Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 6, pp. 650-670 © Pleiades Publishing, Ltd., 2025.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 6, pp. 695-719.

650

REVIEW

The Role of m

A-RNA Methylation

in the Development, Progression, and Treatment

Response of Bladder Cancer

Tatiana Sinyagovskaya

1,a

*, Yuliya Li

, Natalya Vinchevskaya-Khmelnitskaya

Aisha Agabalaeva

, Natalia Ponomareva

, Sergey Brezgin

, Irina Goptar

Vladimir Chulanov

3,4

, Alim Dymov

, Andrey Vinarov

, Dmitry Kostyushev

1,4,5

and Anastasiya Kostyusheva

Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical

and Vector-Borne Diseases, Sechenov First Moscow State Medical University, 119991 Moscow, Russia

Institute for Urology and Reproductive Health,

Sechenov First Moscow State Medical University, 119991 Moscow, Russia

Department of Infectious Diseases, Diseases,

Sechenov First Moscow State Medical University, 119991 Moscow, Russia

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

Faculty of Bioengineering and Bioinformatics,

Sechenov First Moscow State Medical University, 119991 Moscow, Russia

e-mail: tsv.relentless@gmail.com

Received December 11, 2024

Revised April 20, 2025

Accepted April 23, 2025

Abstract— Bladder cancer (BCa) remains a significant clinical challenge because of high recurrence rates

and variable response to immunotherapy and chemotherapy. Recent studies have highlighted the role of

N6-methyladenosine (m

A) modification in RNA in the regulation of various cellular processes, including tumor

progression and drug resistance. The review examines the impact of m

A methylation on BCa pathogenesis,

with a particular special focus on the role of m

A pathway factors and m

A-modified RNAs in tumorigene-

sis, proliferation, invasion, and migration of cancer cells. The mechanisms of m

A-mediated chemotherapy

resistance in BCa cells are discussed, including single nucleotide polymorphisms in m

A-associated patterns.

Significant advances in the high-throughput analysis of m

A methylation have enabled development of novel

A-based biomarkers for the risk assessment, early diagnostics, and prediction of relapse and treatment

response in BCa. The review outlines the prospects of the m

A-based molecular diagnostics in BCa.

DOI: 10.1134/S0006297924604441

Keywords: m

A, N6-methyladenosine, bladder cancer (BCa), biomarker, prognosis

* To whom correspondence should be addressed.

INTRODUCTION

Bladder cancer (BCa) remains the most common

malignancy of the urinary tract. According to the

World Health Organization (WHO) data for 2022, BCa

ranks 6th in prevalence among cancers in men, 9th –

among both sexes, and 13th – in global mortality rates

[1]. In the Russian Federation, it holds the 6th position

in prevalence among cancers in men, 9th – among

both sexes, and 16th – in mortality rates among all

oncological diseases [1].

BCa is categorized according to the depth of in-

vasion into the muscular layer and is subdivided into

non-muscle-invasive BCa (NMIBC) and muscle-invasive

BCa (MIBC). Approximately 75% of newly diagnosed

cases are NMIBC. The overall 5-year survival rate

for NMIBC exceeds 70%, but remains below 6% for

MIBC [2]. The ‘gold standard’ treatment for NMIBC

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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

is transurethral resection of bladder tumor (TURBT),

followed by adjuvant intravesical chemotherapy or im-

munotherapy. MIBC requires more aggressive therapy,

such as trimodal treatment, which combines TURBT,

radiotherapy, and systemic chemotherapy. The recur-

rence rate for NMIBC varies widely, with the reported

rates between 40 and 90% [3-5]. The rate of NMIBC

progression to MIBC ranges from 5 to 55% [6].

One of the cancer biomarkers that has attracted

significant attention is N6-methyladenosine (m

A), a

dynamic and reversible epitranscriptomic RNA mod-

ification involved in various cellular processes. m

methylation occurs exclusively within the context-

specific sequences known as DRA*CH motifs (where

D  =  A, G, or U; R  =  A or G; H  =  A, C, or U). A unique

distribution and density of m

A in RNA molecules is

typically referred to as an m

A pattern. The distribu-

tion of m

A patterns across RNA transcripts is non-

random; they are highly conserved but can vary de-

pending on the RNA type and cellular context, tissue,

developmental stage, and disease. m

A patterns can

be assessed within individual RNA transcripts or in

the entire transcriptome. DRA*CH and alternative m

consensus motifs are typically evolutionary conserved

and appear mostly near 3′- and 5′-untranslated regions

(UTRs), whereas their presence in coding regions is

less frequent [7]. m

A methylation can occur in spe-

cific mRNAs or non-coding RNAs (ncRNAs), affecting

their stability, splicing, translation, and decay. The

presence, location, and density of m

A modifications

in individual transcripts can determine their fate and

functions. Together, these factors influence a complex

network of m

A-mediated regulation, which impacts

all aspects of RNA metabolism.

Abnormal m

A methylation patterns have been

observed in various pathological conditions, such as

neurodevelopmental and neurological disorders [8, 9],

cardiovascular diseases [10,  11], viral infections [12],

metabolic disorders [13], and immune response [14].

Altered m

A methylation patterns have been observed

and extensively studied in various cancers, including

breast, lung, liver, and colorectal cancers, as it was

found that specific m

A modifications correlate with

tumor progression, metastasis, and patient progno-

sis [15].

A modifications have been identified as a sig-

nificant factor in cancer biology, affecting various as-

pects of tumorigenesis and cancer progression. m

methylation of mRNA serves as a gene regulatory

mechanism and influences proliferation and inva-

sion of tumor cell, as well as tumor immune evasion

and metastasis, by coordinating and modulating gene

expression in a reversible and highly dynamic man-

ner [16].

The review discusses the role of m

A methylation

in the development of BCa, the pro- and anti-oncogenic

functions of m

A pathway factors, m

A methylation of

mRNAs and ncRNAs related to oncogenes and tumor

suppressors, and their potential diagnostic and prog-

nostic significance. We also explored how m

A meth-

ylation contributes to the development of resistance

to chemotherapeutic agents.

CURRENT CHALLENGES IN THE DIAGNOSTICS

AND TREATMENT OF NMIBC

At present, BCa diagnosis is confirmed exclusive-

ly by histological examination of biopsies obtained

during the TURBT. Urine cytology is used to detect ex-

foliated tumor cells. This method is highly sensitive in

the detection of carcinoma in situ and high-grade (G3)

tumors, but is much less efficient in the identification

of low-grade (G1) tumors [17]. Thus, the sensitivity of

urine cytology test in detecting carcinoma in situ var-

ies widely, ranging from 28 to 100% [18]. The positive

result indicates the presence of transitional cell carci-

noma, which can originate in any part of the urinary

tract, but the negative result does not rule out the

possibility of BCa [19].

Due to the low sensitivity of urine cytology in

the diagnostics of low-grade BCa, numerous methods

for analyzing BCa molecular markers have been de-

veloped. However, none of these markers have been

widely adopted as a standard with a high prognostic

value [20].

Currently, biomarkers can be categorized into two

distinct groups: (i)  diagnostic markers, which help con-

firm a BCa diagnosis; (ii)  prognostic markers, which

assess the risks of disease progression and recurrence

in patients already diagnosed with BCa [21].

Five diagnostic test systems have been approved

by the FDA: NMP22 test kit, NMP22 BladderChek Test,

BTA TRAK, BTA stat, and UroVysion [22,  23]. Despite

the established efficacy of these test systems in the

identification of BCa markers, their application in clin-

ical practice remains limited [24].

At present, there are no urinary BCa biomarkers

that can substitute for cystoscopy or reduce the fre-

quency of cystoscopic examinations [20], indicating an

ancillary role of non-invasive urine analysis methods

conducted prior to definitive diagnosis.

A standard of treatment for NMIBC is transure-

thral bladder resection followed by adjuvant chemo-

or immunotherapy. The two techniques for bladder

resection are TURBT (resection of the bladder wall

with the tumor as a single block) or piecemeal tumor

resection [19].

