The Emerging Roles of N6‑Methyladenosine Modification in

Hepatocellular Carcinoma

Ruibo Liu

School of Chemistry and Life Sciences, Beijing University of Technology, Beijing, China

Keywords: N6‑methyladenosine (m6A), Hepatocellular Carcinoma, Non‑Coding RNAs.

Abstract: In terms of cancer-related deaths, hepatocellular carcinoma (HCC) ranks high on a global scale. The molecular

processes that cause HCC are still a mystery, even though many treatments have been developed for the

disease in recent years. In mammals, N6-methyladenosine (m6A) methylation is one of the most significant

RNA modifications. It is thought to regulate RNA metabolism and gene expression. Numerous regulatory

variables govern this procedure. Two examples are methylase and demethylase. ncRNA, including circular

RNAs circRNAs, lncRNAs, and miRNAs, are important players in the development and spread of tumours.

They also regulate mRNA production, epigenetic alterations, and other biological processes. The diverse roles

of m6A regulators in HCC include ferroptosis and metabolic reprogramming. In addition, this review

examines inhibitors that target m6A enzymes as potential therapeutic targets for HCC, along with the present

research status of m6A gene editing methods.

1 INTRODUCTION

In the several types of liver malignancies,

hepatocellular carcinoma (HCC) accounts for 75–

85% of all cases and has a high mortality rate. After

lung cancer and stomach cancer, it is the third

leading cause of cancer-related death globally. At

this time, the most effective method for treating

early-stage HCC is surgery. Unfortunately, many

HCC patients already have advanced disease when

they are diagnosed. The first-line chemotherapeutic

treatment for advanced HCC is sorafenib, a kinase

inhibitor. Still, cancer metastasis and drug

resistance affect a small percentage of people. A

better understanding of the molecular pathways that

cause HCC is crucial for effective diagnosis,

therapy, and prevention of the disease. In

epigenetics, the DNA sequence is not changed but

gene expression is regulated through heritable

mechanisms. These processes incorporate

chromatin remodeling, non-coding RNA (ncRNA),

DNA methylation, and histone modifications.

Adenosine undergoes methylation at the sixth

position, resulting in N6-methyladenosine (m6A),

an epigenetic modification. This epigenetic

alteration is found in the vast majority of eukaryotic

RNAs. Enzyme complexes known as m6A

methyltransferases (writers) and m6A demethylases

(erasers) control the reversible and dynamic m6A

modification. Readers are proteins that recognise

and carry out m6A's biological functions. Not only

that, but these regulators have an effect on the

expression and function of circRNA, lncRNA, and

microRNA. Recent studies have shown that

metabolic reprogramming, cancer progression, and

m6A and ncRNA interaction dysfunction can all

play a role in cancer. Therefore, understanding the

etiology, prognosis, and therapy of HCC requires

better clarification of the link between m6A

alteration and ncRNA. This review will summarize

recent findings on the role of m6A modification in

the initiation and development of HCC and

elucidate the impact of the interaction between

ncRNA and m6A in HCC. Lastly, this review will

discuss the therapeutic possibilities of m6A

modifiers as biomarkers and targets for HCC

therapy.

2 THE REGULATORS RELATED

TO M6A

An epigenetic alteration known as m6A attaches a

methyl group to the N6 position of adenine and is

Liu, R.

The Emerging Roles of N6-Methyladenosine Modiﬁcation in Hepatocellular Carcinoma.

DOI: 10.5220/0014486200004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 261-266

ISBN: 978-989-758-789-4

261

quite common. Typically, it is found in the 3'-

untranslated regions (UTRs) of mRNA, close to the

stop codon regions, as well as in 5'-UTRs.

Additionally, m6A is significantly abundant in

ncRNAs, including lncRNAs, miRNAs, and circular

RNAs (Xu et al. 2024). The regulation of m6A is

mediated by a reversible methylation system that

involves three types of proteins: the writer, the eraser,

and the reader.

