAFK, UBALD2, etc.
Hepatitis B METTL3 Enhancing reverse transcription of the HBV and inhibiting the expression of HBc and HBs proteins
METTL14
Hepatitis C YTHDF Inhibiting HCV replication
METTL3 Inhibiting infectious HCV particle production
METTL14 Inhibiting infectious HCV particle production
FTO Enhancing infectious HCV particle production
NAFLD METTL3 Suppressing adipogenesis
FTO Promoting adipogenesis
HCC METTL3 Promoting growth, migration and colony formation of HCC cells
METTL14 Suppressing metastasis of HCC
YTHDF1 Promoting cell cycle progression and metabolism of HCC
YTHDF2 Promoting proliferation of HCC cells
CCA WTAP Promoting TNM stage, lymph node metastasis and vascular invasion of HCC
m6A methylation plays critical roles on the regulation of hepatic diseases, including virus replication, adipogenesis, growth and migration of HCC. Refs: references.
termed pregenomic RNA (pgRNA) [52]. The site of m6A methylation is
located within the epsilon stem loop structure of the 5′and 3′ termini of
the pgRNA as well as the 3′ terminus of all HBV mRNAs [52]. These
modifications are required for reverse transcription of pgRNA and ef-
ficient destabilization of all HBV transcripts [52]. Depletion of METTL3
or METTL14 can reduce reverse transcription of the pgRNA, but lead to
up-regulation of HBc and HBs proteins, suggesting that m6A methyla-
tion of HBV transcripts suppresses HBV proteins expression [52]. De-
pletion of either YTHDF2 or YTHDF3 significantly promoted the ex-
pression of the HBV proteins HBs and HBc, suggesting that the YTHDF
proteins also suppress HBV protein expression [52]. Furthermore, m6A
modification in HBV RNA is critical to modulate the viral life cycle and
its related hepatitis by regulating gene expression and reverse tran-
scription [52].
Apparently, present studies show that m6A modification negatively
regulates HBV/HCV infections. Therefore, m6A modification has a ne-
gative impact on hepatic virus infection but its roles in regulating other
hepatitis viruses need to be further clarified. Especially, because the
functions of m6A in promoting/suppressing HIV infections are still
controversial, its roles in regulating other hepatitis viruses maybe are
different.
5. m6A methylation and non-alcoholic fatty liver disease (NAFLD)
Non-alcoholic fatty liver disease (NAFLD) is characterized by he-
patic steatosis with no history of significant alcohol use or other known
liver disease [53], and hepatic steatosis is caused by metabolic dysre-
gulation of de novo lipogenesis, fatty acid uptake, fatty acid oxidation,
and triglycerides export [54]. Currently, m6A demethylation has been
shown to play a vital role in the regulation of adipogenesis. FTO posi-
tively regulated adipogenesis by m6A demethylation, but METTL3 ne-
gatively correlated with adipogenesis by m6A methylation [55,56].
More importantly, FTO can promote adipogenesis by inhibiting the
Wnt/β-catenin signaling pathway in porcine intramuscular pre-
adipocytes [57], and enhancing RUNX1T1 mediated adipocyte pro-
liferation [58], as well as promoting Cyclin A2 (CCNA2) and cyclin
dependent kinase 2 (CDK2) mediated mitotic clonal expansion at the
early stage of adipocyte differentiation [59]. And YTHDF2 can inverse
FTO-mediated adipogenesis by m6A methylation [59]. In humans, a
significant increase in FTO mRNA and protein levels has been found in
the liver of NAFLD patients [60]. Elevated levels of FTO mRNA and
protein can be also found in a NAFLD rat, which are involved in oxi-
dative stress and lipid deposition [61]. All in all, FTO may be a pro-
misingly therapeutic target to improve hepatic steatosis.
47
Analytical Biochemistry 578 (2019) 45–50
Refs
[17]
[52]
[49]
[56]
[55]
[63]
[64]
[65]
[14]
[67]
6. m6A methylation and liver cancer
Liver cancer is the sixth common cancer worldwide, mainly in-
cluding hepatocellular carcinoma (HCC) and cholangiocarcinoma
(CCA) [62]. Recent studies have suggested that HCC is associated with
m6A “writers”, such as METTL3 and METTL14. METTL3 is up-regulated
in HCC, and it can promote the growth, migration and colony formation
of HCC cells in vitro and facilitate tumorigenicity, growth and lung
metastasis of HCC in vivo through repressing suppressor of cytokine
signaling 2 (SOCS2) mRNA [63]. SOCS2 mRNA stability can be
downregulated through m6A-YTHDF2-dependent pathway [63]. In ad-
dition, overexpression of METTL3 can predict a poor prognosis of pa-
tients with HCC [63]. Down-regulation of METTL14 plays an important
role in tumor metastasis, and it is regarded as a poor prognostic in-
dicator for recurrence-free survival of HCC [64]. METTL14-dependent
m6A methylation positively regulates the primary microRNA 126 pro-
cess by the microprocessor protein DGCR8, whereas microRNA 126 can
suppress the inhibiting effect of METTL14 in tumor metastasis [64].
Moreover, m6A “readers”, mainly including YTHDF1 and YTHDF2, are
also associated with HCC. YTHDF1 is remarkably overexpressed in
HCC, and has been shown to positively correlate with pathology stage
by promoting HCC cell cycle progression and metabolism [65].