Current clinical guidelines recommend a single

instillation of a chemotherapeutic agent (most com-

monly, mitomycin  C, epirubicin, or pirarubicin) after

TURBT, which has been proven to significantly reduce

SINYAGOVSKAYA et al.652

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

the recurrence rates. A comprehensive review by Syl-

vester et al. [25] reported that this treatment lowers

the five-year recurrence rate from 59 to 45%. Such

improvement in the outcome (14%) means that ap-

proximately one in seven patients will benefit from

the therapy, while the remaining six will be unaffect-

ed but still exposed to its potential side effects and

risks. Hence, a single instillation of a chemotherapeu-

tic agent cannot be considered a definitive solution,

thus emphasizing the need for continued research

aimed to further reduce the risk of tumor recurrence

and metastasis.

After obtaining histological results and determin-

ing the degree of tumor differentiation, patients are

stratified into four or three risk groups [19]. Adjuvant

therapy is not recommended for patients in the low-

risk group. Patients with the intermediate-, high-, or

very high-risk NMIBC typically undergo induction

and maintenance courses of intravesical BCG (Bacil-

lus Calmette–Guérin) therapy lasting from one to

three years [26]. Intravesical chemotherapy may be

recommended for intermediate-risk patients with in-

tolerance or contraindications to the BCG therapy [19].

Despite a high efficacy of current treatment op-

tions secured in clinical guidelines, the recurrence

rate of NMIBC remains significant (from 40 to 90%)

[3-5], and the rate of NMIBC progression to MIBC var-

ies between 5 to 55% [6], which emphasizes the ongo-

ing need for new approaches to BCa therapy.

Such high recurrence and progression rates re-

quire a deeper understanding of molecular mech-

anisms driving BCa development. Early detection is

critical for improving the clinical outcomes, which is

why the development of reliable biomarkers capable

of improving the diagnostic and prognostic accuracy

and guiding the treatment decisions, has become a

major research focus.

In summary, the challenges associated with BCa

are multifaceted and include (1)  early BCa diagnosis

and risk assessment; (2)  evaluation of risk of NMIBC

progression to MIBC; (3)  prediction of NMIBC recur-

rence; and (4)  classification of patients into respond-

ers and non-responders to current anti-cancer regi-

mens. While some of these challenges can be partially

addressed through existing methods, such as imaging,

cytological analysis, and genomic mutation profiling,

there is an urgent need to identify novel molecular

biomarkers to enhance the diagnostic accuracy and

treatment efficacy in BCa. One of the promising re-

search areas is the use of m

A modification as a po-

tential biomarker.

Molecular mechanism of m

A-methylation. m

is the most prevalent epitranscriptomic modification

of both mRNAs and ncRNAs, including lncRNAs (long

non-coding RNAs), circRNAs (circular RNAs), miRNAs

(microRNAs), and others. m

A methylation is catalyzed

by the methyltransferase complex (or ‘writer’), con-

sisting of the METTL3/METTL14 (methyltransferase

like proteins  3 and 14) heterodimer  [27] and aux-

iliary components, such as WTAP (Wilms tumor  1

associated protein), VIRMA (KIAA1429; vir like m

methyltransferase associated), RBM15/15B (RNA bind-

ing motif protein 15/15B), ZC3H13 (zinc finger CCCH-

type containing  13), and others, which lack the meth-

yltransferase activity and play a regulatory role in

the complex functioning  [28]. Methyl group can be re-

moved by demethylases (or ‘erasers’), such as ALKBH5

(AlkB homolog  5)  [29] and FTO (fat mass and obesi-

ty-associated protein)  [30], indicating the reversible

and highly dynamic nature of the m

A modification.

A-binding proteins, known as ‘readers’, recog-

nize the m

A mark and determine the fate (stabiliza-

tion/translation or degradation) of the RNA molecule.

Reader proteins belong to various protein fami-

lies and include YTH-domain proteins (YTHDF1/2/3,

YTHDC1/2)  [31], heterogeneous nuclear ribonucleo-

proteins (HNRNPC, HNRNPG, HNRNPA2B1)  [32], in-

sulin-like growth factor  2 mRNA-binding proteins

(IGF2BP1/2/3)  [33], and eukaryotic initiation factors

(elF3)  [34]. The specificity of reader proteins is de-

termined by a combination of biochemical, spatial,

and contextual factor that ultimately make the deter-

mination of RNA fate based on m

A marks a highly

context-dependent process. Understanding this mech-

anism is important for the accurate prediction of the

outcome of downstream processes, which could shed

light on aberrant m

A functions leading to various

diseases.

A modification can influence expression of

both oncogenes and tumor suppressor genes, playing

a dual role in cancer progression, which emphasizes

its importance in a complex network of molecular in-

teractions that sustain tumor growth and determine its

resistance to therapy. m

A modifications can promote

accumulation of oncogenic proteins by stabilizing on-

cogenic mRNAs and inhibiting decay of tumor-promot-

ing mRNAs, which can result in the dysregulation of

cell proliferation, promotion of cell cycle progression,

inhibition of apoptosis, and other cancer hallmarks

[35]. Some studies indicate that m

A modifications can

significantly impact the metastatic potential of cancer

cells by regulating key genes involved in cell adhesion,

migration, and invasion, thus contributing to tumor

spreading. Conversely, m

A modifications can ensure

the stability of tumor suppressor mRNAs by prevent-

ing their degradation, maintaining their inhibitory

effect on cancer cells [36, 37].

A pathway in BCa. m

A modifications modu-

late expression of genes associated with cell prolifer-

ation, apoptosis, and metastasis [15]. m

A methylation

also plays a critical role in the progression of BCa [38].

Alterations in the m

A methylation patterns in BCa

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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

can lead to the upregulation of oncogenes or down-

regulation of tumor suppressor genes, thus contribut-

ing to tumorigenesis [38]. Expression of m

A-pathway

factors in BCa and healthy tissues have been investi-

gated in several transcriptomic studies. For instance,

Chen et al. [39] studied the levels of key m

A reg-

ulators and analyzed correlation of their expression

with clinicopathological variables. Clinicopathological

information for 408  BCa patients and healthy donors

was obtained from the TCGA (The Cancer Genome At-

las) database, allowing for the comparison between

the non-tumor tissue group and BCa samples of vary-

ing grades and stages. It was shown that KIAA1429

(one of the major m

A methyltransferases) and YTHDF

A reader protein) were highly expressed in high-

grade BCa. Expression of ALKBH5 (m

A demethylase)

positively correlated with the tumor grade and M1

stage. Although METTL3 (another major m

A methyl-

transferase) was highly expressed in cancer tissues,

its expression decreased as the grade increased and

was low in high-grade tumors. FTO (an important

A demethylase) was expressed poorly in BCa  [39].

Bioinformatic analysis revealed differential patterns

in the expression of m

A-related genes across tu-

mor tissues. Specifically, expression of FTO, ZC3H13,

YTHDF3, YTHDC1, WTAP, METTL16, and METTL14

was downregulated in cancerous tissues compared

to normal tissues. Conversely, HNRNPA2B1, IGF2BP1,

IGF2BP3, METTL3, YTHDF2, and YTHDF1 demon-

strated high expression levels. Moreover, expression

of HNRNPA2B1, HNRNPC, IGF2BP2, RBM15, YTHDF1,

and YTHDF2 was associated with the advanced clin-

ical stages of BCa [40]. Alterations in the expression

of m

A factors indicate a critical role of m

A methyl-

ation in BCa development. Therefore, both expression

of m

A-related factors and m

A-methylation patterns

could potentially serve as prognostic markers to help

clinicians define the clinical stage of cancer, differ-

entiate cancer subtypes, predict disease progression

and outcome, evaluate the risk of recurrence, and

design the treatment plans accordingly. For instance,

abnormal m

A levels in transcripts associated with

tumor suppression may correlate with the advanced

clinical stages, while changes in the methylation levels

of oncogenic transcripts can indicate aggressive can-

cer subtypes or higher risk of disease progression.

By analyzing the m

A methylation status of key tran-

scripts, clinicians can more accurately define the

clinical stage of BCa, differentiate between the lumi-

nal and basal subtypes, predict the likelihood of dis-

ease progression, and estimate the outcomes. Specific

A methylation signatures could help evaluate the

risk of recurrence and guide personalized treatment

plans, e.g., help in selecting patients who may benefit

from immunotherapy or targeted therapy. While this

approach is still in the realm of clinical theory, ad-

vancements in RNA epigenomics and single-molecule

sequencing technologies are paving the way for its

practical application in clinical practice in the fore-

seeable future.

A factors with the oncogenic role in BCa.

