m⁶A methylation is catalyzed by mRNA writers, including METTL-3, METTL-14, WTAP, VIRMA and RBM15. The core components of the m⁶A methyltransferase complex (METTL-3, METTL-14, WTAP and VIR) are highly conserved in most eukaryotes (15). The study of the m⁶A site on human small nuclear (sn) RNA U6 showed that human cells express at least one activated m⁶A methyltransferase, apart from METTL-14 and METTL-3. However, these enzymes have not been identified due to the limitation in technology.

METTL-3, also known as MT-A70, is the earliest reported m⁶A methylase. Barbieri et al (16) reported that the upregulation of METTL-3 expression significantly promoted the m⁶A methylation of mRNA transcribed by the oncogene SP1, resulting in an increased expression of the SP1 protein, which was associated with the differentiation of hematopoietic stem cells into acute myeloid leukemia (AML) cells. In addition, Vu et al (17). confirmed that downregulation of METTL-3 gene expression increased the phosphorylation of AKT and promoted the differentiation of hematopoietic stem cells into AML cells. The two studies provide novel directions for the diagnosis and treatment of AML.

In addition, some studies have shown that under hypoxia, the transcription factor zinc-finger protein 217 (ZNF217) inhibited the m⁶A modification of KLF4 and NANOG by binding to METTL-3, leading to elevated expression of KLF4 and NANOG, and the promotion of breast cancer (18). Cai et al (19) have found that high expression of METTL-3 in breast cancer cells induced m⁶A modification on HBXIP mRNA. HBXIP promoted the m⁶A modification of METTL-3 by reducing the expression level of tumor suppressor gene LET-7G, which forms a positive feedback pathway with HBXIP/LET-7G/METTL-3/HBXIP and promoted the malignant biological behaviors of breast cancer. These studies provide new approaches for the diagnosis and treatment of breast cancer.

Furthermore, Chen et al (9) found that overexpressed METTL-3 in primary hepatocellular carcinoma (HCC) could change the m⁶A modification of the tumor suppressor gene, SOCS2, leading to degradation of SOCS2 mRNA and promotion of cancer cell proliferation and migration. This study showed that hypermethylation was associated with the progression of HCC. Taketo et al (20) found that, in pancreatic cancer cell lines with low expression of METTL-3, the cancer cells were more sensitive to gemcitabine and other anticancer drugs [Everolimus (21) or Elemene (22)] and external radiation. In addition, METTL-3 was associated with cell cycle regulation, mitogen-activated protein kinase cascade and RNA splicing, suggesting that METTL-3 may be one of the potential targets to improve the therapeutic efficacy in patients with pancreatic cancer.

However, in renal cell carcinoma (RCC), METTL-3 exhibited tumor-suppressing activity (23). In vivo experiments confirmed that lower expression of METTL-3 was significantly associated with tumor histological grade and tumor size. In addition, patients with RCC and overexpression of METTL-3 had a higher overall survival rate and good prognosis. Downregulation of the METTL-3 gene expression in a RCC cell line could promote cell epithelial-mesenchymal transition (EMT), proliferation, invasion and metastasis. It was suggested that METTL-3 could be used as a new marker for the treatment of RCC, however, further studies are required to investigate the role of METTL-3 and related factors in carcinogenesis to further understand the biological mechanism of the occurrence and development of RCC.

The aforementioned studies have investigated the different activities of METTL-3 in various types of cancer, indicating that m⁶A methyltransferase, METTL-3 could be a potential target for developing novel therapeutic strategies, and investigating the mechanism of the occurrence and development of cancer.

METTL-14 is a homologous heterodimer of METTL-3 in the MT-A70 methyltransferase family (24). It has been reported that knockout of the METTL-14 gene in HeLa cells led to a decrease in m⁶A methylation level, suggesting that METTL-14 was an important part of the m⁶A methyltransferase complex (25). Since METTL-3 is a subunit with catalytic activity, METTL-14 is responsible for identifying substrates. The two proteins are combined to form a stable methyltransferase complex with a ratio of 1:1 In vivo, which allows the catalyzation of m⁶A modification in target RNA (26). Weng et al (8) found that the knockout of METTL-14 in AML cell lines could effectively inhibit the proliferation of the AML cells. METTL-14 was negatively regulated by SP1 at the protein level and induced cancer promotion by regulating target genes via m⁶A modification. This study firstly revealed the role of the SP1-METTL-14-MYB/MYC signal axis in the progression, maintenance, and self-renewal of leukemia, providing new ideas for the diagnosis and treatment of AML. In addition, Ma et al (27) proved that the decrease in expression of METTL-14 in HCC tissue was an independent factor in predicting cancer recurrence. The reduction of METTL-14 led to a decreased level of m⁶A methylation, which inhibited cell proliferation and promoted apoptosis of HCC cells. In a HCC cell line, METTL-14 mediated the decreased expression of miR-126, leading to the invasion and metastasis of HCC. Furthermore, Ma et al also found that the expression level of METTL-14 and demethylase WTAP in HCC was decreased, indicating that m⁶A modification has a complicated feedback regulation mechanism. Therefore, the investigation into the interaction between METTL-14 and micro (mi) RNA could provide novel targets for the treatment of HCC.