2.1 Writers

Writers are primarily responsible for the methylation

of m6A with a protein complex. Its main kernel

components, are METTL3 and METTL14. METTL3

is regarded as the main enzyme exerting

methyltransferase activity in the protein complexes.

METTL14 can bind with METTL3 to form a

METTL3-METTL14 complex, which can stabilize

the structure of METTL3. METTL14 improves the

complex's substrate selectivity via binding to RNA.

Although WTAP does not have any enzyme activity,

it does serve as a junction protein that helps

METTL3-METTL14 target RNA. Furthermore, the

amount of components of the writers have been

found: METTL16, a protein that is related to virilizer-

like m6A methyltransferase (KIAA1429;

alternatively called VIRMA). In order to identify the

3' hairpin of U6 snRNA and MAT2A pre-mRNA,

which encode SAM synthase, METTL16 can act as a

catalyst. Specifically, VIRMA directs preferential

mRNA methylation at the 3'UTR and close to stop

codons by enlisting catalytic core components (Ma et

al. 2024).

2.2 Erasers

m6A is a vital reversible change regulated by both

methylase writers and demethylase erasers. Fat mass

and obesity-associated proteins (FTO) can facilitate

the demethylation of the m6A site on RNA, resulting

in alterations to the structure and function of RNA.

Research indicates that FTO significantly influences

the proliferation and spread of HCC cells as well as

lipid synthesis. AlkB homolog 5(ALKBH5) is

predominantly situated in the nucleus and

substantially influences mRNA nuclear export and

metabolism (Xu et al. 2024, Ma et al. 2024).

2.3 Readers

After RNA has been written by writers and cleared by

erasers, methylation modifications of the m6A on it

are recognized by readers and mediate RNA fate. A

significant number of readers have been found in

mammals, such as the YT521-B homology (YTH)

domain-containing protein family. Within the

YT521-B homology (YTH family, YTHDF1

promotes the translation of m6A-modified mRNAs,

YTHDF2 facilitates mRNA degradation, and

YTHDF3 collaborates with YTHDF1 and YTHDF2,

respectively (Ma et al. 2024).

3 THE NCRNA-MEDIATED

REGULATION OF M6A

MODIFICATION IN

HEPATOCELLULAR

CARCINOMA

ncRNAs, which mostly consist of miRNAs,

lncRNAs, and circRNAs, are a subset of RNA

molecules that are capable of transcription but do not

include protein-coding sequences. ncRNAs regulate

gene expression and participate in several biological

processes, especially in tumor malignancies.

Evidence suggests that m6A alterations and ncRNAs

interact in ways that contribute to the

pathophysiology of numerous illnesses and

malignancies. The abnormal expression of ncRNAs

can contribute to hepatocellular carcinoma

progression by modulating the expression levels of

m6A regulators, exhibiting both cancer-promoting

and cancer-inhibiting effects. However, the process is

intricate, and the precise mechanism remains unclear.

Therefore, additional research into the role of

ncRNAs and m6A alteration in tumor growth is

essential.

3.1 m6A and miRNAs in

Hepatocellular Carcinoma

RNA polymerase II produces the primary miRNAs

which are small, non-coding RNAs that consist of 20–

24 nucleotides. Next, precursor miRNAs (pre-

miRNAs) are created when these primary miRNAs

are cleaved in the nucleus. Once in the cytoplasm, the

pre-miRNA continues its maturation into a fully

functional miRNA. In order to degrade or repress the

translation of mRNA, this mature miRNA is bound to

the RNA-induced silencing complex. Research shows

that microRNAs (miRNAs) play a role in the

progression of liver cancer by modulating m6A

writers. Specifically, miR-362-3p/miR-425-5p is a

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miRNA that reduces the expression of the m6A

writing enzyme ZC3H13, which has been identified

as an oncogene in hepatocellular carcinoma (Cui et al.