Inrecent years, the m

A demethylase FTO has gained

significant attention due to its potential role in vari-

ous cancers, including BCa. Several targets of FTO in

BCa have been identified, including PTPN6 (tyrosine

protein phosphatase non-receptor type  6), a non-re-

ceptor tyrosine phosphatase that dephosphorylates

and regulates the activity of numerous proteins in-

volved in signal transduction, cell growth, differenti-

ation, cell cycle, and oncogenic transformation [41].

PTPN6 is aberrantly expressed in various cancers,

such as hepatocellular carcinoma, renal cell carcino-

ma, gastric cancer, and BCa [41]. Wu et  al.  [42] dis-

covered that FTO induces expression of PTPN6 and

stabilizes it, thus promoting proliferation and meta-

static capabilities of BCa cells. The authors analyzed

human BCa and adjacent normal tissues collected

from 20 patients diagnosed with BCa (sex and age of

the patients were not taken into account), as well as

investigated human BCa cell lines (T24, 5637, RT4, J82,

and HTT1378) and normal bladder epithelial cell line

SV-HUC-1 in in  vitro experiments The samples were

not stratified into groups based on the disease stage or

tumor grade, limiting the generalizability of the study.

While m

A-modified PTPN6 mRNA was detected, no

specific m

A sites were mapped, which hinders the

understanding of how FTO regulates this transcript.

Despite the limitations, the findings of this study sug-

gest that the FTO-PTPN6 axis may serve as a potential

prognostic marker in BCa.

Metastasis-associated lung adenocarcinoma tran-

script  1 (MALAT1), an lncRNA involved in the regu-

lation of gene expression through epigenetic mecha-

nisms, has been associated with the tumorigenesis in

most cancer types, including BCa [43]. It was found

that in BCa, FTO acts as an oncogene by reducing the

A methylation of the 5′-UTR of MALAT1, leading

to the increased stability and upregulated expression

of this ncRNA. Elevated MALAT1 then functions as a

sponge for miR-384, reducing its availability, which

increases MAL2 expression and promotes cancer cell

viability and tumor growth. This study was conduct-

ed in human BCa paired tissue samples (tumor and

adjacent noncancerous) collected from 25 patients;

144 cancer tissue microarrays were used for the val-

idation. Human BCa cell lines (5637, J82, 253J, T24,

SCABER) and SV-HUC-1 cells were investigated in the

in vitro experiments. The authors did not stratify pa-

tients by a specific BCa type or distinct m

A modifica-

tion profile; tissue samples were analyzed primarily

for the overall pathologic stage (pTa-T1 vs. pT2-T4). Up-

regulated FTO expression was found to be associated

SINYAGOVSKAYA et al.654

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

with more advanced pathologic stages (pT2-T4), while

no statistically significant association between the FTO

or MAL2 levels and patients’ sex or age was observed.

FTO has been shown to modify the maturation

of pri-miR-576 in an m

A-dependent manner and to

promote proliferation, migration, and invasion of

BCa cells by regulating the miR-576-CDK6 pathway

[44]. The authors examined BCa and adjacent nor-

mal tissues from 20 patients who underwent radical

cystectomy. BCa cell lines (T24, 5637, UM-UC-3) and

SV-HUC cells were used in in  vitro experiments. Tis-

sue microarrays from 67 BCa patients were analyzed.

It was found that FTO expression did not differ sig-

nificantly between males and females or different age

groups (p  =  0.755 for sex, p  =  0.995 for age), but was

associated with a higher TNM stage (p  =  0.035). The

authors provided detailed insights into the m

A mod-

ification-based mechanisms by which FTO regulates

the miR-576-CDK6 axis and contributes to BCa pro-

gression. By identifying FTO as a potential prognos-

tic biomarker, the study facilitates future diagnostic

or therapeutic advancements in BCa. The correlation

between the FTO expression and higher TNM stages

suggests a potential use of FTO for predicting cancer

progression.

Another key m

A-related regulator is the reader

protein IGF2BP3 (insulin like growth factor 2 mRNA

binding protein  3) that may play an oncogenic role

in BCa progression. Expression of IGF2BP3 was high-

er in BCa cells compared to adjacent healthy tissues

[45] and was associated with several factors indicat-

ing more advanced stages of BCa: (1)  advanced tu-

mor characteristics and (2)  more aggressive disease

progression [45]. BCa samples were collected from

95 patients who underwent resection; SV-HUC-1 cells

and BCa cell lines (5637, J82, UMUC3, and T24) were

used for in vitro analysis. The study included the data

for 414 BCa patients from the TCGA, categorized by

the IGF2BP3 expression levels (high and low). The pa-

tients were classified as ≤70 years vs. >70 years old,

but no significant correlation between the IGF2BP3

expression and age was found (p  =  0.372). The ele-

vated IGF2BP3 expression levels correlated with poor

overall survival (p  =  0.015) and were associated with

more advanced tumor stages (T3/T4) and high tumor

histologic grade, reinforcing the role of IGF2BP3 as

a marker of BCa progression. However, no correla-

tion between the IGF2BP3 expression and nodal (N)

or distant metastasis (M) status was observed, which

was unexpected given the role of IGF2BP3 in cell

migration and invasion. This discrepancy suggests

that IGF2BP3 may be more involved in the local

tumor progression rather than in metastasis. The

study highlighted a strong correlation between the

IGF2BP3 expression and immune cell infiltration in

BCa. High IGF2BP3 levels were associated with an

increased infiltration of macrophages, neutrophils,

and CD8

  T  cells, suggesting a potential immuno-

modulatory role of IGF2BP3. Moreover, the elevated

IGF2BP3 levels have been linked to poorer outcomes

for BCa patients, suggesting that IGF2BP3 may partic-

ipate in determining the severity of BCa and influ-

ence patients’ survival. In a recent study  [33], it was

suggested that IGF2BP family proteins can recognize

A in the transcripts and stabilize such transcripts,

thus increasing their half-life time and duration of

protein expression. For example, stabilization of the

neuropilin1 (NRP1) mRNA led to the M2 macrophage

polarization associated with BCa progression

[46].

High-mobility group protein  B1 (HMGB1) was

identified as a target of IGF2BP3. HMGB1 is a nucle-

ar DNA-binding protein integral to numerous cellular

processes, including inflammation, cell differentia-

tion, and apoptosis. Interestingly, the levels of IGF2BP3

were notably elevated in the individuals who showed

positive response to immunotherapy in contrast to

those who did not respond. Moreover, increased ex-

pression of IGF2BP3 and its m

A-mediated interaction

with HMGB1 mRNA correlated with the improved

overall survival, suggesting that IGF2BP3 may im-

pact the immune microenvironment of BCa, thus in-

fluencing the efficacy of immunotherapy [47]. Using

bioinformatic methods, the authors identified multi-

ple m

A motifs in HMGB1 mRNA that could serve as

IGF2BP3-binding sites. IGF2BP3 expression and func-

tion was investigated in BCa cell lines RT112/84 and

BFTC905.

Another gene of significant interest is ITGA6,

which encodes integrin alpha-6 subunit. ITGA6 is a

cell surface receptor involved in the cell–cell and cell–

substrate interaction, cell migration, differentiation,

tissue repair, and regulation of cell growth [48,  49].

The association between the ITGA6 expression and

cancer progression is rather complex. The upregula-

tion of ITGA6 has been linked to the enhanced inva-

sion, metastasis, and poor prognosis in various can-

cers [50-52]. Thus, ITGA6 was found to maintain cell

adhesion-mediated drug resistance in ovarian cancer

and leukemia [53, 54].

Recently, a mechanism of ITGA6 regulation by the

A modification has been proposed [55]. Tissue sam-

ples from of 186 BCa patients who underwent radical

cystectomy and bladder biopsies were investigated for

A methylation. It was confirmed that the 3′-UTR

of ITGA6 mRNA contains four m

A motifs methylat-

ed by METTL3. Methylation promoted translation of

the ITGA6 transcript through binding of the YTHDF1

and YTHDF3 reader proteins in vitro and in  vivo.