YTHDF2 can enhance mRNA degradation cells by identifying mRNA
m6A sites, resulting in promoting proliferation of HCC [14]. However,
miR-145 can suppress proliferation of HCC cells by down-regulating
YTHDF2 through targeting its mRNA 3′UTR [14]. Therefore, WTAP
may be a promising target for the treatment of CCA.
WTAP is associated with cell proliferation and apoptosis in glio-
blastoma [66]. WTAP is also overexpressed in CCA, which plays a po-
sitive role in CCA TNM stage, lymph node metastasis and vascular in-
vasion [67]. The migration, invasion and tumorigenicity of CCA cells
can be inhibited by WTAP siRNA, but the proliferation is insignificantly
affected by WTAP siRNA or overexpression [67]. Moreover, WTAP
could promote the expression of metastasis-related genes, including
MMP7, MMP28, and Muc1 [67]. Therefore, WTAP may be a promising
target for the treatment of CCA.
7. Clinical significance of m6A
The m6A methylation of RNAs plays important roles in maintenance
and progression of liver diseases, and its invention may be a potentially
effective treatment. For example, the curcumin has protective effects in
the LPS-induced liver injury and disruption of hepatic lipid metabolism,
probably owning to the increasing level of m6A RNA methylation [68].
Furthermore, recent studies have suggested that betaine has the hepa-
toprotective effects in NAFLD through improving hepatic m6A
K. Xu, et al.
methylation status by decreasing FTO expression [69,70], and recti-
fying the decreased m6A methylation in adipose tissue by promoting
FTO expression [71]. However, natural products like curcumin and
betaine, have various effects such as antioxidant, anti-inflammatory,
and anticancer properties [72,73]. Therefore, it is difficult to evaluate
the role and effect of m6A modification in treatment of related liver
diseases by curcumin or betaine.
Moreover, inhibition of the RNA demethylase, FTO, can suppress
the vital activity of HBV/HCV and their hepatitis progression [49,52].
Therefore, FTO inhibitors may provide potential therapeutic agents for
viral hepatitis such as rhein, αKG analogues, CHTB, meclofenamic acid,
4-chloro-6-(6′-chloro-7′-hydroxy-2′,4′,4′-trimethyl-
Chroman-2′-yl), benzene-1,3-diol (CHTB), N-(5-Chloro-2,4-dihy-
droxyphenyl)-1-phenylcyclobutanecarboxamide (N-CDPCB), nafamo-
stat mesylate, and Radicicol and on forth [74–82]. However, the clin-
ical application or off target effects of these inhibitors may be further
identified.
Besides, S-adenosylhomocysteine (SAH) can be hydrolyzed by SAH
hydrolase into adenosine and homocysteine, and it is reported as a
competitive inhibitor of adenosylmethionine-dependent methyl-
transferases [83]. 3-Deazaadenosine (DAA), a SAH hydrolysis inhibitor,
can inhibit m6A modification of mRNA substrates [84]. Furthermore,
DAA and its analogues can inhibit virus replication by editing m6A-
containing mRNA [85,86]. In addition, IGF-1 can direct nuclear 26 S
proteasome mediated WTAP degradation by IGF-1 receptor-phospha-
tidylinositol 3-kinase-Akt signaling [87], and it may be a novel ther-
apeutic agent for treatment of CCA.
8. Conclusions and perspectives
In conclusion, m6A modification is highly involved in the regulation
of hepatic growth and hepatic diseases by modifying mRNAs and
ncRNAs (Table 1). m6A-mediated gene post-transcriptional modifica-
tion plays physiological roles in regulation of proteins production, cir-
cadian rhythms, cell cycle, reprogramming, cell differentiation, state
transitions, stress responses and cancer [88]. And the expressions of
ncRNAs (miRNAs, lncRNAs and circRNAs) are closely associated with
the development of liver diseases, and they are regarded as potential
targets for early diagnosis and therapeutic intervention [89]. In addi-
tion, RNA m6A methylation has multi-functions in regulation of hepatic
growth, HBV and HCV replication, adipogenesis, as well as HCC and
CCA progression. Many promising agents can be used to target m6A in
the treatment of liver diseases such as FTO inhibitors, SAH hydrolysis
inhibitors, WTAP inhibitors and other natural productions.
In summary, methylated m6A level may be potential indicator for
early diagnosis of hepatitis and liver cancer, and m6A may be a pro-
mising target for the treatment of liver diseases. In addition, the func-
tion of m6A in promoting/suppressing viral infections exists different
views. Therefore, the pro/antivirus effects of m6A methylation on he-
patic viruses need to be identified. Recent reports have suggested that
m6A RNA methylation plays important roles in self-renewal, tumor-
igenesis and radio-resistance of glioblastoma stem cells by METTL3
[90,91]. The effect of m6A on liver cancer stem cells needs to further be
identified. Furthermore, the effects of ncRNAs by methylated m6A on
liver diseases remain unclear. The further studies on methylated
ncRNAs would help us to better understand their roles in hepatic dis-
eases or other diseases.
Authors contribution
Writing-Original Draft preparation, Kechen Xu and Yuchao Sun,
Baixiang Sheng, Yiwen Zheng, Xiaoli Wu; Writing-Review & Editing,
Keyang Xu.
48
Analytical Biochemistry 578 (2019) 45–50
Acknowledgments
None.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ab.2019.05.005.
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