METTL-16 is a newly discovered m⁶A methyltransferase (28). The downregulated expression of METTL-16 led to a decrease in the level of m⁶A methylation in cells. Warda et al (29) found that METTL-16 could bind to snRNA U6, long non-coding (lnc) RNA and pre-mRNA via cDNA cross-linking analysis, which deepened the understanding of the interaction between m⁶A and other RNA.

S-adenosylmethionine (SAM) is an important methyl donor of DNA methylation and acts as a key regulator controlling gene expression (30). It has previously been reported that SAM played an important role in RNA methylation (31). The study suggested that METTL-16 maintained the stability of intracellular SAM by regulating the alternative splicing of MAT2A. The absence of SAM increased the residence time of METTL-16 in the hairpin of MAT2A 3′ untranslated region (UTR) and promoted the alternative splicing of MAT2A, subsequently regulating the homeostasis of intracellular SAM content (32). The association between RNA modification and alternative splicing was established by this mechanism. However, the association between METTL-16 and the occurrence and development of cancer remains unclear. Therefore, further studies are required to investigate the role of METTL-16 in cancer development.

WTAP is an essential component in m⁶A methylation modification. Ping et al (33) proved that WTAP assisted with the accurate location of the METTL-3-METTL-14 heterodimer and promoted m⁶A methylation. In addition, either knockdown or overexpression of METTL-3 led to an elevation of WTAP expression, indicating that METTL-3 plays an important role in the regulation of WTAP function (34). However, the upregulation of WTAP could not promote cancer cell proliferation in the absence of METTL-3. Therefore, the carcinogenic effect of WTAP is associated with the m⁶A methyltransferase complex. The association between WTAP and the occurrence and development of cancer is unclear. Xi et al (35) found that WTAP was highly expressed in glioma tissue and was associated with pathological grade and poor postoperative survival rate. Li et al (36) confirmed that the expression level of WTAP was significantly increased in both the cytoplasm and nucleus of pancreatic ductal adenocarcinoma (PDAC), whereas the high expression level in the nucleus was significantly associated with sex and tumor stage and was considered to be an independent prognostic factor of PDAC. Tang et al (37) reported that the high expression level of WTAP in patients with RCC was associated with poor overall survival rate and prognosis. The study also found that WTAP may promote the proliferation of RCC cells by regulating the stability of CDK2 mRNA, leading to the occurrence and development of cancer. Therefore, WTAP may become a new target for the diagnosis and treatment of RCC.

α-ketoglutarate-dependent dioxygenase FTO protein (FTO) encoded by the obesity gene, FTO was the first demethylase found in mammals, which proved that m⁶A modification was dynamically reversible (38). Similarly, AlkB homologous protein 5 (ALKBH5) in mammals could also catalyze the restoration of m⁶A methylation (39). Currently, it is not clear whether demethylases exist in lower-grade eukaryotes. Some studies have found that when the first nucleotide adjacent to the cap in the nucleotide sequence is adenosine, FTO cannot induce demethylation, but the specific mechanism is unclear.

