Introduction

According to the central principles of molecular biology, genetic information progressively flows from DNA to RNA to proteins. Epigenetics is a branch of genetics that studies heritable changes in gene expression with unchanged DNA sequences, including chromatin remodeling, DNA methylation, RNA methylation, histone modification, and other modifications.1,2 These modifications, like nucleosome remodeling, can regulate the expression of genes, thereby determining cell state and influencing cell differentiation and development. The role of DNA modifications in regulating gene expression is well understood. In recent years, with the development of immunoprecipitation technology and second-generation sequencing technology, the diversity of RNA has been reflected, and the role of RNA in transcription and translation and the expression of other genes has been gradually understood.

More than 170 forms of modification are known to exist in various coding and non-coding RNA in cells, and most RNA species contain more than one chemical modification.3,4 m6A, m5C, and m7G are the three most representative modifications in addition to m1A,5 Ψ,6 and A-to-I,7 among others. Different RNA modifications are connected and transformed through gene transfer processes and cell networks, forming dynamic regulation. Based on epigenetic modifications, the targeted regulation of RNA in tumors has gradually become a new method for understanding tumors’ occurrence, development, and treatment. With bioinformatics entering the post-genomic era, Chinese scholars have established RMVar: a database of RNA modifications (RM) - associated variants that hosts existing RNA modifications as much as possible, which is a useful resource for researchers to understand the basic functions of RNA modifications and its relationship with diseases.8

The treatment and metastasis of tumors in clinical practice have always been the focus and difficulty. Recently, researchers established RMDisease: a genetic variation database that can influence RNA modifications, providing a research platform for clinical workers to explore the pathogenesis of various diseases, including tumors, at the extra-transcriptome level.9 Tumors are difficult to be found in the early stage because of theirs limited volume and no metastasis. Most of the current tumor treatments are surgical resection, radiotherapy and chemotherapy. Such treatments not only have many side effects, but also lead to tumor recurrence and tolerance to treatment, and ultimately failure. Research shows these modifications can affect tumor development, metastasis, and drug resistance and have a certain relationship with the tumor microenvironment, including immune cells, stromal cells, extracellular matrix (ECM), other secretory molecules, blood, and lymphatic networks.10–13

RNA modifications can improve the therapeutic prognosis of tumor patients by regulating the malignant biological behavior of tumor to achieve precise surgical resection and improve the sensitivity of radiotherapy and chemotherapy. Moreover, RNA modifications associated with the tumor microenvironment are especially becoming the current focus of the scientific community, offering hope for the targeted treatment of tumors.

In this review, we summarized the composition and function of RNA modifications in normal cells such as m6A, m5C, m7G, m1A, Ψ, and A-to-I and their association with the tumor microenvironment. Therefore, we can understand RNA modifications in the development and treatment of tumors in depth and provide scientists with ideas for future research on RNA modifications.

The Function of RNA Modifications from Transcription to Translation N6-Methyladenosine (m6A)

In 1975, the concept of m6A modification in RNA was first reported.14 m6A is one of the most common modifications in the messenger RNA (mRNA) of eukaryotic cells, with S-adenosyl-L-methionine (SAM) as the methylated donor.15 m6A methylation can be catalyzed by methyltransferases such as methyltransferase-like 3/14/16 (METTL3/14/16) (“writers”), removed by demethylases such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) (“erasers”), and interacts with m6A binding proteins. The m6A modification takes part in the shearing process of primary mRNA (pre-mRNA) and participates in the maturation of mRNA from the cell nucleus. The role of m6A modification in the downstream regulation of mRNAs, such as translation, export, and stability, is mainly determined by its reader, which recognizes target-modifying mRNA, such as YTH domain family proteins (YTHDCs and YTHDFs) and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) (“readers”)16,17 (Figure 1B).

Figure 1 RNA modifications in tumor cells interact with each other to maintain the dynamic balance of the tumor microenvironment. (A) m1A is generally located in the 5’-UTR of mRNA and can be converted to m6A by Dimroth rearrangement. (B and C) m6A and m5C modifications mediate mRNA processing, stability of RNA, post-translational protein modification, etc. (D) m6A modification of carRNA reversely regulates chromatin. (E) Ψ in rRNA aggregated at the binding site of tRNA and mRNA and thus participated in RNA translation. m7G modification mainly affects tRNA and rRNA and mediates post-translational protein modification. (F) RNA modifications play an important role in the material exchange and information transmission between tumor cells and other cells through the cell-cell “internet” by EVs.

In order to better explore the function of m6A modification, researchers created an online database-DRUM, to combine the association between disease, gene, m6A-RNA methylation sites and gene expression, RNA methylation, disease similarity data of the Random Work with Restart (RWR) algorithm through the method of multi-layer heterogeneous network, to support the query of disease related RNA m6A methylation points.18 Another comprehensive online platform, m6A-TSHub, is used to reveal context specific m6A methylation and gene mutations that may regulate m6A epigenetic markers.19 The establishment of these databases provides a convenient platform for researchers to study m6A mechanism in tumors.