Interestingly, mutation of a single m

A site did not

decrease the translation rate of the transcript, whereas

introduction of several mutations into multiple m

sites noticeably slowed it down [55]. Inanother study,

A-RNA METHYLATION IN BLADDER CANCER DEVELOPMENT 655

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

removal of m

A from several positions in the ITGA6

transcript by the designed multisite editor dCasRx-m

led to the reduction in the transcript translation and

inhibition of BCa progression in vitro and in  vivo,

suggesting a significance of cooperative transcript reg-

ulation by multiple m

A sites. The off-target activity of

the dCasRx–m

A editor has been assessed and proven

to be limited [56]. These findings highlight a potential

importance of m

A methylation of the ITGA6 mRNA

in tumorigenesis and suggest that further research

in this area could lead to the identification of novel

therapeutic targets for BCa.

The CUB domain-containing protein 1 (CDCP1) is a

transmembrane protein that acts as a protein-protein

interaction hub for proteins regulating cell–cell and

cell–substrate adhesion through proteolytic process-

ing and tyrosine phosphorylation  [57]. Dysregulation

of CDCP1 expression in cancer cells has been observed

in colon [58,  59], lung  [60], and kidney  [61] cancers.

CDCP1 was found to induce cell detachment and

promote metastasis in breast cancer  [57]. In general,

an elevated expression of CDCP1 is associated with

the enhanced migratory capabilities of cancer cells,

which facilitates tumor dissemination and metastasis

and leads to poor disease outcome  [62]. The oncogenic

role of CDCP1 in BCa, including promotion of cancer

development and progression, has been demonstrated

in several studies.

The METTL3-m

A-CDCP1 axis was also found to

be implicated in BCa oncogenesis, suggesting a regula-

tory role of m

A modification in the CDCP1 expression

and translation in aberrant uroepithelial cells [63].

The authors utilized 114 BCa and 30 cystitis samples

from patients who underwent radical cystectomy and

bladder biopsies and identified specific m

A modifi-

cation sites in the 3′-UTR and coding region of the

CDCP1 mRNA, which were observed in both control

and malignant BCa cells, whereas CDCP1 expression

itself was barely detectable in cystitis tissue. Methyl-

transferase METTL3 facilitated methylation at these

sites, which served as a signal for the YTHDF1 reader

protein to bind to the CDCP1 transcript, thus stabiliz-

ing it and promoting its translation. Conversely, de-

pletion of METTL3 moderately inhibited proliferation,

invasion, and migration of BCa malignant cells [63].

Another research confirmed these data by demon-

strating that the targeted RNA methylation system

RCas9-METTL3 introduced m

A modification to the

3′-UTR of the CDCP1 transcript, leading to its enhanced

translation and promotion of tumor growth in  vitro

and in  vivo. The system used exhibited satisfactory

on-target activity [64].

As key factors in cell adhesion, ITGA6 and CDCP1

are linked to cancer progression, drug resistance, and

poor prognosis in BCa [55, 63]. Both are regulated by

the METTL3-mediated m

A modification, which sug-

gests a common regulatory axis for potential thera-

peutic targeting. While these findings are promising,

several challenges related to the specificity, resistance,

and clinical implications must be addressed. Although

the analyzed samples were from post-radical cystecto-

my BCa patients, the data on the comorbidities were

lacking and no stratification based on the tumor stage,

grade, or molecular subtype was performed, raising

concerns about generalizability and interpretation of

the study results. The possibility of using ITGA6-CDCP1

and related m

A factors (e.g., METTL3) as therapeu-

tic targets is questionable, since their inhibition may

disrupt the downstream pathways, potentially, even in

healthy cells. Utilizing m

A sites as direct prognostic

markers is a novel approach [65-67]. The use of ad-

vanced molecular tools has demonstrated a potential

for precise RNA editing and its therapeutic applica-

tions (for instance, removal of aberrant modifications

to nullify the negative effects of said modifications).

However, challenges remain, such as predicting long-

term effects of RNA editing and ensuring safe and ef-

ficient delivery of editing systems via nanoparticles,

exosomes, or viral vectors, [68-70]. The cooperative

regulation of ITGA6 by multiple m

A sites compli-

cates the development of the targeted therapy. Ab-

errant m

A profiles correlating with the tumor stage

and grade, could serve as prognostic markers for dis-

ease progression, help stratify patients into high- and

low-risk groups, and guide the treatment decisions.

The studies have provided mechanistic insights into

the regulation of ITGA6 and CDCP1 by m

A modifica-

tions, linking RNA methylation to cancer progression.

Development of targeted tools, e.g., molecular editors,

offers exciting possibilities for precision medicine, but

further research is needed to validate these findings

and translate them into effective therapies for BCa

patients.

A factors acting as tumor suppressors inBCa.

The effects of m

A modifications are diverse and may

include inhibition of tumorigenesis. NOTCH1 (Notch

receptor  1), a component of the Notch pathway, is

responsible for the self-renewal of tumor-inducing

cells (TICs) and oncogenesis [71]. Primary BCa sam-

ples (ranging from early non-invasive to advanced

invasive BCa) from 6 patients have been studied in

order to confirm the role of the NOTCH1-m

A axis in

tumorigenesis. It was found that the m

A modification

of the NOTCH1 transcript by METTL14 attenuated its

expression [71], but the exact mechanism of this pro-

cess remains largely unknown and requires further

research. These findings revealed a novel METTL14-

A-Notch1 regulatory axis in BCa, highlighting a

possible implementation of the m

A modification as

a therapeutic target and biomarker of BCa progres-

sion. However, the study used only six BCa samples

for the initial analysis of the m

A content. Such small

SINYAGOVSKAYA et al.656

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

sample size limited the generalizability of the findings;

moreover, the samples varied significantly in terms

of age, sex, tumor size, stage, and metastasis status.

Also, no tissues from healthy controls were analyzed,

which could potentially affect the results of m

A map-

ping.

A may also act as an mRNA decay signal, as

it was shown for the tumor-suppressor factors SETD7

(SET domain containing 7), KLF4 (Krüppel-like fac-

tor 4), and SYTL1 (synaptotagmin-like protein 1). The

roles of SETD7 and KLF4 in cancer still remain con-

tradictory. Downregulation of SETD7 (lysine methyl-

transferase involved in histone and non-histone pro-

tein methylation) has been linked to the enhanced

migration and invasion of lung cancer cells [72] and

to correlate with a poor outcome in patients with

gastric cancer [73]. A large body of evidence suggests

that SETD7 acts as an oncogene, based on the positive

correlation between its expression and cancer stage

in hepatocellular adenocarcinoma [74]. Conversely,

SETD7 has been associated with the prevention of

the epithelial-to-mesenchymal transition in various

cancer types, indicating its potential involvement in

the inhibition of metastasis [75]. KLF4 was found to

act as a tumor suppressor by preventing metastasis

in colorectal cancer [76]), but also as an oncogene in

osteosarcoma by promoting tumorigenesis invivo [77].

KLF4 is significantly downregulated in urothelial BCa

cells, indicating poor overall survival and risk for re-

currence [78, 79]. KLF4 overexpression induced by the

CRISPR-ON transcriptional activation system inhibited

cell proliferation and promoted cell cycle arrest in G1

phase via regulation of the AKT-p21 signaling path-

way [79]. The data obtained suggest that SETD7 and

KLF4 mRNAs are the downstream targets of METTL3,

which installs m

A in their transcripts. The modified

transcripts are recognized by the YTHDF2 reader pro-

tein, resulting in mRNA degradation. Repression of

METTL3 correlated with the cell cycle arrest in G1

phase and reduction in cell proliferation and migra-

tion invitro [80].

KLF4-m

A and SETD7-m

A axes represent prom-

ising but complex targets for cancer therapy. While

the contradictory roles of SETD7 and KLF4 as both

tumor suppressors and oncogenes in different cancers

pose serious challenges, advancements in understand-

ing molecular mechanisms underlying their activity

could lead to novel therapeutic and prognostic strate-

gies. Future research should focus on resolving these

contradictions, identifying context-specific factors, and

developing targeted interventions with minimal tox-

ic effects. However, the implication of the aforemen-

tioned axes yields a prognostic value, since aberrant

A profiles, as well as SETD7 and KLF4 expression

levels, may correspond to specific tumor stages and

grades.

