For m⁶A-modified mRNA to perform specific biological functions, a specific RNA-binding protein, methylation reader protein, is required. It mainly includes YTH m⁶A RNA-binding protein (YTH) domain proteins (including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) (9), heterogeneous nuclear ribonucleoproteins (HNRNPC, HNRNPG, and HNRNPA2B1) and eukaryotic initiation factor (eIF). The functions of these reader proteins are mainly to alter protein-RNA interactions by impairing the homologous binding of m⁶A to RNA-binding proteins and altering RNA secondary structure (10). Studies have found that YTHDF1 has a clear oncogenic role, and its high expression in cancer genes can accelerate the transformation of important oncogenic drivers in cancer via numerous methods, impacting cancer progression and prognosis. For example, in gastric cancer progression, mutated YTHDF1 enhances the expression of the key oncogenic factor Wnt receptor Frizzled7 (FZD7), leading to gastric cancer progression and poor prognosis (11).

The m⁶A methyltransferase consists of methyltransferase-like 3 (METTL3), METTL14, WTAP, RBM15, ZC3H13, VIRMA, and the newly discovered METTL16, which are also called writers. Its primary function is to catalyze the m⁶A modification of adenylate on mRNA. METTL3 and METTL14 have a critical catalytic domain (12), and the METTL3-METTL14 methyltransferase complex assembly during m⁶A modification is a crucial factor initiating the modification. In addition, methyltransferases also include associated protein (WTAP), RBM15, ZC3H13, vir-like m⁶A methyltransferase associated (VIRMA), and METTL16. These methyltransferases all play an important role in forming METTL3-METTL14 complexes in different links, affecting cancer cell proliferation and migration (13–16).

m⁶A modification is the earliest reversible modification found among many RNA modifications, and the reversibility of its modification is due to the existence of demethylases. In addition, their encoding genes are called “erasers”. Fat mass and obesity-associated protein (FTO) is the first demethylase discovered and is a member of the Alkb protein family, which can affect the RNA-binding ability of the splicing factor serine and arginine-rich splicing factor 2 (SRSF2), thereby regulating the splicing process of pre-mRNA (17). ALKBH5, another member of the Alkb protein family, was found to have a demethylation function, which can directly catalyze m⁶A-methylated adenosine to remove methyl groups, different from the oxidative demethylation of FTO (18).

LncRNAs, a type of ncRNAs of 200 or more nucleotides, can regulate gene expression at multiple levels (21). Many m⁶A-modified lncRNAs have been found recently, and they can control gene expression and function in a variety of ways. They can act as transcriptional regulators, acting in cis or trans, regulating the transcription of adjacent genes (22). XIST, one of the first functionally annotated lncRNAs, plays a key role in X chromosome inactivation by recruiting multiple factors (23). A study shows that in head and neck squamous cell carcinoma, METTL3- and METTL14-mediated m⁶A methylation contributes to the stability of LNCAROD, and LNCAROD overexpression promotes the malignant development of HNSCC by promoting YBX1-hspa1a interaction, thereby enhancing the stability of the YBX1 protein (24). Another study found that modifying lncRNAs with m⁶A had the opposite effect on cancers. METTL14 suppressed colorectal cancer growth and metastasis by downregulating oncogenic long non-coding RNA XIST in the METTL14-YTHDF2-lncRNA regulatory axis (25). LncRNA GAS5 binds directly to Yes-associated protein (YAP), promoting its phosphorylation and ubiquitin-mediated degradation, thereby attenuating YAP-mediated transcription of YTHDF3 and inhibiting the progression of rectal cancer (26). LincRNA-p21 acts as a tumor inhibitor in the development of esophageal squamous cell carcinoma (27). In hepatocellular carcinoma, the m⁶A methylation modification of LINC00152 is involved in the prognosis of LIHC patients through the cytoskeleton regulation pathway (28). Furthermore, in rectal cancer, m⁶A modification of lncRNA RP11 can upregulate the translation of Zeb1 to trigger cancer cell dissemination (29); lncRNA-THOR enhances IGF2BP1-targeted mRNA expression and promotes human osteosarcoma cell survival and proliferation (30).