With further study of m6A modification in mRNA, it was found that m6A modification can regulate the stability and translation of mRNA in various biological processes. However, RNA stability mediated by m6A modification is a complex process involving crosstalk with other mRNA modifications, RNA species, or decay pathways. The regulation of mRNA degradation is the main factor affecting the overall mRNA abundance in cells. As efficient transcription results in a relatively long poly (A) tail, m6A modification was not detected in the poly (A) tail; therefore, it may not be involved in polyadenylation-dependent mRNA degradation, which indirectly maintains mRNA stability.20 Just as identified using high-throughput sequencing analysis, m6A modification is enriched in the 3’-UTR region, which contains several important functional domains required for mRNA degradation, such as AU-rich elements (ARE), iron reaction elements (IRE), and cytoplasmic polyadenylation elements (CPE). Moreover, the 3’-UTR is a region targeted by microRNAs (miRNAs); therefore, the possibility that m6A modification is involved in regulating mRNA stability cannot be ruled out.21,22 This characteristic that the mRNA stability is maintained by m6A-dependent mode has been identified in tumors; for example, Wilms’ tumor 1-like protein (WTAP) interacts with its m6A target, long non-coding RNA (lncRNA) DIAPH1-AS1, to promote the growth and metastasis of nasopharyngeal carcinoma in an m6A-dependent manner.23 Another study on head and neck tumors found that the m6A reader-IGF2BP2 could maintain the mRNA stability of Slug in head and neck squamous cell carcinoma cells in an m6A-dependent manner, thereby promoting lymphatic metastasis and epithelial-to-mesenchymal transformation (EMT) of tumors.24 In contrast to m6A enhancing mRNA stability, FTO, as an m6A demethylase, reduces the mRNA stability of apolipoprotein (APOE) by reducing its m6A modification, eventually inhibiting glycolysis and growth in papillary thyroid carcinoma.25 Similarly, ALKBH5, another m6A demethylase, also reduces the mRNA stability of PKMYT1 by reducing its m6A modification, thereby inhibiting the invasion of gastric cancer.26

Precursor proteins are notoriously inactive and often undergo a series of post-translational processes before transforming into mature functional proteins. Studies on the post-translational modification of proteins and m6A in tumor cells have made some achievements. The relationship between the interaction, localization, and phosphorylation of the METTL3-METTL14-WTAP complex produced by m6A modification has been recognized.27 Accordingly, phosphorylated-YTHDF2, mediated by EGFR/SRC/ERK signaling, is located at serine 39 and threonine 38 sites. In order to stabilize the YTHDF2 protein and promote the invasive growth of glioblastoma,28 phosphorylation of AKT in prostate cancer has also been shown to be downstream of METTL3/YTHDF2/LHPP/NKX3-1 to induce tumor proliferation and migration.29 The ubiquitination and SUMOylation of proteins are reversible post-translational modifications that play an important role in molecular regulation.30 Furthermore, the roles of m6A and ubiquitin post-translational modifications have been demonstrated in different tumor subtypes, such as clear cell carcinoma of the kidney and stomach and ovarian cancer.31–33 Moreover, m6A methylation promotes cancer development by ubiquitination of histones.34 Also, the small ubiquitin-like modifier (SUMO) catalytic cycle includes maturation, activation, binding, linking, and modification. Disorders of the SUMO system are associated with many diseases, particularly tumors. SUMOylation is widely involved in carcinogenesis, DNA damage response, proliferation, metastasis, and apoptosis of cancer cells.35 The roles of m6A modification and SUMOylation in tumors are also being studied. For example, METTL3 is modified by SUMO1 primarily at lysine residues, including K177, K211, K212, and K215, which the SUMO1-specific protease, SENP1, can reduce. SUMOylation of METTL3 significantly inhibits its m6A methyltransferase activity, thereby decreasing m6A mRNA levels.36 The influence of SUMOylation on the ability of YTHDF2 to bind m6A modified mRNA and the regulation of mRNA after binding have also been found. Therefore, a new mechanism by which the m6A reading protein, YTHDF2, promotes cancer development was revealed.37 The SUMO-modified METTL3 made CirC_0000677 promote proliferation and drug resistance in colorectal cancer (CRC)38 (Figure 1B).

Finally, the one-way flow of genetic instructions from DNA to RNA to protein is now the dominant idea in many studies. However, in a recent study, we found that the knockout of m6A methylation-related enzymes, METTL3 or YTHDC1, in embryonic stem cells from mice increased chromatin openness and activated transcription through chromosome-associated regulatory RNA (carRNA) in an m6A-dependent manner. m6A acts as a switch that influences the abundance of carRNAs, thereby regulating nearby chromatin status and downstream transcription39 (Figure 1D). Therefore, the process of methylation in cells is reversible and dynamic. None of the writers, the erasers, or readers are isolated from each other, which can be explored in terms of their functions.

There have been far fewer studies on m5C, m7G, m1A, Ψ, and A-to-I compared to those on m6A;40 however, this does not mean that these RNA modifications are not important. Therefore, these RNA modifications are discussed in this review.

5-Methylcytosine (m5C)

The mRNA distribution characteristics of m5C modifications are highly conserved in mammals; however, the genes modified by m5C modifications in different tissues are specific. A recent study has suggested that m5C modification is mainly distributed in CG-enriched regions.41 Studies have found that NOP2/Sun domain family member 2 (NSUN2) (“writer”) protein is the main mRNA m5C methyltransferase, and its activity depends on C271 and C321 sites. Loss of NSUN2 prevents mRNA from being transported from the cell nucleus.42 The nucleation regulator protein, Aly/REF export factor (ALYREF) (“reader”), specifically binds to the m5C modified site through lysine 171, thereby promoting mRNA nucleation43 (Figure 1C).

The m5C modification is distributed with mRNA, enriched around the 5’-UTR and 3’-UTR, and conserved in transfer RNA (tRNA) and ribosomal RNA (rRNA). It is dynamically regulated by related enzymes, including methyltransferases (members of the NSUN, DNMT, and TRDMT families), demethylases (the Ten-eleven translocation family and AlkB homolog 1, ALKBH1), and binding proteins (ALYREF and YBX1). Previous studies have indicated that the m5C modification is involved in multiple RNA metabolic processes, including mRNA output, RNA stability, and translation44,45 (Figure 1C).