SYTL1 plays a critical role in enhancing the an-

ti-tumor immune response. Thus, the overexpression

of SYTL1 was found to activate natural killer (NK)

cells, promoting the effect of the anti-tumor immunity

both in vitro and in  vivo. [81] The m

A methyltrans-

ferase WTAP downregulates SYTL1 expression via m

methylation of the SYTL1 mRNA, which marks it for

degradation after recognition by the YTHDF2 reader

protein. The authors analyzed 35 BCa and adjacent

tissue samples collected from patients who underwent

radical cystectomy. BCa cell lines (5637, T24, SW780,

J82), SV-HUC-1 cells, and NK-92 cells were used in the

in vitro experiments, and the results of these experi-

ments were validated in vivo. No sex, age, or disease

stage were taken into account in this work, as the

study was primarily focused on BCa samples and

cell lines. The authors did not investigate the SYTL1

expression in healthy individuals and patients with

other diseases or comorbidities. However, analysis

of bioinformatics databases allowed them to identify

broader expression patterns, in particular, reduction

in the SYTL1 expression in BCa cells compared to ad-

jacent normal tissues. No changes in the SYTL1 ex-

pression over time or in response to treatment were

studied. Although the authors suggested that modu-

lation of SYTL1 expression could enhance the tumor

suppression mediated by NK cells, they did not pro-

vide therapeutic strategies for clinical use.

Ferroptosis and m

A modifications in BCa. Fer-

roptosis, a form of regulated cell death characterized

by iron-dependent lipid peroxidation, has been in-

creasingly recognized for its role in various patholog-

ical conditions. Ferroptosis is driven by peroxidation

of phospholipids in cellular membranes, resulting in

oxidative stress and accumulation of lipid peroxides

followed by membrane damage and cell lysis [82]. Cell

death occurring exclusively by ferroptosis correlates

with the accumulation of lipid peroxidation markers

and can be suppressed by iron chelators, lipophilic

antioxidants, inhibitors of lipid peroxidation, and de-

pletion of polyunsaturated fatty acids (PUFAs).

Ferroptosis play a particularly important role in

BCa due to the unique metabolic and oxidative en-

vironment of tumor cells. Several ferroptosis induc-

ers, such as erastin, artemisinin, conjugated polymer

nanoparticles, and quinazolinyl-arylurea derivatives,

have been shown to sensitize BCa cells to anti-cancer

treatment [83]. A combination of these compounds

with standard anticancer drugs, along with the ferro-

ptosis-related m

A factors and genes, could overcome

the resistance to therapy by targeting ferroptosis-relat-

ed vulnerabilities of BCa cells. This approach, which

leverages both conventional and ferroptosis-inducing

agents, presents a promising direction in improving

the efficacy of treatment, especially in patients with

therapy-resistant BCa.

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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

Ferroptosis is regulated by various pathways

and factors, including NRF2 (nuclear factor eryth-

roid  2- related factor  2), a transcription factor that

plays an essential role in cellular defense against

oxidative stress [84]. NRF2 regulates expression of

antioxidant proteins, such as solute carrier family  7

member 11 (SLC7A11)  [85] and glutathione peroxi-

dase  4 (GPX4) [86], that can prevent ferroptosis. Con-

versely, NRF2 has been shown to sensitize cancer cells

to ferroptosis by upregulating genes involved in iron

metabolism and redox homeostasis, e.g., the gene for

the multidrug resistance-associated protein  1 (ABCC1-

MRP1) [87]. Ferroptosis can be a mechanism of cell

death in tumors. Tumor cells, which often contain

high levels of reactive oxygen species (ROS), can un-

dergo ferroptosis due to the iron reaction with exces-

sive hydrogen peroxide, leading to the production of

hydroxyl radicals [88]. For example, activation of the

p62-Keap1-NRF2 pathway promoted resistance to fer-

roptosis in hepatocellular carcinoma cells, highlighting

a complex regulatory mechanism involved in the cell

death pathways in cancer [89]. In BCa, the NRF2-de-

pendent antioxidant response promotes tumor growth

through the p62-KEAP1-NRF2 pathway [90]. A num-

ber of m

A-associated ferroptosis-related genes (FRGs)

linked to BCa have been identified. Thus, myosin bind-

ing protein H (MYBPH), sclerostin (SOST), small pro-

line-rich protein 2A (SPRR2A), and cornulin (CRNN)

were suggested as potential oncogenes, while CYP4F8

(cytochrome P450 family  4 subfamily  F member  8),

PDZD3 (PDZ domain containing  3), CRTAC1 (cartilage

acidic protein  1), and LRTM1 (leucine rich repeats and

transmembrane domains  1) were proposed as tumor

suppressors. The mechanisms of interaction between

these proteins and NRF2 remain largely unknown

[91]. An aberrant expression of NRF2 in BCa is medi-

ated in an m

A-related manner, resulting in resistance

to ferroptosis. WTAP introduces m

A modifications

in the 3′-UTR of the NRF2 mRNA, making it a target

for YTHDF1, which stabilizes this transcript and pro-

motes its translation [92]. This process is associated

with the accelerated cell proliferation and repression

of the erastin-induced ferroptosis, thus ensuring poor

disease prognosis. The authors used BCa and adjacent

non-cancerous tissues from 45 paired samples ob-

tained from patients who underwent nephrectomy, as

well BCa cell lines (J82, UM-UC-3) and SV-HUC-1 cells

(control). Neither sex or age of patients, nor the ex-

pression of WTAP or NRF2 in healthy individuals were

taken into account in this study The authors focused

on the mechanistic aspects of m

A modification and

did not explore potential therapeutic interventions

targeting WTAP or NRF2 in vivo.

As reported in [93], WTAP is upregulated in BCa

cells and its high expression correlates with a poor

disease prognosis. The study included 48 males and

14 females in the BCa group and 14 males and 6 fe-

males in the control group. The average age in the

control group was 57  ±  19 years vs. 52  ±  13 years in

the BCa group. Statistical comparisons indicated no

significant differences in sex and age between the

groups (p  >  0.05), suggesting that sex and age were not

confounding factors in the analysis. Normal bladder

mucosa tissue was used as a control. The authors did

not differentiate between the BCa molecular subtypes,

which made it unclear whether the expression of

WTAP varied between them, neither did they consid-

er potential confounding variables, such as smoking

status, comorbidities, or genetic predisposition, all of

which could influence WTAP expression. The down-

stream targets modified by WTAP and the potential of

specific m

A sites to serve as independent prognostic

markers were not discussed as well. Complementary

evidence comes from study [92], which elucidates the

WTAP-induced m

A-modification of NRF2 and links

this axis to ferroptosis resistance and adverse out-

come.

A methylation in BCa-related ncRNAs. Vari-

ous ncRNAs can also undergo m

A methylation. Only

around a third of transcribed genes in the human ge-

nome encode proteins, while the remaining two-thirds

are non-coding genes. Some genes are transcribed into

lncRNAs that regulate transcriptional and post-tran-

scriptional processes by modulating gene expression

via affecting chromatin remodeling. lncRNAs can

serve as signal, decoy, guide, or scaffold molecules

[94]. lncRNAs and circRNAs (covalently closed con-

tinuous loops) interact with miRNAs, resulting in the

so-called ‘sponge’ effect, a specific mechanism of RNA

interference in the regulation of gene expression [95].

Both lncRNAs and circRNAs have been implicated in

the development and progression of various diseases

due to their ability to act as miRNA sponges, which

emphasizes their significance in biological process-

es and as potential therapeutic targets. Because of

their abnormal expression, lncRNAs are of great sig-

nificance in the early diagnostics of cancer. For ex-

ample, lncRNA CASC11 (cancer susceptibility  11) has

been identified as an oncogenic lncRNA at the early

stage of BCa development. It was found to activate

cell proliferation through miRNA‐150 [96] and to pro-

mote tumor cell growth and metastasis in colorec-

tal cancer by activating the WNT-β-catenin signaling

pathway [97].

Huang et al. [98] identified 50 m

A-lncRNAs with

a potential prognostic value that were methylated

mostly by METTL3 and RBM15 methyltransferases.