The FTO gene is located on chromosome 16 (16q12.2) and is widely expressed in all stages of human growth (40). Its main functions are to regulate the rate of fat consumption, promote the overall metabolic rate, and ensure the energy balance of the body (41). Jia et al (42) first confirmed that the FTO protein was a crucial demethylase in both DNA and RNA modification, especially in m⁶A demethylation. This report ushered in the era of m⁶A research. Selberg et al (26) confirmed that the level of m⁶A in mRNA was increased in FTO knockout leukemia cells or gastric cancer cells and vice versa. However, the expression of m⁶A methylase METTL-3 was not affected. Based on these results, researchers preliminarily proved that the methylation process of m⁶A was reversibly and dynamically regulated. Previous studies have shown that the expression of the FTO gene was associated with breast cancer (41), thyroid cancer (43), endometrial cancer (44), gastric cancer (45), and other types of cancer (46,47). Li et al (48) found that FTO increased leukemia oncogene-mediated cell transformation and leukemogenesis by reducing the m⁶A modification of ASB2 and RARA genes, which led to the inhibition of AML cell differentiation induced by all-trans retinoic acid. In another study, Zhou et al (49) found that the expression of the FTO gene was significantly increased in patients with cervical squamous cell carcinoma (CSCC), and the increased expression of FTO and β-catenin indicated a poor prognosis. Therefore, the expression level of FTO and β-catenin could predict the prognosis of CSCC. In short, few studies have focused on the mechanism of FTO-induced m⁶A modification in the carcinogenesis and development of cancer. More studies are required to clarify the relevant molecular biological mechanisms involved in FTO-induced m⁶A modification. However, it remains controversial whether the activity of methylase and demethylase is limited to catalyzing m⁶A modification on RNA.

ALKBH5 belongs to the AIkB family, but unlike other family members, ALKBH5 only has demethylation ability on single-stranded RNA/DNA (50). With the participation of hypoxia-inducible factors (HIF), ALKBH5 can induce the transformation of breast cancer cells into tumor stem cells by reducing the m⁶A methylation of NANONG, which improves the stability of NANONG mRNA and elevates its expression (51). Similarly, Zhang et al (52) found that ALKBH5 was significantly overexpressed in glial stem cell-like cells (GSCs) and the interference of ALKBH5 could inhibit the proliferation of GSCs. In addition, the study also found that lncRNA FOXM1-AS promoted the interaction between ALKBH5 and FOXM1, indicating that m⁶A demethylase ALKBH5 acted as an oncogene in glioma. Recently, low expression of ALKBH5 in pancreatic cancer cell lines was found to promote the m⁶A demethylation of lncRNA KCNK15-AS1, resulting in a decreased ability of cancer invasion and metastasis (53). This provided a new direction for the diagnosis and treatment of pancreatic cancer.

In summary, further studies are required to investigate whether ALKBH5 is associated with the occurrence and development of other types of cancer and whether these key demethylation modifications are associated with the stability, translation, and alternative splicing of mRNA.

The term, mRNA readers, refers to proteins that can specifically bind to mRNA with m⁶A methylation. The YTH domain is the marker of m⁶A binding protein on mRNA. Their affinity with m⁶A methylated mRNA is higher than that of unmethylated mRNA (54). The carboxyl terminal domain of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) selectively binds to the m⁶A modified mRNA, which assists the YTHDF2-mRNA complex to move to the RNA decay site of the cell, thus inducing the degradation of mRNA. The degradation of mRNA plays an important role in stem cell differentiation by regulating key pluripotent factors (55). In different situations, the YTH protein can interact with different subsets of the m⁶A locus and induce different effects on gene expression. Insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) is another m⁶A reader using the Khomlog (KH) domain to selectively bind m⁶A modified RNA and promote mRNA translation, which is different from proteins with the YTH domain (56). This discovery increased the understanding of the mechanism and function of the m⁶A binding protein. In addition, m⁶A modification could destroy the complementary pairing of nucleotides, improve the accessibility of single-stranded RNA motifs, and promote the recognition of m⁶A binding proteins heterogeneous nuclear ribonucleoprotein C and G.

The YTH domain recognizes m⁶A methylation in a methylation-dependent manner (57). A total of five proteins in the human body contain YTH domains. YTHDC1 can regulate the expression of mRNA in the nucleus by affecting the alternative splicing of mRNA precursors (58). Zhao et al (35) found that the expression of YTHDF1 was significantly increased in patients with advanced HCC. In addition, potential target genes regulated by the YTHDF1 protein may be associated with the cell cycle of the tumor, degradation of different amino acids and metabolism of various lipids. Li et al (59) reported that overexpression of miR-493-3p in YTHDF2 knockdown prostate cancer cell lines promoted m⁶A modification and thereby inhibited the proliferation and migration of the cancer cells. These findings lay a foundation for further investigation of the biological function of m⁶A and RNA epigenetics and provide a new direction for investigating the underlying mechanism of cancer development. Currently, the role of YTH family members in m⁶A methylation has become a hot topic, which provides novel approaches for investigating cancer-related mechanisms.