The role of high-throughput technology in m5C modification is also reflected. Researchers predicted the prognosis of m5C regulator-NSUN3 and NSUN4 on lung squamous cell carcinoma (LUSC) by combining the public LUSC data set of LUSC.46 CIBERSORT tool and TIMER database were used to evaluate the relationship between the risk model of m5C-related lncRNA in lung adenocarcinoma and tumor infiltrating immune cells;47 Scientists obtained the expression matrix of m5C lncRNA in breast cancer (BC) from the cancer genome atlas database, and finally constructed a BC-specific m5C lncRNA risk model.48 What’s more, researchers obtained triple negative breast cancer (TNBC) data from cancer genome map and gene expression summary, and analyzed the specificity and role of m5C regulator-NSUN2 and NSUN6 in tumor immune microenvironment.49 Through TCGA database, NGS analysis and other means, the role of m5C modification in different types of tumors has been gradually understood.

Similar to m6A modification, m5C modification is also involved in the post-protein modification; ubiquitination and SUMOylation of m5C contribute to tumor development. The reaction between ubiquitin modification and m5C-modified mRNA in epithelial ovarian cancer (EOC) can lead to mRNA instability.50 In a study of gastric cancer, the SUMO2/3 promoted the carcinogenic activity of NSUN2 by stabilizing NSUN2 and mediating its nuclear transport by directly interacting with NSUN2.51 Moreover, the role of m5C modification in non-coding RNA has also been discovered by scientists. It was recently found that the lack of m5C in mitochondrial RNA (mtRNA) in oral cancer cells caused functional changes in the mitochondria, severely impairing their metabolic plasticity and promoting tumor progression. We believe that with technological progress, researchers will have a deeper understanding of m5C52 (Figure 1C).

N7-Methyladenosine(m7G)

m7G RNA methylation can regulate mRNA transcription, miRNA biosynthesis, biological functions, tRNA stability, and 18S rRNA nuclear processing and maturation53,54 (Figure 1E). m7G RNA methylation is present in various molecules, including the mRNA 5’cap, mRNA interior, pre-miRNA, tRNA, and rRNA. First, RNA phosphatase cuts off the last phosphate group at the 5’ end of the mRNA, and then with the assistance of mRNA guanylyltransferase, GTP is linked to the 5’ end of the mRNA in the form of a 5’-5’ triphosphate bond. Subsequently, guanine-N7-methyltransferase transfers the methyl group on S-adenosine to the N7 position of guanine to complete the m7G modification, and the 2’-O-ribose methylation modification of adjacent nucleotides is catalyzed by a specific methyltransferase.54,55 Furthermore, RNA methyltransferase, m7G methyltransferase-like 1 protein (METTL1), catalyzes m7G modification of tRNA. METTL1 depletion leads to a decrease in the abundance and cell cycle of m7G modified tRNA, especially for TCT-4-1-Arg,56,57 and inhibition of carcinogenicity such as intrahepatic cholangiocarcinoma,58 lung cancer59 bladder cancer.60 A recent study showed that increased m7G modification of tRNA subsets by the METTL1/WD repeat domain 4 (WDR4) complex stabilizes these mRNAs to improve the efficiency of protein translation and reduce ribosomal dormancy, leading to higher malignancy in human cancers.61 In hepatocellular carcinoma, m7G methylation and ubiquitination of p53 lead to decreased expression of p53 and promote tumor progression62 (Figure 1F). In addition, scientists recently found that m7G methylation inhibits lung cancer cell migration by promoting the maturation of miRNA let-7,63 thereby discovering the role of m7G in non-coding RNA and providing new ideas for future research.

N1 - Methyladenosine (m1A)

m1A is produced by adding methyl groups to the N1 position of adenosine. Under physiological conditions, m1A is positively charged and may influence local RNA structures or protein-RNA interactions. Its methyl group is located at the Watson-Crick base-pairing interface, thus blocking normal base pairing with thymine nucleoside or uridine. Alternatively, m1A can form hydrogen bonds with other nucleosides through Hoogsteen base pairing, which is relatively unstable compared to the normal A:U or A:T base pairing. In the case of chemical transformation, m1A can undergo Dimroth rearrangement under alkaline conditions to form m6A5,40,64 (Figure 1A). However, recent studies mainly focused on m6A, unlike m1A, which was relatively poorly studied.

m1A is distributed among mRNA, tRNA, rRNA, and mtRNAs. Unlike m6A, Ψ, and other RNA modifications, m1A is mostly distributed in the 5’-UTR of mRNA (Figure 1A). The locus distribution of m1A in tRNA was 9,14,22,57,58. The expression of m1A modification was low at sites 14 and 22 and caused negligible biological effects. The modification of site 57 occurs as an intermediate of 1-methylinosine (m1I). The most abundant m1A modifications are at sites 9 and 58, which play an important role in tRNA’s tertiary structure and stability maintenance. There are three m1A sites in the nuclear-coding rRNA, all located on the large subunit rRNA. Although mtRNA lacks the 5’-UTR, m1A was detected in its CDS coding region. In addition, the mitochondrial methyltransferases TRMT61B and TRMT10C can target mtRNAs.5,65