A risk score model has been developed based on 11

lncRNAs with the prognostic significance. The knock-

down of METTL3 and RBM15 in BCa cells inhibited

proliferation, invasion, and migration of tumor cells

in vitro and in vivo. The study did not fully address

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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

the heterogeneity of BCa, as the authors did not dif-

ferentiate between the tumor molecular subtypes,

which may impact the generalizability of the devel-

oped prognostic model. Also, no specific m

A sites

have been identified in the lncRNAs.

miRNA-221 and miRNA-222 play the oncogenic

role in BCa by binding to the 3′-UTR of the tumor

suppressor PTEN (phosphatase and tensin homolog)

mRNA, inhibiting its expression and promoting cell

proliferation in  vitro and in  vivo  [99]. METTL3 was

found to induce miRNA221/222 maturation in an

A-dependent manner, which correlated with can-

cer progression [99]. The authors used BCa and paired

normal tissues from patients who had been diagnosed

with BCa and underwent surgery (180 cases), two BCa

cell lines, and normal urinary epithelial cell line (con-

trol) for in vitro experiments; the results if these ex-

periments were validated in vivo. The patients were

categorized based on age and sex (<65 and ≥65 years

old; male and female), however, the relation of the

obtained data to those factors was not analyzed. The

study was focused exclusively on BCa and did not de-

tail whether the patients had any non-cancerous con-

dition; METTL3 expression and miR221/222 maturation

were studied exclusively in the context of BCa devel-

opment. Moreover, no multivariate analysis was per-

formed to determine whether the METTL3-miR221/222

axis could be used as an independent prognostic fac-

tor when adjusted for confounding variables, such

as tumor stage, grade, or molecular subtype. While

technically challenging, targeting ncRNAs has become

increasingly feasible with the CRISPR RNA-targeting

systems like Cas13. However, low delivery efficiency,

required specificity, and small size of miRNA targets

remain significant obstacles.

BCa-associated lncRNA BLACAT3 (BLCa-associated

transcript 3) is implicated in the promotion of angio-

genesis and hematogenous metastasis by activating

the downstream NF-κB signaling in an m

A-dependent

manner [100]. The authors assessed clinical samples

of tumor and adjacent normal tissues from 107 pa-

tients with MIBC who underwent radical cystectomy,

although no stratification by sex or age was per-

formed. Invitro experiments were carried in RT4 cells

(NMIBC cell line), MIBC-derived cell lines (UM-UC-3,

5637, T24, J82, SW780), and SV-HUC-1 cells (control).

The study provided experimental evidence linking

epitranscriptomic modifications (m

A) to cancer pro-

gression via lncRNAs (an emerging field in oncology).

The authors demonstrated the following mechanis-

tic pathway: m

A-modified BLACAT3 recruits YBX3

(DNA- and RNA-binding protein involved in the tran-

scription regulation, RNA stabilization, and transla-

tion), leading to the upregulation of NCF2 (a subunit

of the NADPH oxidase complex), which promotes an-

giogenesis and metastasis. The study suggested that

BLACAT3 might be specific to MIBC, as no significant

differences were observed between the RT4 cells

(NMIBC) and normal urothelial cells.

A recent study by Liu et al. [101] unveiled a

molecular mechanism of the interaction between

LINC01106 (long intergenic non-protein coding RNA

1106), miR-3148, and adaptor protein DAB1 (Dis-

abled-1). It was found that LINC01106 and miR-3148

competitively bind to the DAB1 mRNA. LINC01106

stabilizes the transcript, leading to a better disease

prognosis, while miR-3148, on the opposite, inhibits

its translation, resulting in a poor overall outcome of

BCa. Interestingly, CRISPR-mediated hypermethylation

of LINC01106 facilitated by dCas13b-METTL3-METTL14

increased its affinity to DAB1. The authors analyzed

four BCa cell lines and SVHUC-1 cells (control), as well

as 30 paired BCa tissue samples (malignant and adja-

cent normal tissues) obtained from BCa patients that

underwent surgery.

circRNAs are generally known to play the on-

cogenic role. Thus, the IGF2BP3 reader protein was

found to stabilize m

A-methylated circPSMA7, result-

ing in the enhanced expression of mitogen-activated

protein kinase  1 (MAPK1) and BCa progression and

metastasis. circPSMA7 acts a sponge for miR-128-3p

that exhibits the tumor suppressor activity by target-

ing the MAPK1 mRNA [102]. The study analyzed 33

paired samples of fresh BCa and adjacent normal tis-

sues. BCa cell lines UM-UC3 and T24, SV-HUC-1 cells,

and 293T human embryonic kidney cells were used

in in vitro experiments. The authors predicted eight

potential m

A modification sites in circPSMA7 and

carried out methylated RNA immunoprecipitation

(MeRIP) assay to confirm the presence of m

A modifi-

cations in the transcript. However, no mutations were

introduced in the identified sites to validate each site

separately and to determine which sites contributed

to the IGF2BP3-mediated stabilization of circPSMA7.

It also remained unclear whether individual sites

could be used independently as prognostic markers.

METTL14 plays a significant role in the advance-

ment of BCa by promoting expression of lncDBET

(D4Z4 binding element transcript lncRNA). Five m

sites in lncDBET methylated by METTL14 were pre-

dicted in [103]. The elevated levels of lncDBET trig-

ger a cascade of events that ultimately contribute to

the development of BCa, in particular, activation of

the peroxisome proliferator-activated receptor (PPAR)

signaling pathway through recruitment of fatty acids,

which has a profound impact on the lipid metabo-

lism in cancer cells. The mechanism involves a direct

interaction between lncDBET and fatty acid-binding

protein  5 (FABP5). The knockdowns of METTL14,

lncDBET, or FABP5 suppressed tumor growth in vi-

tro and invivo. The study was conducted in SVHUC-1

cells and BCa cell lines (UMUC3, 5637, T24, J82,

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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

and EJ-M3), as well as in fresh BCa tumor tissues

and adjacent non-tumor specimens collected from

patients subjected to radical cystectomy or transure-

thral resection and were used to evaluate the levels

of METTL14, lncDBET, and FABP5. The patients were

not stratified based on the cancer stage or subtype.

Because of cancer heterogeneity, it is unclear whether

all BCa subtypes exhibited similar METTL14-lncDBET-

FABP5 expression patterns. The authors proposed the

METTL14-lncDBET-FABP5 axis as a potential thera-

peutic target, but did not address if such approach

would be clinically feasible without disruption of es-

sential pathways and unwanted off-target effects. The

prognostic value of this axis still has to be elucidated,

as m

A patterns and expression profiles of m

A fac-

tors may help stratification of patients into subgroups

and prediction of disease progression.

Interestingly, an elevated expression of METTL14

was discovered to be characteristic of more ‘aggres-

sive’ cell lines [104]. The authors analyzed 120 forma-

lin-fixed paraffin-embedded bladder urothelial carci-

noma samples (60 NMIBC and 60 MIBC), 40 normal

urothelial tract samples from nephrectomy specimens,

and 7  BCa cell lines. The knockdown of METTL14

caused a significant decrease in the m

A modification

levels in the cells, leading to a substantial inhibition

of cancer cell migration and invasion. Since METTL14

and METTL3 form a heterodimer that acts as a key

A ‘writer’ complex, downregulation of METTL14 led

to the disruption of its catalytic activity  [104]. How-

ever, others authors have reported that the METTL14

upregulation in BCa cells suppressed cell prolifera-

tion and migration of BCa cell lines [105,  106]. This

discrepancy may arise from the tumor heterogeneity,

as bladder tumors contain subpopulations of cells

with different molecular profiles  [107], i.e., different

regions of the same tumor may have varying levels

of METTL14. In  [104], the samples were not strati-

fied into subgroups based on the m

A profiles, which

makes it unclear whether METTL14 had a stronger

impact in certain groups of patients. The authors did

not explore potential downstream targets of METTL14,

so it remains obscure how METTL14 contributes to

the tumor progression or aggressiveness in BCa.

In summary, m

A plays a multifaceted role in the

regulation of both oncogenes and tumor-suppressors

in BCa. The levels of m

A methylation influence the

transcriptome fate in a diverse and somewhat unpre-

dictable way: either high or low methylation levels

may either up- or downregulate transcript expression.

A is a highly dynamic modification, and methyl-

ation patterns may vary at different disease stages,

which can be potentially used to precisely differenti-

ate the stage of disease development and predict its

progression, outcome, and possibility of recurrence.

Figure  1 illustrates m

A-related factors that play onco-

genic role in the development of BCa, as well as the

mechanisms of their action.

Factors associated with m

A that exert tumor sup-

pressive functions in the BCa development are illus-

trated in Fig. 2.

This novel approach has been extensively stud-

ied in a number of works that have proposed various

targets and potential applications in cancer therapy.