MiRNAs are non-coding single-stranded RNAs of 21-25 nucleotides in length that regulate gene expression at the post-transcriptional level by building RNA-induced silencing complexes (RISCs) that bind to the 3’untranslated region of target mRNAs (3’UTR) to regulate gene expression (31). In the nucleus, miRNAs are first transcribed into longer primary miRNAs (pri-miRNAs) and subsequently processed into precursor miRNAs (pre-miRNAs). It is then cleaved into mature single-stranded miRNAs by Dicer in the cytoplasm, and the participation and processing of such pri-miRNAs are m⁶A-dependent. METTL3 tags pre-miRNAs through m⁶A modification, enabling DGCR8 to recognize and bind its specific substrates, thereby promoting miRNA maturation and increased miRNA levels in cells (32).

Circular RNAs (circRNAs) are a class of single-stranded covalently closed RNA molecules that participate in many physiological processes, including competing with endogenous RNAs as sponge miRNAs, forming RNA-protein complexes, regulating gene transcription, and even encoding proteins (33). In most cases, abnormal m⁶A modification contributes to tumorigenesis and tumor progression. However, m⁶A modification on circRNAs can suppress innate immunity; YTHDF2 sequesters m⁶A-circRNA and is essential for suppressing innate immunity (34). Chen et al. found that m⁶A modification of circNSUN2 promoted liver metastasis of colorectal cancer by promoting cytoplasmic export and forming a circNSUN2/IGF2BP2/HMGA2 RNA-protein triple complex to stabilize HMGA2 mRNA (35).

m⁶A methyltransferases (“Writers”) are an essential class of catalytic enzymes. METTL3 is a key regulator that promotes m⁶A modification, and the abnormal regulation of METTL3 is also inextricably linked to tumor development. Some studies have confirmed that the overexpression of METTL3 may be an important factor in promoting the development of common malignant tumors in women. Pan et al. found that the expression of RBM15 and METTL3 in CESC (cervical squamous cell carcinoma) tissues was higher than that in normal tissues (49). In addition, Hua et al. found that METTL3 promoted the epithelial-to-mesenchymal transition of ovarian cancer cells and the proliferation, invasion, and tumor formation of ovarian cancer cells, affecting their prognosis and overall survival (50). Moreover, Ma et al. compared the expressions of METTL14, WTAP, and METTL3 in ovarian cancer and found that METTL3 independently regulates m⁶A modification and thus affects the proliferation and metastasis (51). Besides, Li et al. found that METTL3 inhibited the viability of cervical cancer cells and enhanced their sensitivity to the chemotherapeutic drug cisplatin by downregulating the expression of the receptor for advanced glycation and its product in cervical cancer tissues (40); METTL3 modulated the m⁶A modification of MALAT1. The expression of MALAT1 is upregulated, and MALAT1 can promote the expression of high mobility group A2(HMGA2) by sponge miR-26b, thereby promoting the development of breast cancer (41). Regarding this, Wang et al. proposed that WTAP may promote the proliferation, invasion, and migration of ovarian cancer through two gene sequences of FAM76A and HBS1 (42).

As m⁶A methylation reading proteins, “Readers” can recognize the information of RNA methylation modification and participate in downstream mRNA translation, degradation, and miRNA processing. It mainly changes the interaction between protein and RNA by weakening the homologous binding of m⁶A to RNA-binding protein and changing the secondary structure of RNA.

YTHDF1 acts as an important reading element in m⁶A modification by recognizing m⁶A-containing mRNAs and promoting their translation initiation and elongation (52). Wang et al. applied online data analysis to identify RANBP2 as a critical target of YTHDF1 in cervical cancer cells, and subsequent reduction of RANBP2 decreased cervical cancer cell proliferation, migration, and invasion (43). Overexpression of YTHDF1 promoted the growth, migration, and invasion of Hela and Siha cells. At the same time, knockdown of RANBP2 reversed the effect of overexpression of YTHDF1 on cervical cancer progression, indicating that YTHDF1 promotes cervical cancer progression by regulating RANBP2 expression in an m⁶A-dependent manner. Some scholars have proposed that YTHDF1 directly targets eIF3C (a subunit of EIF3) and promotes ovarian cancer’s occurrence, metastasis, and prognosis (51).