As mentioned before, m1A modification in tumors is rarely studied compared with other RNA modifications. One of the main reasons is that m1A modification is rare in RNA.66 Previous technical limitations were mainly based on the normal physiological effects of m1A modifications in cells under cell culture conditions. However, with the progress of high-throughput sequencing technology, the role of m1A in different types of tumors, such as hepatocellular carcinoma,67 glioma68,69 and bladder cancer70 has been gradually clarified. It has been found that the upregulation of m1A expression by TRMT6 and other methyltransferase family members promotes the malignant behavior of these tumors. Therefore, m1A can be regarded as a new tumor marker for monitoring tumor development and the prognostic evaluation of tumor therapy in these tumors.67–70

Pseudouridine (Ψ)

The ribosome is the site of protein synthesis, and rRNA is the most abundant type of RNA in cells. Ψ, the most abundant post-transcriptional modification of RNA, is the fifth nucleotide. Ψ is the C-C glycoside isomer of uridine (U). It binds the C5 atoms of the base to the glycoside bond, which allows Ψ to have an additional hydrogen bond donor at the non-Watson-Crick edge and then contributes to maintaining the stability of its host RNA.6 In addition, Ψ aggregates in important functional regions of rRNA, especially at the binding sites of tRNA and mRNA, and thus participates in RNA translation modifications71 (Figure 1E).

With the development of high-throughput assays, thousands of Ψ sites have been dynamically distributed on the coding sequence and 3’-UTR of mRNA, and Ψ has been identified in the positions associated with the alternative splicing region in the newly born pre-mRNA. PUS1, PUS7, and RPUSD4, three tissue-specific pre-mRNA-modified Ψ synthases, control a wide range of changes in pre-mRNA splicing and 3’-terminal processing.72 The increase in the expression of PUS7 inhibits pseudouridylation of tRNA in glioblastoma stem cells (CSCs) and leads to the generation of glioblastoma.73 Similarly, HSP90 can rely on the overexpression of PUS7 to promote CRC metastasis.74 Recent research on Ψ has revealed new findings. In neuroblastoma cells, SNHG25 promotes the accumulation of SNORA50C and the assembly of the associated small nucleolar ribonucleoprotein (snoRNP) through pseudouridine synthase 1 (DKC1), which ultimately promotes the growth and migration of neuroblastoma (NB) cells. Eventually, there will be more functions of Ψ in more RNA types that will be recognized.

Adenosine-to-Inosine (A-to-I)

The conversion of adenosine to inosine by hydrolysis has become the most common RNA editing in higher eukaryotes, which can be considered a special type of RNA “modification” and plays a vital role in coding RNA and non-coding RNA.7 Such irreversible adenosine deamination occurs in double-stranded RNA (dsRNA), which is highly conserved in humans and is mediated by the RNA-acting adenosine deamination enzyme family, ADAR.75 A-to-I plays different roles in different organelles. When identified as guanosine, it can lead to recoding and alternative splicing in proteins or make some changes in the specificity of miRNA; when recognized by inosine-binding proteins, its transcripts can be retained in the nucleus or degraded. Recent studies have found that specific dsRNA sensors in cancers induce antiviral responses that inhibit cell division and promote apoptosis, a new addition to RNA-targeted therapy.75,76 In addition, an increasing number of studies have focused on the RNA editing enzyme, ADARs, and found that ADAR1-mediated A-to-I editing can effectively maintain the stability of tumor stem cells in glioblastoma and cell immortalization in breast cancer as a proto-oncogene.77,78 In addition, when the expression of ADAR1 is downregulated, some tumor suppressor miRNA are activated to inhibit the growth and invasion of triple-negative breast cancer cells.79

RNA Modifications in Antisense RNA in Tumor Cells

Reviewing the above types of RNA modifications, we can clearly understand that these modifications exist in both coding and non-coding RNAs, playing their respective functions. Here, we discuss the RNA modifications of a more overlooked RNA, the antisense RNA.

An RNA molecule complementary to mRNA and other RNA antisense RNA in eukaryotes has been found.80,81 Since ribosomes cannot translate double-stranded RNA, the specific complementary binding of antisense RNA to mRNA inhibits the translation of the mRNA.82 Based on this principle, in recent years, antisense RNA genes have been synthesized artificially and transcribed into antisense RNA by introducing them into cells,82,83 which can inhibit the expression of a specific gene and block the function of the gene, suggesting that the role of RNA modifications in antisense RNA is worth studying in tumors, such as lncABHD11-AS84 and lncMAPKAPK5-AS.85 For example, m6A reader YTHDF3 enhances its interaction with DICER1-AS1 and participates in the transcription of the sense gene DICER1, thereby regulating glycolysis and tumor development in pancreatic cancer.86 Although there are few studies on RNA modifications in antisense RNA, and almost all of them focus on m6A, antisense RNA still needs to be given attention because it can suppress the expression and function of target genes like cancer-promoting genes. Moreover, RNA modifications can enhance the function mentioned above, providing the prospect for targeted tumor therapy in the future.82,83

RNA Modifications in the Tumor Microenvironment

The tumor microenvironment refers to tumor cells’ internal and external environments during tumorigenesis, growth, and metastasis. This finding is clinically significant for tumor prevention and treatment.87 The complex tumor microenvironment contains various cells and components: (1) immune cells: T and B lymphocytes, tumor-associated macrophages (TAM), dendritic cells (DC), natural killer cells (NK), neutrophils, myeloid-derived inhibitory cells (MDSC), etc.; (2) stromal cells: cancer-associated fibroblasts (CAF), pericytes, mesenchymal stromal cells, etc.; (3) extracellular matrix (ECM) and other secretory molecules: growth factors, cytokines, chemokines, and extracellular vesicles (EVs); (4) Blood and lymphatic vascular networks.10–13 The relationship between RNA modifications and the tumor microenvironment, especially the role of m6A and m5C modifications, has been described in many kinds of cancer subtypes such as lung adenocarcinoma,88,89 pancreatic carcinoma90,91 and CRC;92,93 however, these studies are not in-depth and very few in number (Figure 1).