2020). The Wnt/β-catenin signaling pathway is

inhibited and an aggressive tumor phenotype is

caused by miR-186, another microRNA, which

reduces METTL3 expression. Furthermore,

microRNAs regulate m6A readers to impact HCC

progression. One example is the effect of

overexpressed miR-145 on hepatocellular carcinoma

cells, which causes a rise in m6A levels and a

downregulation of YTHDF2 expression. This is

because miR-145 targets the 3'-UTR of YTHDF2

mRNA. Hepatocellular carcinoma cells show a

marked decrease in miR-145 expression and an

increase in m6A levels when 3'-UTR of YTHDF2

mRNA is targeted. The ability of microRNAs to

influence m6A writers and readers means that they

may influence the advancement of cancer (Cui et al.

2020, Qiu et al. 2024).

3.2 m6A and lncRNA in Hepatocellular

Carcinoma

LncRNAs interact with DNA, RNA, and proteins on

multiple levels to regulate gene expression. Their

length exceeds 200 nucleotides. They can disrupt

transcription factor binding, serve as scaffolds for

transcriptional regulators, or sequester miRNAs.

Moreover, lncRNAs can directly bind to proteins,

influencing their activity, stability, and cellular

localization, thereby impacting signaling pathways.

For instance, the upregulation of LINC01273 in HCC

enhances tumor growth and resistance to sorafenib by

modulating the miR-600/METTL3 axis to regulate

m6A levels. Conversely, the downregulation of the

tumor suppressor lncRNA AC1156199 in HCC

impedes oncogene expression, cell proliferation, and

invasion by disrupting the assembly of the m6A

methyltransferase complex through WTAP targeting.

Additionally, lncRNA FTO-IT1 stabilizes FTO

mRNA, promoting HCC cell proliferation and

glycolysis by reducing m6A modification of

glycolysis-related genes through recruitment of the

ILF2/ILF3 protein complex (Qiu et al. 2024).

3.3 m6A and circRNAs in

Hepatocellular Carcinoma

CircRNA is a circular, single-stranded RNA molecule

that ranges in length from 100 nucleotides to over 4

kilobases. Its circular shape enhances its stability,

making it highly resistant to degradation by nucleic

acid exonucleases. CircRNA functions as a molecular

sponge, effectively sequestering miRNAs, and also

interacts with a variety of RNA-binding proteins,

potentially altering their typical functions. Lin et al

discovered that RERE overexpression boosts cell

viability and invasiveness while decreasing

apoptosis. RERE facilitates the progression of

hepatocellular carcinoma by increasing the

expression of ZC3H133, which subsequently

mediates the m6A modification of GBX2. 2) . Liu et

al noted a marked reduction in circRNA-GPR137B

expression in hepatocellular carcinoma tissues.

GPR137B can suppress cancer cell proliferation by

adsorbing miR-4739 to enhance FTO (Lin et al. 2022,

Qiu et al. 2024).

4 THE ROLE AND

SIGNIFICANCE OF M6A

METHYLATION IN

HEPATOCELLULAR

CARCINOMA

In order to meet the needs of survival, HCCs have

evolved a variety of mechanisms such as metabolic

reprogramming and anti-ferroptosis. Metabolic

reprogramming enables HCC to better adapt to the

tumor's special microenvironment. Anti-ferroptosis

can reduce cell death caused by abnormal iron ions

and lipid-reactive oxygen species. Innovative

approaches to treating HCC can be derived from a

more thorough comprehension of these pathways.

4.1 m6A Modification in Metabolic

Reprogramming

One of the significant features of tumor cells is their

metabolic reprogramming, which fulfills their

biosynthetic, bioenergetic, and redox requirements.