Eukaryotic initiation factors (eIF3). There are numerous and complex eIFs. Up to now, a total of 13 eIFs have been identified (60). eIF3 is the most complex factor in eIF translation and plays an important role in the initiation of protein translation. Li et al (61) first found that eIF3e was an independent prognostic factor for overall survival and disease-free survival time in patients with colon cancer. Downregulation of eIF3e expression could inhibit proliferation and promote apoptosis of colon cancer cells. Furthermore, the interaction between METTL-3 and eIF3h could increase mRNA translation and form dense polyribosomes, which was necessary for carcinogenic transformation (62). The study by Chao et al (39) revealed the regulatory mechanism of protein translation based on the mRNA cycle and indicated that METTL-3-eIF3h could be a potential therapeutic target for patients with lung cancer.

Breast cancer stem cells (BCSCs) can proliferate indefinitely via self-renewal and forming recurrent or metastatic tumors (87). In the hypoxic tumor microenvironment, ALKBH5 could reduce m⁶A methylation in NANOG mRNA, increase the expression of NANOG mRNA and mediate the enrichment of BCSCs in a HIF-dependent manner (51). Zinc-finger protein 217 (ZFP217) and ALKBH5 play complementary roles in regulating m⁶A methylation in RNA (18). Hypoxia-induced ZNF217 inhibited m⁶A methylation of NANOG mRNA, whereas ALKBH5 induced m⁶A demethylation. Taken together, they can increase the expression of NANOG mRNA and protein and enrich BCSCs. In addition, ZFP217 and ALKBH5 were associated with a more malignant phenotype of breast cancer by inhibiting m⁶A methyltransferase-related modifiers or inducing HIF-dependent hypoxia (88). A recent study reported that m⁶A modification regulated the expression of early polyadenylation (premature polyadenylation; PPA), which blocked the expression of tumor suppressor genes and lead to carcinogenesis (89). In breast cancer cells, premature polyadenylation causes oncogenic truncations of the tumor suppressor genes MAGI3 (90), LATS1 (91) and BRCA1 (92). The activation and truncation of PPA in tumor suppressor genes was regulated by m⁶A modification. Compared with that in normal breast cells, m⁶A methylation, activated by PPA significantly, was decreased in tumor suppressor gene-related exons. However, there are no conclusions on how breast cancer cells regulate the level of m⁶A in exons to trigger PPA.

As an ATP-dependent RNA helicase and a member of the YTH family, YTHDF2 promoted initial translation by unlocking the 5′-UTR of mRNA, and the transcription and translation of metastasis-related factors by inducing m⁶A methylation, thereby enhancing the metastasis of cancer cells (93). In colon cancer, YTHDF2 promoted metastasis by promoting the translation of HIF-1α. Knockdown of the YTHDF2 gene could reduce the expression level of metastasis-related genes, such as HIF-1α and inhibit the metastasis of colon cancer cells in vitro and In vivo (94). In addition, the expression level of YTHDF2 was positively associated with the stage of colon cancer. At present, few studies have identified the function and target of YTHDF2 in the progression and metastasis of colon cancer. However, these findings will provide new insights into the role of RNA demethylase in tumorigenesis.

Hou et al (95) have revealed that YTHDF2 was positively associated with the malignant grade of HCC. miR-145 could increase the level of m⁶A methylation by targeting the 3′-UTR of YTHDF2 mRNA in HCC cells, leading to the malignant progression of HCC. In addition, YTHDF1 was highly expressed in human HCC tissues and associated with the regulation of the cell cycle and metabolism of HCC cells. Furthermore, the deletion of m⁶A methylation was associated with the metastasis of HCC with the downregulation of METTL-14. In HCC, METTL-3 mediated the methylation of m⁶A in the mRNA of the chromosome (or critical) region in DiGeorge syndrome (96). METTL-14 could significantly upregulate the level of miR-126 modified by m⁶A methylation, thus promoting the maturation of miR-126 and inhibiting the metastasis of HCC cells (97). At present, the mechanism of how METTL-14 has a low expression in liver cancer cells remains unclear. More in-depth investigation is required to clarify the structure and biological function of METTL-14 in cancer to determine whether METTL-14 can be used as a therapeutic target for the treatment of liver cancer. However, the interaction between METTL-14 and YTHDF1/2 with miRNA still provides clues for identifing new approaches to treat liver cancer.

The role of METTL-14 in pancreatic cancer has also been confirmed. The methylase, METTL-14 was highly expressed in pancreatic cancer tissues. METTL-14 could promote the proliferation, invasion, and metastasis of pancreatic cancer cells by increasing the level of m⁶A methylation, inhibiting the expression of miR-1-3p, and activating the mitogen-activated protein kinase (MAPK) pathway (98). In addition, a new mechanism of lncRNA with m⁶A methylation was found. ALKBH5 inhibited the progression of pancreatic cancer by promoting m⁶A demethylation of lncRNA potassium two-pore domain channel subfamily K member 15 (KCNK15) and WNT1-induced signal pathway protein 2 (WISP2) antisense lncCNK15-AS1 (53). This finding reveals a new area for investigating the role of lncRNA methylation in cancer development.