Similar to solid tumors, studies of RNA modifications in hematologic malignancies have been conducted.94,95 For example, lncRNA UCA1 can promote the progression of acute myeloid leukemia (AML) by increasing the expression of METTL14.96 Furthermore, the level of m6A expression in follicular lymphoma and AML may be used to monitor the tumor microenvironment for targeted treatment. Similarly, detecting m6A modification levels in lncRNA helps evaluate the prognostic level of patients with acute and chronic myelogenous leukemia.97,98

Tumor Hypoxia and Biotransformation

The main reason why carcinomas are difficult to cure is that tumor cells will continue to adapt to the adverse tumor microenvironment quickly. Tumor hypoxia is mainly caused by an imbalance between low oxygen supply caused by abnormal vascularization and high oxygen consumption of tumor cells.99 The rapid proliferation of tumor cells accelerates oxygen consumption and prevents the amount of oxygen available for further diffusion into tumor tissues. For example, hypoxia-mediated reader-YTHDF2 overexpression of m6A results in the activation of the mTOR/AKT axis, thereby promoting the proliferation and invasion of lung squamous cell carcinoma.100

In hypoxic environments, tumor cells mainly absorb energy through glycolysis. Some recent studies have shown the role of RNA modifications in hypoxia and glycolysis in tumor cells. They also show that the dysregulation of energy metabolism caused by abnormal RNA modifications like m6A may be related to the malignant biological behavior of tumors.101 Furthermore, among solid tumors, pyruvate kinase muscle isozyme M2 (PKM2) is involved in tumor metabolism and growth as a rate-limiting enzyme in glycolysis, while overexpression of ALYREF promotes bladder cancer cell proliferation through glycolysis mediated by indirect upregulation of PKM2 expression through activation of ALYREF and hypoxia-inducible factor-1 α (HIF-1α);102,103 FTO can reduce the mRNA stability of APOE and inhibit glycolysis in thyroid papillary carcinoma (PTC) by reducing m6A modification.25 Furthermore, RNA modifications also act on glycolysis in liquid tumors. For example, phosphofructokinase PFKP and lactate dehydrogenase B (LDHB), two key glycolytic genes, were effectively regulated by FTO/m6A/YTHDF2. R-2-hydroxyglutaric acid (R-2HG) restored glycolysis in AML cells by inhibiting the expression of PFKP/LDHB mediated by FTO/m6A/YTHDF2.104

Moreover, most studies on RNA modifications in the biotransformation of tumor cells have focused on the energy metabolism of tumors, especially glycolysis and lactic acid synthesis, in the anoxic environment of tumor cells. However, fat metabolism, deoxycholic acid metabolism, and other bio-transformations are equally important and can be regulated by RNA modifications. The increase in R-2HG produced by IDH1/2 inhibited adipogenesis and decreased the activity of the adipose-related protein, FTO, thereby increasing the level of m6A in leukemia cells. The MYC/CEBPA signaling pathway was then inhibited to prevent leukemia progression.105 In addition, m6A modification can regulate deoxycholic acid in deoxycholic acid metabolism to inhibit gallbladder cancer.106

In summary, the studies of m6A modification in hypoxia and biotransformation have been considered for both solid and liquid tumors. However, it is undeniable that other RNA modifications, except m6A, have not been sufficiently reported to date, which needs to be considered by the scientists in future.

Immune Escape and Cell Networks in Tumors

Currently, many studies on RNA modifications have focused only on tumor cells. However, tumor cells are not isolated, and their growth and metastasis are influenced by the assistance of other cells in the body or EVs in the intracellular environment. The complex interactions between cancer and immune cells are also important in researching new cancer treatments. Immune cells can also stimulate the growth of tumor cells instead of protecting the body. For example, neutrophils can promote the circulation of the cell cycle of circulating tumor cells in breast cancer.107

Recent studies have suggested that the deletion of METTL3 in macrophages reshaped the tumor microenvironment by enhancing tumor invasion by M1- and M2-like TAM and regulatory T Cell (Treg) and had a synergistic effect on the growth and invasion of tumor cells, leading to high malignancy108 However, deleting the mRNA m6A-binding protein YTHDF1 in dendritic cells enhanced anti-tumor immunity109,110 Similarly, METTL3-mediated methylation of m6A RNA in NK cells promotes anti-tumor immunity.111 Finally, ALKBH5 promotes the assembly of hypoxia-induced paraspeckles and secretion of IL8 to generate an immunosuppressive tumor microenvironment.112 For example, intrinsic ALKBH5 inhibits T-cell proliferation and cytotoxicity by maintaining PD-L1 expression in tumor cells, which promotes immune escape.113 In addition to m6A, METTL1 and the m5C modulator-NOP2 are associated with immune invasion in many tumor subtypes.114,115 As a piece of evidence, upregulation of METTL1 enhances TGF-β2 translation by inducing myeloid-derived suppressor cells to form an immunosuppressive environment, thereby leading to HCC recurrence.116 Also, the NSUN2 promotes the progression of NPC by regulating immune infiltration.117 Therefore, we can clarify the role of RNA modifications in tumor immunity.

EVs are increasingly regarded as important mediators of intercellular communication and the transport of macromolecules. As a piece of evidence, cells communicate with each other using some miRNAs transported by EVs, which constitutes a cell-cell “internet”.118 RNA modifications are efficiently delivered on such “internet.” Information and materials can be exchanged between tumor cells and immune cells through EVs.119 Furthermore, M2-like TAM can secrete lncMAPKAPK5-AS1(MAAS)-carrying exosomes (a type of EVs). These exosomes act as a medium for communication between TAM and tumor cells, which can be transferred to HBV + HCC cells and promote the proliferation of HCC cells85 (Figure 1F).