However, a deeper understanding of m

A modifica-

tion mechanisms and the role of this modification as a

prognostic biomarker is still required, e.g., for the use

of m

A biomarkers for correcting existing manage-

ment of patients. Many ongoing studies have limita-

tions that need to be resolved. For example, no links

have been established between the m

A profiles or

expression patterns of m

A factors and tumor stage,

grade, or other risk factors, even if it could help clini-

cians to stratify patients into groups. In most studies,

no association between the expression of m

A factors

and patients’ age and sex have been investigated (al-

though many authors state that these parameters do

not confound the analysis) [44, 93]. Often, the studies

do not specify whether the patients had chronic dis-

eases, comorbidities, or polymorbidities, which raises

the possibility that the expression of m

A factors could

be associated with such concurrent conditions. Most

studies are performed in BCa tissues and do not ex-

plore the possibility of m

A factor assessment in the

blood, urine, or other biological fluids, which would be

more clinically relevant for non-invasive diagnostics.

A-mediated drug resistance. Several studies

have highlighted the role of m

A modification in drug

resistance, including resistance to chemotherapy, im-

munotherapy, and specific drugs (e.g., cisplatin and

anthracyclines) in different cancers, including BCa

[108-110].

With the discovery of cancer immune checkpoints

and checkpoint inhibitors, such as programmed cell

death protein 1 (PD-1) and programmed death-ligand1

(PD-L1), the possibilities for using novel treatment

strategies have considerably expanded. For example,

METTL3 and METTL14 were reported to suppress in-

nate immune response to cancer cells in colorectal

carcinoma and melanoma by introducing m

A in the

3′-UTRs of transcripts of key factors involved in the

immune response signaling pathways, namely STAT1

(signal transducer and activator of transcription pro-

tein family) and IRF1 (interferon regulatory factor1).

METTL3-14 depletion caused a noticeable increase in

the production of cytokines and chemokines, as well

as activation of IFN‐γ signaling, suggesting m

A to

be a key factor in the regulation of cell sensitivity

to immunotherapy. Moreover, METTL3-14-depleted

cells also exhibited profound sensitivity to anti-PD-1

treatment [111]. It was suggested that METTL3 ex-

pression is regulated by JNK1, a core component

SINYAGOVSKAYA et al.660

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

Fig. 1. Oncogenic effect of m

A modification of RNA in bladder cancer biology. a) m

A methylation of ITGA6 tran-

script mediated by METTL3 and YTHDF1/YTHDF3 reader proteins, promotes its translation and tumor progression [55].

b)METTL3-m

A-CDCP1 axis regulates CDCP1 expression and translation and promotes tumor progression, cell migration, and

metastasis [63]. c)m

A-eraser FTO demethylase regulates expression and stability of PTPN6, a key phosphatase involved in

cell signaling and cancer progression, suggesting its prognostic potential in BCa [42]. d) NRF2 promotes tumor growth and

resistance to ferroptosis through the m

A-mediated stabilization by WTAP and YTHDF1 [92]. e)FTO acts as an oncogene by

reducing m

A methylation of the MALAT1 lncRNA, enhancing its stability and expression, which promotes tumor growth,

since MALAT1 sponges miR-384 leading to the increased MAL2 levels; higher FTO expression correlates with advanced

cancer stages [43]. f) IGF2BP2 stabilizes m

A-modified NRP1 transcript, leading to the M2 macrophage polarization and

promotion of BCa progression [46]. g) FTO promotes BCa progression by modifying the maturation of pri-miR-576 through

the m

A-dependent mechanisms and regulates the miR-576-CDK6 pathway; higher FTO expression correlates with advanced

TNM stages [44]. h)METTL3 promotes miRNA221/222 maturation through m

A modification, facilitating cancer progression

via inhibition of the tumor suppressor PTEN [99] i) METTL14 promotes BCa progression by increasing lncDBET expres-

sion, which activates the PPAR signaling pathway and alters lipid metabolism through direct interaction with FABP5 [103].

j) m

A-methylated circPSMA7 is stabilized by IGF2BP3, which enhances MAPK1 stability and promotes tumor progression;

miR-128-3p can potentially reverse this effect [102]. k) IGF2BP3 interacts with m

A-methylated HMGB1 mRNA, influencing

the immune microenvironment of BCa tumor and affecting immunotherapy outcome [47].

of the JNK signaling pathway, which results in the

A methylation of PD-L1 mRNA, leading to its in-

creased expression in BCa cells [112].

Casein kinase 2 (CK2) is a serine/threonine pro-

tein kinase that regulates glycolysis in cancer cells.

Increased lactate production and glucose utilization

contribute to cancer progression. Demethylase ALKBH5

decreases the stability of the CK2 mRNA by removing

A from the transcript. ALKBH5 is downregulated in

BCa cells, resulting in more pronounced cell prolifer-

ation and tumor growth. Overexpression of ALKBH5

increases the sensitivity of BCa cells to cisplatin via

the CK2a-mediated glycolytic pathway [113, 114].

Some circRNAs play a major role in facilitating

chemoresistance in cancers (including BCa) in an

A-dependent manner. Xu et  al. [115] demonstrat-

ed that the expression of circ104797 in BCa tissues

was elevated compared to adjacent healthy tissues,

and the underlying mechanism of this upregulation

is m

A-dependent. The content of m

A-methylated

circ104797 was increased in cisplatin-resistant cell

lines, while targeted demethylation using the CRISPR-

dCas13b-ALKBH5 system significantly reduced it.

circ104797 is known to act as a sponge for miR-103a

and miR-660-3p that modulate cell sensitivity to drugs

by controlling the ability to expel chemotherapeutic

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BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

Fig. 2. Tumor suppressive effect of m

A modification of RNA in bladder cancer biology. a) METTL14-m

A-Notch1 axis in-

hibits self-renewal of tumor-inducing cells (TICs) [71]. b) SYTL1 enhances anti-tumor immune response by activating NK

cells; m

A methyltransferase WTAP downregulates SYTL1 expression through m

A modification, leading to the transcript

degradation by YTHDF2 [81]. c) METTL3-introduced m

A modification is recognized by YTHDF2 and leads to the decay

of mRNA for the tumor-suppressor factor SETD7 [80] d) KLF4 is a tumor suppressor downregulated in BCa cells and

linked to poor survival and recurrence risk. KLF4 overexpression inhibits cell proliferation and induces cell cycle arrest

in G1 phase. METTL3 m

A-methylates the KLF mRNA, causing it recognition by the YTHDF2 reader protein and further

degradation [80]. e) LINC01106 stabilizes DAB1 mRNA, improving BCa prognosis, while miR-3148 inhibits its translation,

leading to poorer disease outcome. CRISPR-mediated hypermethylation of LINC01106 enhances its affinity to DAB1 [101].

agents and regulating genes involved in the DNA dam-

age response and DNA repair, respectively. The authors

suggested that circ104797 can be used as a biomarker

for predicting cell response to the anti-cancer treat-

ment by measuring its expression levels or levels of

its m

A-methylation, or as a target of treatment aimed

to disrupt circ104797 interaction with miRNAs to im-

prove the treatment outcome [115].

Another circRNA upregulated in BCa cells is

circ0008399. It binds WTAP to promote formation

of the WTAP-METTL3-METTL14 m

A methyltransfer-

ase complex that induces m

A modification in tumor

cells. Moreover, it facilitates the posttranscriptional

regulation of TNFAIP3 (tumor necrosis factor, alpha-

induced protein  3), an anti-apoptotic protein that in-

hibits TNF-induced apoptosis [116], thus promoting

cell resistance to chemotherapy [117].

Interestingly, key m

A regulators may also act as

tumor suppressors. Thus, lower levels of METTL16

were found to correlate with a poor outcome in BCa

patients [118]. Furthermore, METTL16 exerted a po-

tent anti-cancer effect in BCa cells both in in vitro

and invivo by reducing cell proliferation and increas-

ing the sensitivity to drugs (cisplatin). These effects

were achieved through a complex molecular path-

way involving HIF-2α (hypoxia-inducible factor  2α),

PMEPA1 (prostate transmembrane protein, androgen

induced 1), and autophagy regulated by the m

methylation of the respective mRNAs. These findings

open new prospects for developing targeted therapies

against BCa and selecting prognostic biomarkers for

prediction of patients’ response to chemotherapeutics.