The metabolism of glucose has been most intensively

studied. To promote tumor growth, cancer cells

frequently utilize glycolysis to break down glucose

and maintain their metabolic energy, referred to as the

Warburg effect (Warburg, 1925). By shifting glucose

synthesis from oxidative phosphorylation to aerobic

glycolysis, the Warburg effect promotes cancer

growth via the MTORC1 pathway. Overexpression of

METTL3 enhances glycolysis in HCC cells, which

speeds up cancer progression, according to the

current study. Two mechanisms have been identified

so far: (1) METTL3 can cause metabolic

The Emerging Roles of N6-Methyladenosine Modiﬁcation in Hepatocellular Carcinoma

263

reprogramming by way of overexpression of pyruvate

dehydrogenase kinase 4 (PDK4). METTL3 can

accelerate the translation of PDK4 as well as increase

the sexual stabilization of mRNAs by recruiting the

YTHDF1/eEF-2 complex and IGF2BP3 by adding an

m6A modification to the 5'UTR of PDK4. PDK4 has

a critical role in glycolysis. Overexpression of PDK4

shifts carbon flow from oxidative phosphorylation to

glycolysis. (2) The METTL3 m6A subunit

methylates HIF-1α to improve glycolysis, while the

Hepatitis B Virus X Protein Interacting Protein

(HBXIP) acts as a positive regulator of METTL3.

Additionally, it is crucial for the progression of lipid

metabolism HCC. Scientists have found that

METTL5 is essential for the progression of HCC in

an in vitro model. Reduced translation of genes

implicated in fatty acid metabolism and defective 80S

ribosome assembly is observed in the absence of

METTL5-m6A-methylated 18S rRNA (Peng et al.

2022).

4.2 m6A Modification in Ferroptosis

The buildup of intracellular iron-dependent lipid

peroxides—linked to iron ions, lipid reactive oxygen

species (L-ROS), and glutathione peroxidase 4

(GPX4)—defines ferroptosis, a non-apoptotic kind of

cell death. In order to keep up with the demands of

fast growth, tumor cells typically have a higher

metabolic activity and iron need. For example,

increased synthesis of PUFA-PL (unsaturated ether

phospholipids) in cancer cells leads to destabilization

of the iron pool, thereby increasing susceptibility to

Ferroptosis. In addition, disturbances in lipid

metabolism in tumor cells can also trigger

Ferroptosis. However, hepatocellular carcinoma cells

usually show some resistance to Ferroptosis. Some

recent studies have shown that the m6A mechanism

affects the susceptibility of hepatocellular carcinoma

cells to Ferroptosis. The researchers found that

hepatocellular carcinoma tissues exhibiting elevated

expression of METTL16 and SENP3 correlated with

worse prognostic outcomes. The m6A mutation of

METTL16 resulted in elevated production of SENP3,

which, by stabilising LTF proteins by de-

SUMOylation, augmented their ability to sequester

free iron. The METTL16-SENP3-LTF axis is

implicated in modulating ferroptosis and

hepatocellular carcinoma progression, as indicated by

these studies (Wang et al. 2024). Shuwei Chen and

colleagues discovered that WTAP can m6A-modify

circCMTM3 to facilitate iron mortality in

hepatocellular carcinoma (HCC). CircCMTM3

reduces ferroptosis by engaging IGF2BP1 to enhance

the stability of PARK7, a crucial antioxidant protein,

in hepatocellular carcinoma (HCC). A study by Z.

Fan et al. revealed that METTL14 specifically targets

m6A methylation of the SLC7A11 mRNA 5'UTR,

and the methylation-altered SLC7A11 mRNA is

subsequently recognised by YTHDF2 (readers) for

destruction. The depletion of SLC7A11 ultimately

enhances ROS production and triggers Ferroptosis

(Chen et al. 2023).

5 THE CLINICAL

APPLICATIONS OF M6A

MODIFICATION IN

HEPATOCELLULAR

CARCINOMA

Considering the various pathways outlined in several

research related to hepatocellular carcinoma, the

modification of m6A RNA is anticipated to serve as

a significant target for prognosis and treatment of the

disease. There are many aberrant m6A regulators that

have been identified as oncogenic factors, and

targeting these factors for the treatment of HCC has

been given high hopes. Small molecule inhibitors,

m6A gene editing systems, and other approaches can

all be utilized as targeting strategies.