Therefore, this provides a new idea for future studies on RNA modification in the tumor microenvironment.

Tumor Invasion and Metastasis Epithelial-to-Mesenchymal Transformation (EMT)

EMT is an important biological process by which epithelial-derived malignant tumor cells acquire the ability to migrate and invade. Through EMT, epithelial cells lose their polarity and connection to the basement membrane and other epithelial phenotypes and acquire a higher ability to migrate, invade, resist apoptosis, and degrade the extracellular matrix. EMT is characterized by the decreased expression of cell adhesion molecules (such as E-cadherin), the transformation of the cytoskeleton from keratin to vimentin, and mesenchymal cell morphology.120,121 The role of m6A modification in EMT has been extensively described in different tumors,122 such as glioblastoma,123 lung adenocarcinoma, breast cancer,124 head and neck cancer,24 etc. In addition, METTL3 is an important promoter of EMT and metastasis of gastric cancer cells. It is an important downstream target gene that plays an important regulatory role in the metastasis of gastric cancer cells and breast cancer.125 m5C modification and m7G methyltransferase- WDR4 can enhance EMT in lung adenocarcinoma102 and hepatocellular carcinoma cells.62,126

In addition to EMT, the extracellular matrix also plays an important role. It has been found that the upregulation of m5C regulatory factor- NSUN2 is closely related to spliceosome, RNA degradation, cell cycle signaling pathway, and RNA polymerase in TNBC.49 Moreover, another m5C regulatory factor-NSUN6 downregulation is associated with extracellular matrix receptor interactions, metabolism, and cell adhesion. m5C-related-lncRNAs in pancreatic ductal adenocarcinoma and lung adenocarcinoma can regulate the tumor immune microenvironment,47,127 and METTL1-mediated m7G tRNA modification promotes bladder cancer development through extracellular matrix regulation,60 thereby inspiring subsequent studies on the role of RNA regulation in tumors.

Angiogenesis

Angiogenesis has been recognized as a marker of cancer. Newborn blood vessels can provide energy to tumors, promote tumor growth and metastasis, and lead to drug resistance and recurrence.128,129 Moreover, the employment of anti-angiogenesis drugs is becoming a new method of tumor therapy, aiming to cause less harm to tumor patients and have a better prognosis than traditional therapy.130 The role of RNA modifications in angiogenesis has also gradually been recognized. For example, the increased m6A can promote angiogenesis in glioblastoma and head and neck squamous cell carcinoma.123,131 Furthermore, m7G Methyltransferase METTL1 promotes angiogenesis in peripheral vascular diseases.132 However, the detailed mechanism of m6A in tumor angiogenesis is unclear, and the role of other RNA modifications in tumor angiogenesis has not been clarified. Therefore, these are areas that scientists should explore in the future.

In summary, RNA modifications in tumor cells are not isolated, static, and simple but occur in an interconnected, dynamic, and complex tumor microenvironment. Therefore, the existing research on RNA modifications and tumor microenvironment is insufficient, and future studies can be more detailed, systematic, and comprehensive to provide new ideas for tumor treatment.

RNA Modifications Regulate Tumor Treatment Resistance

It is well known that drug resistance in tumor cells is an important factor leading to tumor treatment failure. Recently, RNA modifications have been found to play a unique role in regulating tumor drug resistance. Therefore, it is necessary to summarize the regulatory effects of RNA modifications on tumor treatment, such as 5-fluorouracil (5-FU), cisplatin, and other drugs, to improve tumor resistance and explore new options for tumor treatment in the future.133 (Figure 2, Table 1).

Table 1 Comparison of Different Therapeutic Methods in RNA-Modified Tumors

Figure 2 Overview of RNA modifications in different therapies. (A) Chemotherapy drugs-5-FU and CDDP regulate the signal pathways/axis of lncRNA, circRNA and miRNA by regulating the regulators of m6A, m5C and m7G (“Writer”-METTL1, METTL3, “Reader”-YTHDF1), thus inducing tumor chemotaxis. (B) Radiotherapy antagonizes the action of m6A modification by destroying the stability of mRNA in tumor cells. (C) ATF3 and AK4 regulated by m6A “Writer”-METTL3 can regulate the resistance of breast cancer cells to tamoxifen. (D) The signal pathways/target axis mediated by RNA modification can affect the biological behavior of different types of tumors; m6A, m7G “Writer”-METTL3, WDR4 can induce sorafenib resistance in HCC cells. (E) Some active components in traditional Chinese medicine have also been found to interact in RNA modification. In the figure, the upward black arrow represents “upward adjustment”, and the downward black arrow represents “downward adjustment”.