There has long been a demand for more effi-

cient methods for predicting potential therapeutic

resistance and evaluating the treatment outcomes.

Abnormal expression of m

A regulators is typically

associated with various forms of resistance, offering

a basis for predicting cancer response to treatment.

First, there is a number of effective small-molecule

inhibitors for almost all major groups of m

A path-

way factors [109], which can be used to overcome

the drug resistance caused by the expression of these

factors. Second, expression levels of the m

A pathway

factors and the levels of m

A methylation of certain

transcripts associated with the drug resistance can be

used as diagnostic markers for predicting resistance

to therapy.

Mutations in m

A motifs and m

A factors.

A modifications are prevalent in certain mRNA re-

gions, such as stop codons, 3′-UTRs, and long inner

SINYAGOVSKAYA et al.662

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

Fig. 3. The workflow for developing personalized treatment strategies based on the m

A RNA modification profiling in a pa-

tient population. a)Tissue samples are collected from the patients, including those with different BCa stages. m

A sequencing

is performed to assess m

A modification patterns in RNA, which may vary depending on the disease severity. b) Based on

A patterns, patients are classified into distinct subgroups. c) Patients’ survival probability is assessed based on the m

profiles to provide insights into the correlation between the m

A modification of RNA and clinical outcomes and to help

predict patients’ response to specific treatments. d)Personalized treatment strategy is designed based on the assessed risks.

exon regions. They are typically located in the con-

sensus motifs including UGACA, RRACH (R  =  G or A;

H  =  A, C, or U), and DRACH (where D  =  A, G or U;

R  =  A or G; H  =  A, C or U), as has been verified by

high-throughput sequencing. m

A modification is a

major molecular mechanism regulating the fate of

RNAs in response to various stimuli [119-121]. Func-

tional m

A sites are highly evolutionary conserved,

whereas “accidental” nonfunctional m

A modifica-

tions occurring as a result of methyltransferase off-

site targeting, are typically purged by natural selection

[122]. Mutations within the consensus motifs can be

detrimental, as they could potentially prevent meth-

ylation of adenosines and attenuate or even abolish

the enhanced translation of the corresponding tran-

scripts. Cancer cells seem to be generally resistant

to mutations within m

A consensus motifs [123].

SNPs (single nucleotide polymorphisms) affect-

ing m

A-methylation can occur in two primary loca-

tions – near and directly at m

A-modification sites –

and significantly impact various biological processes

and pathways. Some SNPs cause mutations, potential-

ly interfering with the transcription and translation;

others are located in the UTRs or near stop codons,

affecting mRNA interaction with transcriptional reg-

ulators and RNA-binding proteins. These alterations

can influence mRNA stability and efficiency of its

nuclear transport [124]. As a result, gene expression

and overall cellular functions can be affected. The ef-

fects of these m

A-SNPs can vary depending on their

exact location and genes involved [125]. Further re-

search is needed to fully understand the implications

of these genetic variations in cellular processes and

disease mechanisms.

Identification of m

A-related SNPs can be partic-

ularly important in molecular diagnostics. One of the

examples of such m

A-SNPs is rs3088107 in the RNFT2

(ring finger protein, transmembrane  2) oncogene. Nor-

mally, expression of RNFT2 is elevated in BCa cells,

which promotes proliferation and metastasis of cancer

cells. rs3088107 SNP abolishes RNFT2 expression in

an m

A-mediated manner [125].

Mutations in the genes coding for m

A-related

factors may not only affect m

A methylation and

the fate of respective transcripts, but also result in

the production of proteins with an altered substrate

specificity. Indeed, the gain-of-function missense mu-

tation R298P in METTL14, which is responsible for the

recognition of the non-canonical sequence of GGAU

instead of canonical GGAC, is frequently found in can-

cer patients. The effects of the R298P mutation are

similar to those of the METTL3-METTL14 complex

overexpression linked to cancer cell growth and pro-

liferation [126].

In conclusion, since the m

A methylation pathway

plays a role in both tumorigenesis and tumor suppres-

sion at the levels of mRNA expression and ncRNAs,

and because mutations in m

A domains lead to

changes in the expression of m

A factors, m

A meth-

ylation can serve as a diagnostic marker of disease

A-RNA METHYLATION IN BLADDER CANCER DEVELOPMENT 663

BIOCHEMISTRY (Moscow) Vol. 90 No. 6 2025

progression and development rate. Figure 3 presents

a potential workflow for m

A profiling as a novel

prognostic biomarker for the stratification of BCa pa-

tients. RNAs or m

A-methylated RNAs can be extracted

from the patients’ tissues or blood using specific m

A-

targeting antibodies, sequenced, and subjected to bio-

informatic analysis, that would allow for the assess-

ment of expression levels of mRNAs and ncRNAs, as

well as the levels of m

A methylation of target RNAs.

Based on the results of analysis, predictions can be

made regarding the rate of disease progression, like-

lihood of tumor recurrence, and therapy efficacy to

help with the development of disease management

strategy for individual patients.

CONCLUSION

BCa presents a significant challenge to healthcare

because one of the highest prevalence among cancers,

as well as high recurrence and mortality rates. Timely

and accurate diagnostics and selection of appropriate

treatment significantly affect the BCa prognosis in pa-

tients. While the m

A-based molecular diagnostics is

still at its inception, the usefulness of m

A biomarkers

have been demonstrated for many cancers. Numer-

ous prospective m

A biomarkers of BCa have been

described, although in a limited number of patients.

There is a current need for identification of new BCa

biomarkers that could be conveniently analyzed, and

not only in tumor cells, but also in patients’ urine,

blood cells, or circulation (e.g., exosomal biomarkers

or free nucleic acids). The lack of valid and reliable

molecular biomarkers for predicting BCa development,

NMIBC-to-MIBC progression, and response to cisplatin

and other drugs (including PD-1/PD-1L) negatively af-

fects the prognostics in BCa patients.

The levels of m

A methylation and SNPs in mR-

NAs and ncRNAs, as well as the expression levels of

A factors, may serve as valuable biomarkers to

shed light on tumor behavior and patient prognosis

and to define the optimal treatment options. Dis-

tinct m

A patterns may act as early biomarkers for

cancer detection and risk assessment. Identification

of new m

A-related genes and evaluation of their

significance through the assessment of m

A scores,

clustering, enrichment analysis, and disease outcome

prediction could help to pinpoint potential targets for

precision therapy, evaluate the risk of cancer relapse,

and diagnose BCa at the earliest possible stage to en-

sure high survival rates. m

A profiling could be used

for stratifying the patients according to cancer sub-

types and clinicopathological characteristics, predict-

ing their response to treatment, and evaluating the

outcomes. The use of m

A machinery for therapeutic

and prognostic purposes is currently at its infancy.

However, this topic has been studied extensively,

and several prognostic models based on m

A profiles

have been already developed. A proposed prognostic

A-driven marker panel for renal cell carcinoma

(RCC) based on the transcriptome-wide m

A-seq data

can identify dysregulated m

A-modified target genes,

such as NDUFA4L2 (NDUFA4 mitochondrial complex

associated like  2, 4-like), NXPH4 (neurexophilin  4),

and UMOD (uromodulin), associated with significantly

poorer overall survival in RCC patients [127]. A novel

A classifier has recently been designed to predict

the treatment efficacy and response to treatment in

BCa patients [128]. All things considered, m

A meth-

ylation may serve as a prospective non-invasive diag-

nostic biomarker in the diagnostics and prognostics

of BCa.

Abbreviations. BCa, bladder cancer; METTL,

methyltransferase like protein; MIBC, muscle-invasive

bladder cancer; NMIBC, non-muscle-invasive bladder

cancer.

Acknowledgments. The authors thank Priority

2030 Research Program (Sechenov University).

Contributions. T.S., D.K., and A.K. developed the

study concept; T.S., Y.L., N.V-K., A.A., N.P., S.B., and I.G.

wrote the original draft; T.S., A.D., A.V., D.K., and A.K.

edited the manuscript; T.S. prepared the figures; V.C.,

A.D., A.V., and D.K. supervised the project; D.K. and

A.K. provided project administration; A.K. acquired

the funding. All authors have read and agreed to the

published version of the manuscript.

Funding. The study was supported by the Russian

Science Foundation (project no.22-75-10032).

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest.

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