Heterogeneous ribonucleoproteins (hnRNPs) are a diverse family of RNA-binding proteins that function in most stages of RNA metabolism (53). Studies have shown that HNRNPC regulates target transcripts’ abundance and alternative splicing through mA binding to RNA. Other researchers have proposed that hnRNPA2B1 can inhibit the growth of ovarian cancer cells, reduce the mobility of ovarian cancer cells in vitro, and hinder the formation of xenograft tumors in vivo. In addition, hnRNPA2B1 promotes the occurrence and development of malignant phenotypes of ovarian cancer by activating the expression of Lin28B (54). Moreover, Shi et al. found that lobaplatin induced apoptosis and cell cycle arrest by downregulating hnRNP A2/B1 in cervical cancer cells, and knockdown of hnRNP A2/B1 significantly reduced tumor growth in nude mice xenografts and increased cervical cancer Cellular sensitivity to lobaplatin and irinotecan (55). Eukaryotic initiation factor 3 (eIF3) can bind to m⁶A-modified bases in the 5’ UTR of RNA, promoting mRNA translation (56). The high expression of eLF3 in ovarian cancer is closely related to its poor prognosis (44); Zhu et al. concluded that eIF3B is highly expressed in cervical cancer tissues and is closely related to advanced FIGO in cervical cancer patients staging, shorter overall survival and lymph node metastasis (57).

Demethylase is an integral part of the reversible modification of m⁶A, and FTO, as the first discovered demethylase, is widely present in adult and embryonic tissues, and its expression is exceptionally high in the brain. Moreover, recent studies have shown that FTO has an important effect on glioblastoma growth and self-renewal (58).

The expression level of FTO is also elevated in cervical squamous cell carcinoma, which can enhance chemoradiotherapy resistance in vitro and in vivo by reducing m⁶A-regulated β-catenin expression (59). Zhao et al. believed that FTO accelerated the growth of cancer cells by promoting proliferation, inhibiting apoptosis, and activating autophagy in ovarian cancer (60). In the latest study, Huang et al. found that FTO expression in high-grade serous ovarian cancer (HGSOC) tumor cells were significantly lower than that in other tissues, and it had a significant inhibitory effect on ovarian cancer cells (47). It can be seen that FTO may have a bidirectional regulatory impact on ovarian cancer tissue, and the specific mechanism needs to be further studied. Furthermore some researchers believe that FTO primarily stimulates the oncogenic activity of breast cancer cell invasion and migration through the FTO/miR-181b-3p/ARL5B signaling pathway, promoting tumor proliferation (61). The tumor suppressor BNIP3 is a downstream target of FTO-mediated m⁶A modification. FTO mediates m⁶A demethylation in the 3’UTR of BNIP3 mRNA and induces its degradation through a YTHDF2-independent mechanism, promoting breast cancer cell proliferation, colony formation, and in vitro and in vivo transfer (46).

Among them, ALKBH5, another member of the Alkb protein family, was found to have demethylation, which can directly catalyze m⁶A-methylated adenosine to remove methyl groups is different from the oxidative demethylation of FTO (62). ALKBH5 affects tumor growth by regulating cell proliferation, migration, invasion, and metastasis. Recently, some scholars have proposed that ALKBH5 has dual roles in various cancers. ALKBH5 can reverse METTL3 autophagy in cells through down-regulation of mRNA stability. In epithelial ovarian cancer cells (63), overexpressed ALKBH5 in human ovarian cancer (SKOV3) cells enhanced the stability of BCL-2 mRNA and inhibited tumor cell growth and metastasis. In contrast, overexpressed ALKBH5 in A2780 cells had the exact opposite effect. In cervical cancer tissues, GAS5-AS1 was also low-expressed and inhibited the proliferation and metastasis of cervical cancer cells. And regulate GAS5 expression by interacting with RNA demethylase ALKBH5, thereby inhibiting CC cell proliferation, migration, and invasion (64).