Chemotherapy 5-Fluorouracil (5-FU)

5-FU is commonly used to treat squamous cell carcinomas.134 As an anti-metabolite that prevents cell proliferation, 5-FU mainly inhibits thymidine synthase and blocks the formation of thymidine required for DNA synthesis. Although it has a relatively short half-life (< 30 min), it can easily enter the brain through passive diffusion. Clinically, it can be used either as a single agent or in combination with other chemotherapeutic therapies.135

LncRNA -LBX2 antisense RNA1 (LBX2-AS1) is an important regulator of cancer progression. The increase in LBX2-AS1 expression in CRC is mediated by METTL3-depended m6A methylation, which promotes CRC progression and leads to resistance to 5-FU in CRC.136 Similarly, circPTK2 can mediate the m6A “reader” protein YTHDF1, leading to the 5-FU resistance on CRC.137 Exosomes are naturally occurring EVs that carry biomolecules such as proteins, miRNAs, and metabolites.138 Its properties suggest special effects for the treatment of tumors. Exosomes secreted by CAF from CRC inhibit the efficacy of 5-FU by transporting miRNA-miR-181D-5p, which m6A modifies. Therefore, a new discovery has been made regarding the mechanism of chemotherapeutic resistance in CRC.139

Furthermore, METTL3 is an effective target for enhancing the therapeutic effects in patients with pancreatic cancer. METTL3-depleted pancreatic cancer cells showed higher sensitivity to anti-cancer agents than those without the regulation of METTL3, such as gemcitabine, 5-FU, cisplatin, and radiation.140 In addition to m6A modification, other RNA modifications such as m5C and m7G have not been studied in relation to 5-FU, which provides a new idea for future research on RNA modifications such as m5C and m7G and tumor chemotherapy resistance. (Figure 2A)

Cisplatin (CDDP)

Cisplatin is another chemotherapy drug that has also been commonly used and has good efficacy in treating tumors, with poor efficacy after radiotherapy and surgical resection, especially in head and neck tumors.141 Its mode of action is associated with its ability to cross-link purine bases on DNA. Furthermore, it can interfere with DNA repair mechanisms, leading to DNA damage and the subsequent apoptosis of cancer cells.142,143

The combined effect of RNA modifications and cisplatin showed that increased METTL1 results in increased modification and expression of m7G-modified tRNA, which leads to enhanced codon recognition during mRNA translation, upregulation of the WNT/β-catenin signaling pathway, promotion of EMT, and chemoresistance to cisplatin and docetaxel in vitro and in vivo.144 The combined role of cisplatin and RNA modifications in tumors other than head and neck tumors is also widely recognized. The overexpression of METTL1 in m7G regulation makes colon cancer cells sensitive to cisplatin by regulating the miR-149-3p /S100A4/p53 axis.145 m5C-related lncRNA can regulate the microenvironment of low-grade glioma;146 m5C-modified mRNA can also be unstable owing to YBX-1 ubiquitination by SIAH1 at Lys304, thus making EOC cells sensitive to intracellular cisplatin (CDDP). In addition, the overexpression of SIAH1 enhances the anti-tumor efficacy of cisplatin in vitro and in vivo.50 Certainly, there has been some progress in studying m6A modifications and drug resistance in tumors. m6A demethylase-ALKBH5 leads to drug resistance in EOC, inhibits bladder cancer proliferation through glycolysis, and enhances sensitivity to cisplatin.147,148 IGF2BP1 and YTHDF1, as m6A readers, are both vital in cisplatin’s resistance. CircMAP3K4 modified by IGF2BP1 can protect HCC cells from cisplatin.149 Similarly, YTHDF1 can also mediate m6A modifications in breast cancer cells, leading to cisplatin resistance150 (Figure 2A).

Radiotherapy

Radiotherapy, as an important treatment of tumor, targets cancer cells by damaging DNA, which has been found to be related to tumor immune microenvironment.151 It has been shown that radiation in GBM (glioblastoma) cells enhances the expression of METTL3, thereby enhancing the m6A level. By recruiting HuR (human antigen R), m6A modification can improve the stability of SOX2, then resisting radiotherapy.152 Similarly, in pancreatic cancer (PC), MAPK, ubiquitin and RNA splicing pathways can rely on m6A modification to promote radio-resistance.140 The point that m6A antagonizes the maintenance of mRNA stability and the effect of radiotherapy has been gradually recognized by researchers, which has an impact on the improvement of tumor treatment.153 (Figure 2B).

Endocrine Therapy

Some specific tumors have been proved to be related to hormone levels, for example, breast cancer. Tamoxifen (TAM) is the most prescribed selective estrogen receptor modulator (SERM) for hormone receptor positive breast cancer patients.154 A recent study found that the protein expression level of ATF3 in tamoxifen resistant (TamR) MCF-7 cells was significantly higher than that in corresponding parent cancer cells. Further research found that m6A-modified ATF3 could regulate the resistance of breast cancer cells to tamoxifen.155 Similarly, another study found that the protein levels of adenylate kinase 4 (AK4) and m6A “writer” METTL3 in tamoxifen resistant (TamR) MCF-7 cells were significantly higher than those in their parent cells, and found a new m6A-mediated external transcriptome mechanism regulating AK4.156 Through RNA modifications, tamoxifen, a traditional chemotherapy drug for breast cancer, has achieved better efficacy (Figure 2C).

Targeted Therapy

Increasing tumor therapies can be reflected in targeted therapy owing to the increasingly thorough study of the role of RNA modifications. Various signaling pathways have been further studied, except for the above traditional treatment methods, whose role has gradually been recognized. Esophageal squamous cell carcinoma (ESCC) cells downregulate APC by METTL3/YTHDF coupling, thus increasing the activity of the Wnt/β-catenin pathway.157 CircRNA can act as miRNA sponges to regulate cancer progression. For example, circORC5-mediated m6A modification regulates the miR-30C-2-3p /AKT1S1 axis to inhibit gastric cancer tumor growth.158 Moreover, several known molecularly targeted drugs are involved in RNA modifications, mainly m6A modification.159 For example, sorafenib, a multi-kinase inhibitor, is a first-line targeted agent approved by the US Food and Drug Administration for advanced HCC.160 Sorafenib can prolong the survival of patients with HCC by inhibiting tumor growth and angiogenesis by targeting the RAF/MEK/ERK pathways and receptor tyrosine kinases.161 However, drug resistance limits their efficacy. The m6A methyltransferase METTL3 promotes m6A modification of the lncRNALINC01273, resulting in sorafenib resistance.162 In sorafenib-resistant HCC, the increased level of circRNA-SORE is caused by increased m6A. These results revealed a new mechanism of sorafenib resistance and provided evidence for circRNA-SORE as a new drug target for sorafenib treatment in patients with advanced HCC.163 m7G methyltransferase-WDR4 promotes mRNA stability and translation of CCNB1 to enhance HCC progression and contribute to sorafenib resistance.62 However, the role of sorafenib in regulating m5C has not yet been studied (Figure 2D).