WTAP, as an m⁶A demethylase, plays a carcinogenic role in AML. Both In vivo and in vitro research has proved that WTAP was associated with cell transformation and all-trans retinoic acid (ATRA)-mediated leukemia cell differentiation (48). In addition, METTL-3 inhibited the differentiation of hematopoietic stem/progenitor cell in patients with AML by inducing m⁶A methylation, which maintained the undifferentiated phenotype of the leukemia cells and promoted the occurrence of AML. On the other hand, the knockdown or deletion of METTL-3 could activate a translation process to promote cell differentiation and apoptosis, leading to the suppression of leukemia cells without affecting normal hematopoietic cells (17). Similarly, METTL-14 plays a key role in both normal myelopoiesis and pathogenesis of AML (8). METTL-14 could block normal myeloid differentiation and promote malignant bone marrow formation by mediating m⁶A methylation. These studies provide new insights into the molecular mechanism of hematological tumorigenesis, suggesting that inhibition of METTL-3/14 may be used as a strategy for the treatment of malignant myeloid tumors.

In 2018, a study found that m⁶A methylation in mRNA played a crucial role in endometrial carcinogenesis with the activation of protein kinase B (PKB) signal (99). m⁶A methylation reduced the expression of PKB negative regulator PH domain and leucine-rich repetitive protein phosphatase 2, whereas the expression of the positive PKB regulator mammalian target of rapamycin c2 was elevated, which promoted the proliferation and invasion endometrial cancer cells (49).

Previous studies have found that a low level of m⁶A was associated with the occurrence of cervical cancer. In addition, the decrease in m6A level was positively associated with The International Federation of Gynecology and Obstetrics stage, tumor size, degree of differentiation, lymph node invasion and tumor recurrence (100). The results suggested that m⁶A methylation site in mRNA may serve as a potential therapeutic target for cervical cancer and could be used as an independent prognostic factor for predicting disease-free survival and overall survival times in patients with cervical cancer (101,102).

In gastric cancer, the expression level of ALKBH5, a m⁶A ‘eraser’, was significantly decreased in highly invasive diffuse gastric adenocarcinoma compared with that in adjacent tissues. The knockdown of ALKBH5 could decrease the mRNA and protein expression levels of E-cadherin and increase the expression level of interstitial markers, such as snail (103) and N-cadherin (104). Further investigation showed that the downregulation of ALKBH5 could decrease the ability of mRNA demethylation and promote the methylation level, which reduced the stability of E-cadherin mRNA and promoted the invasion of tumor cells. Furthermore, ALKBH5, as a tumor suppressor gene in gastric cancer (105), could suppress EMT, migration, and invasion of gastric cancer cells by inhibiting the mRNA and protein expression levels of MMP-2 and MMP-9. In addition, WTAP was found to play an important role in the progression and metastasis of gastric cancer, and was associated with poor differentiation, lymph node metastasis, high TNM stages and poor prognosis (106). M⁶A may also be an important molecular marker for monitoring gastric cancer. Furthermore, the expression level of METTL-3 was positively associated with the prognosis, tumor grade and tumor stage in patients with gastric cancer (107). In addition, METTL-3 was associated with the mRNA and protein expression levels of a-smooth muscle actin to regulate the proliferation and migration of gastric cancer cells, which could be a potential target for the treatment of gastric cancer in the future (108,109).

They regulate the level of m⁶A through direct or indirect modification and participate in tumor progression. WTAP enhanced the expression of marrow zinc finger 1 (MZF1) by reducing the level of m⁶A and destabilizing MZF1 mRNA in bone, thus promoting the progression of lung squamous cell carcinoma. YTHDF2 and miR-495 inhibited the progression of prostate cancer by indirectly downregulating the level of m⁶A methylation (110). ALKBH5 maintained the expression level of fork box protein M1 mRNA by promoting m⁶A demethylation, which retained the tumorigenicity of glioblastoma stem cells (52). The aforementioned studies revealed the importance of m⁶A modification in different types of cancer. The dynamic change of m⁶A methylation has various regulatory effects on cancer cells (111). By revealing the previously unidentified regulation mechanism in tumors, further studies will provide bases for exploring the pathogenesis of tumors and developing new potential targets for cancer treatment (112,113).