Targeted therapy is at the cellular and molecular level to design corresponding therapeutic drugs for the already defined carcinogenic sites. The drug enters the body and will specifically select the carcinogenic sites to combine and act so that the tumor cells specifically die instead of normal tissue cells surrounding the tumor are affected. Early studies of targeting strategies based on m⁶A modulators have focused on demethylases. Besides, previous studies have shown that m⁶A plays an essential role in the occurrence and development of tumors. Therefore, it is of great scientific significance and clinical value to develop specific inhibitors of m⁶A-related proteins. As the first discovered RNA-modifying demethylase, FTO is widely involved in various physiological processes, and its dysregulation is associated with multiple human diseases.

Due to its involvement in obesity and obesity-induced metabolic diseases and the occurrence, development and prognosis of various cancers, such as melanoma, acute myeloid leukemia, glioblastoma, lung cancer, hepatocellular carcinoma and breast cancer, studies have shown that rhein can induce apoptosis (65). Huang et al.. systematically investigated the effect of rhein on adipogenesis by transcriptional and post-transcriptional approaches and found that rhein regulates m⁶A methylation rearrangement and adipogenesis in an independent manner, inhibiting fat mass and obesity-related (FTO) demethylase activity (66). It is indicated that rhein can inhibit the demethylation activity of FTO on m⁶A on mRNA in vitro and in vivo, thereby increasing the level of m⁶A in cells. ALKBH5 and FTO are both m⁶A demethylases. Studies have found that in ovarian cancer, the core cytokine NANOG is a key target to promote the development of ovarian cancer (48). On the contrary, there is much evidence to prove the overexpression of METTL3 in tumor tissues (67), while studies targeting METTL3 have shown that it can effectively inhibit tumor growth, proliferation, and metastasis (68).

In the progression of common malignant tumors in women, drug resistance that often occurs in the later stage is also a significant difficulty in its treatment. Chemotherapy resistance, especially platinum resistance, is a major cause of poor prognosis in ovarian cancer. Bowen Li et al. found that m⁶A can modulate the modification of anticancer drug resistance by modulating drug-target interactions and drug-mediated cell death signaling (69). The ethyl ester form of the FTO inhibitor Meclofenaic (MA2) inhibits FTO and enhances the effect of the chemotherapeutic drug temozolom by targeting the MYC-miR-155/23a cluster-MXI1 feedback circuit in gliomas anti-tumor effects (45) (Table 2).

The tumor microenvironment (TME) is primarily responsible for mediating immunotherapy responses in tumor progression, and bioinformatics research has shown that m⁶A alteration and its regulators may regulate the TME and are linked to immune checkpoint inhibition (ICB) (7, 72).

Yi et al. systematically studied head and neck squamous cell carcinoma (HNSCC) compared with adjacent normal pairs, concluded that m⁶A regulators were upregulated in HNSCC, and found that m⁶A regulators were associated with PDL in the tumor immune microenvironment (TIME) (73). The expression of -1 was positively correlated, which may provide a promising target for improving the responsiveness of HNSCC to immunotherapy. In addition, He et al. systematically analyzed RNA-sequencing data of 24 major m⁶A methylation regulators in 775 breast cancer patients from the TCGA database and classified them for overall survival in the lower RNA methylation status group (RM1). The higher methylation status (RM2) group was significantly reduced (70). Moreover, the RM2 group displayed higher expression and higher numbers of tumor-infiltrating CD8⁺ T cells, helper T cells, and activated NK cells. The expressions of PD-L2, TIM3, and CCR4 were lower than those of the RM1 group, so it can be considered that the regulator of m⁶A is closely related to the malignant degree, prognosis, and anti-tumor immune response of breast cancer and can be used as a potential target and biological target for breast cancer immunotherapy.

In addition, anti-PD-1 immunotherapy is effective initially, but its efficacy is significantly reduced later due to FTO-mediated resistance (71). However, recent studies have shown that FTO knockdown can increase tumor sensitivity to anti-PD-1 immunotherapy, thereby improving efficacy (69). Therefore, the combined use of ICB and FTO inhibitors may block the development of drug resistance in individuals who develop adaptive immunity.