Traditional Chinese Medicine

In addition to traditional chemotherapy drugs and molecular targeted drugs, some effective components extracted from traditional Chinese medicine have also been proved to have multiple targets, slight side effects and good tumor treatment effects.164,165 Recently, some scholars found that Chinese herbal extracts can be regulated by m6A to play a role in the treatment of tumors.159 Humantenine, an alkaloid isolated from Gelsemium elegans, was found to have an impact on the m6A-modified colon cancer cell line HCT116, through bioinformatics analysis.166 Another study on liver cancer patients with type-2 diabetes (T2D) found that Baicalin significantly inhibited the epigenetic modification of m6A-modified HKDC1 in HepG2 tumors, thereby inhibiting T2D induced liver tumors.167 These studies not only proved the anti-tumor effect of these traditional Chinese medicine preparations, but also improved their efficacy by exploring the influence of RNA modifications on their anti-tumor effect (Figure 2E).

In conclusion, these recent studies have highlighted the important role of RNA modifications in cancer therapy.

RNA modifications play different roles in various tumors. For example, regarding m6A modification, METTL14 plays the role of tumor inhibitor in CRC,168,169 bladder cancer,170 and breast cancer.171 It is a carcinogenic factor in cervical cancer,172 EOC,173 pancreatic cancer174 and leukemia175,176 during their development and incidence.177,178 Therefore, studies on RNA modifications should be carried out extensively and comprehensively in various types of tumors.

Discussion

Tumor growth and development are complex processes. Under hypoxic conditions, tumor cells mediated by RNA modifications can still obtain energy through glycolysis to satisfy their growth needs.25,101,104,105,179 When the surrounding nutrients are exhausted, tumors can generate blood vessels under the regulation of RNA modifications, which can connect them with the vascular system (blood vessels and lymph vessels) and membrane blood vessels in the human body to obtain energy and other materials elsewhere or be carried to obtain a better living environment in the distance.123,128–132 Furthermore, tumor and immune cells in the tumor microenvironment can form a cell-cell “internet”, and immune cells that should protect the human body help tumor cells grow and metastasize well with the participation of RNA modifications. Therefore, RNA modifications are vital in many aspects of tumor cell growth and development.107–115,180

Of course, it is undeniable that although RNA modifications have received unprecedented attention, the role of DNA and Histone modifications as members of epigenetic modifications in tumor growth and development cannot be ignored. Tumor cells are in the proliferation phase of the cell cycle for a long time, and explosive growth after cell dormancy makes tumor treatment difficult.181,182 It was recently found that histone modification could inhibit the transcription of lncRNA miR100HG, thereby inhibiting its role in inducing cell cycle G0-G1 arrest in CRC and promoting cell proliferation.183 Similar to RNA modifications, m6A modifications are also present in DNA. N6AMT1, a functional methyltransferase modified by m6A in genomic DNA, inhibits tumor proliferation through the transcription of cell cycle inhibitors.184 However, unlike RNA, DNA, as the source of gene expression, is particularly “unstable” in the cell cycle. DNA damage can cause genomic instability, threatening the maintenance of cellular homeostasis and allowing cells to escape programmed death and immortalize.185

RNA modifications occur through the entire process of transcription, translation, and post-translational modification. The relationship between RNA and DNA modifications is a direction for future research. A recent study on leukemia revealed that m6A readers in RNA modifications could regulate DNA replication and play a key role in the occurrence and development of leukemia.186 As mentioned earlier, m6A modification occurs in both DNA and RNA. Both are of great significance in the occurrence and development of tumors. However, research has not clarified the relationship between the two to date.184 Currently, scientists have found that m6A, as a multifunctional checkpoint, can couple different levels of gene regulation through non-coding RNA as an intermediary.187 Furthermore, changes in DNA, such as base modification and mutation, will cause relevant changes in the activity and function of corresponding transcription factors.188 As a piece of evidence, p53, as a tumor suppressor gene in common transcription factors, can effectively maintain genomic stability. Post-translational modification is the most effective way to regulate its activity.189,190 Activated p53 can quickly be recruited at damaged DNA sites to guide DNA repair,191 and the post-translational modification of p53 involves m6A modification.176 In conclusion, RNA modifications can affect DNA modifications through non-coding RNA, transcription factors, and other factors. Scientists have focused on studying the repair effect of tumor suppressor genes in DNA to treat tumors. Therefore, many signal transduction pathways have been explored, and many studies have proposed RNA-modified pathways to provide tumor-specific therapeutic strategies.

However, studying RNA modifications in tumors is bound to be a long way off for several reasons. First, the dynamic expression patterns of writers, readers, and eraser proteins complicate the identification of the precise functional consequences of modified abnormal deposition in RNA metabolism and tumor cell fate determination. Previous studies have been limited to the role of individual writers, readers, or eraser proteins in RNA modification in specific tumor subtypes. Currently, it is not clear how specific RNA modifications af