5.1 m6A Small Molecule Inhibitors

m6A small molecule inhibitors are compounds that

inhibit the catalytic function or interfere with protein-

RNA interactions by specifically binding to the active

sites of m6A-related regulatory enzymes (writers,

erasers, and readers). These inhibitors regulate the

expression of downstream oncogenes or tumor

suppressor genes by reducing or enhancing the level

of m6A modification. Several small molecule

inhibitors have been developed to target m6A

enzymes (METTL3, ALKBH5, and IGF2BP1) by

disrupting their catalytic function or structure.

Quercetin, an inhibitor of METTL3, blocks the

adenosine pocket of SAM, leading to enzyme

inhibition and suppression of HCC cell proliferation

(Du et al. 2022). Cucurbitacin B (CuB), an inhibitor

of IGF2BP1, induces apoptosis in HCC cells through

a metastable inhibitory effect, enhances immune cell

infiltration, and reduces PD-L1 expression (Ma et al.

2024).

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5.2 m6A Gene Editing Systems

In addition, m6A gene editing systems have received

widespread attention. The traditional CRISPR system

achieves gene editing by targeting DNA, while the

m6A editing system uses catalytically inactive Cas

proteins (such as dCas9 or dCas13) as positioning

modules, and m6A regulatory factors such as writers

and erasers as action modules, which are guided to the

target RNA sequence through sgRNA to methylate

the RNA. The CRISPR/Cas9 m6A editing system

allows for site-specific m6A modification through the

modification of the m6A "writer" complex. A dCas9

mutant with the RNA-targeted catalytic domains of

METTL3 and METT14 (M3-M14) can have a fusion

protein with the M3 and M14 domains linked to its N-

terminus. By doing so, particular RNA sequences can

be targeted by the dCas9-M3-M14 complex. Using

this technique, Liu et al were able to cause RNA

degradation by installing an m6A alteration at the

3′UTR of ACTB mRNA and a m6A modification at

the 5′UTR of Hsp70, both of which enhance protein

translation (Liu et al. 2019). A different study created

dCas13b-YTHDF1 and dCas13b-YTHDF2 proteins

by joining the N-terminal part of YTHDF1 or

YTHDF2 with inactivated dCas13b. Regardless of

the target RNA's m6A modification state, these

proteins can attach to particular RNA targets through

complementary sequences on the gRNA (Rauch et al.

2018, Chen & Wong 2020).

6 CONCLUSION

m6A is among the most prevalent RNA epigenetic

alterations. Its impact on HCC has been examined in

recent years. This review examines the controls and

roles of m6A alteration in hepatocellular carcinoma

(HCC). Writers, erasers, and readers are the primary

determinants of m6A alterations; the aberrant

expression of m6A enzymes precipitates

hepatocellular carcinoma (HCC). Nonetheless, the

functions of certain m6A regulators, such as FTO,

remain contentious. Researchers believe that the

inconsistency may stem from cancer heterogeneity

and cellular context. ncRNA also can effects through

the interaction of the m6A. ncRNA through

interaction with the erasers and writers, which affects

m6A modification and the readers in regulating

signaling pathways and metabolic processes

downstream of ncRNAs. Also, m6A modification can

affect ncRNA expression. This would complicate the

effect of m6A on HCC. The fact that Azza and other

medications that target DNA methylases or histone-

modifying enzymes have received clinical approval

for the treatment of cancer is well-known. Despite

various attempts, treatments targeting m6A alteration

in HCC have not been evaluated. In addition, gene

editing systems targeting m6A are a potential therapy

that can directly target RNA to modify it. Overall,

therapies targeting m6A have potential clinical

applications, and combining them with

immunotherapy is essential to improve clinical

outcomes in HCC. However, the current

understanding of m6A modifications is still in its

infancy, and more valuable evidence on the effects of

m6A modulation patterns on ncRNA biosynthesis

and function deserves further investigation in future

studies.

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