RNA methylation in hepatocellular carcinoma: from metabolic reprogramming and immune escape mechanisms to small molecule inhibitor development.

Introduction
Hepatocellular carcinoma (HCC), the sixth most common malignancy and a major contributor to cancer-related mortality globally, is categorized into primary liver cancer (PLC) and secondary liver cancer (SLC), and has high incidence and fatality rates. HCC is the predominant subtype of PLC, accounting for approximately 75–85% of cases [1, 2]. HCC is more common in people who have chronic infections with the hepatitis B and C viruses, bibulosity, aflatoxin, smoking, are overweight, or have metabolic disorders like type II diabetes mellitus [3]. And hepatitis B vaccination, antiviral therapy, and lifestyle modifications have significantly reduced the precipitating factors for HCC [4]. The fast growth of high-throughput sequencing technology has revealed a close connection present between epigenetic abnormalities and the progression of HCC. These abnormalities include changes in DNA methylation, RNA methylation, histones, and chromatin remodeling [5]. At present, HCC can be treated through surgery (liver transplantation and resection), localized therapies (like radiofrequency ablation and chemoembolization), systemic therapies, and immunotherapy. Systemic therapies comprise single-agent targeted treatments (like sorafenib and levatinib) and checkpoint inhibitors in conjunction with targeted therapies (like atezolizumab plus bevacizumab) [6, 7]. Despite considerable progress in HCC treatment modalities, the prognosis for HCC patients remains inadequate because of issues such as early surgical recurrence and resistance to targeted therapy. Given the characteristics of HCC, such as significant heterogeneity, elevated recurrence rates, and treatment resistance, dynamic therapeutic strategies tailored to the patient’s case and individual risk are more advantageous [8]. So, being able to perform a complete epigenetic analysis of HCC may help us better understand its pathogenic mechanisms better and make it easier to create therapies that target specific molecules.
RNA modifications significantly influence gene expression by altering the chemical properties of RNA bases and ribose. As of now, over 170 chemical changes have been recognized in different RNA classes among prokaryotes and eukaryotes [9, 10]. RNA methylation accounts for almost 60% of all RNA modifications and is critical to post-transcriptional gene regulation [10]. The principal forms of RNA methylation encompass N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), N7-methylguanosine (m7G), 3-methylcytosine (m3C), pseudouridine (Ψ), and 2′-O-methylation (Nm), among others. This highlights its extensive prevalence and importance in gene regulation [11]. RNA methylation is facilitated and regulated by three distinct categories of proteins: writers, which catalyze RNA methylation; readers, which identify methylation; and erasers, which remove methylation (Fig. 1). These proteins function in their own ways [9]. RNA methylation occurs in almost all RNA types, including mRNAs, tRNAs, LNCRNAs, sRNAs, siRNAs, snRNAs, and snoRNAs [12]. As a dynamically reversible process, it plays a crucial role in regulating numerous features such as RNA transcriptome processing, splicing, and export. Increasing numbers of research prove that RNA methylation is crucial in carcinogenesis and progression, with RNA methylation dysregulation has been found in several tumors, including HCC. And aberrant RNA modifications promote translation of oncogenic RNA transcripts in HCC. (Table 1) [13, 14].
This review examines the role of RNA methylations in regulating gene expression, immune cell function, and tumor immune evasion, particularly in the context of HCC tumor immunity and their potential application in combination therapy. We summarize recent studies and seek to offer novel insights and strategies for developing innovative targets for the diagnosis, treatment, and prognosis of HCC.

RNA methylation classification

N6-methyladenosine
N6-methyladenosine (m6A) refers to the methylation of adenosine at the N6 position. The m6A represents the predominant form of RNA methylation, comprising approximately 60% of such modifications [13]. This modification is prevalent in mRNA sequences and can also occur in non-coding RNA sequences, such as rRNAs, tRNAs, lncRNAs, snRNAs, miRNAs, snoRNAs, and circRNAs [12]. Long-stranded non-coding RNAs are subject to methylation modifications and also play a role in mediating these modifications [60]. The m6A significantly influences RNA stability, translocation, splicing, and translation, consequently impacting the overall expression of RNA [9]. Interestingly, hepatitis B virus (HBV) transcripts have the capacity to modify host gene expression through the regulation of m6A modifications in homologous RNAs [61]. The latest studies have shown that HBV proteins promote KIAA1429 expression and HSPG2 expression through its mediated m6A modification, which in turn drives HCC development [62]. The m6A methyltransferase complex (MTC), including METTL3, METTL14, WTAP, RBM15/15B, ZC3H13, VIRMA, and KIAA1429, is essential for catalyzing various m6A alterations [26, 63]. The process of demethylation is catalyzed by demethylases, including FTO and ALKHB5. The m6A methylation readout proteins include various molecules such as IGF2BP1/2/3, YTHDF1/2/3, and heterogeneous ribonucleoproteins (HBRNPA2/B1/C), among others [64]. They mediate RNA m6A modifications, regulating RNA stability and translation, thereby influencing HCC tumorigenesis and progression. The latest studies, KIAA1429 regulates RND3 mRNA stability by targeting its 3′-UTR and vicinity of the termination codon, thereby promoting HCC migration and invasion [65]. YTHDF1 similarly enhances HCC cell growth and invasion by facilitating the translation of GNAS mRNA, which subsequently interacts with STAT3 [66]. Research indicates that there is no statistically significant difference in the expression of ZC3H13 as m6A writers between HCC and paracancerous tissues [67]. However, it has also been indicated that reduced ZC3H13 expression is associated with unfavorable the Overall survival (OS) in patients with HCC [68]. This may require more in-depth studies to determine whether it has prognostic predictive value.
The m6A participate in numerous biological processes. For example, METTL14 enhances the stability of EGFR mRNA in a m6A-dependent manner and suppresses the metastasis of HCC cells via the EGFR/PI3K/AKT signaling pathway [69]. In addition, the silencing of WTAP makes LKB1 mRNA more stable, which helps the process of cellular autophagy [70]. METTL3 facilitates the upregulation of cyclic RNA hsa_circ_0058493 and promotes its cytoplasmic export through a m6A-dependent mechanism, resulting in enhanced progression of HCC [71]. FTO, functioning as a m6A demethylase, enhances its expression by decreasing IL-17RA methylation levels, thereby promoting liver inflammation and fibrosis [72]. IFN-γ induced an increase in m6A modification in HCC cells, which was significantly inhibited by Abrine. Indicating that Abrine may be involved in the regulation of abnormal m6A modifications in tumor cells [73]. Anti-angiogenic therapies are notable due to the heightened tissue vascularity observed in HCC and metastatic HCC. YTHDF2-deficient HCC cells exacerbate inflammation and vascular abnormalities by mediating the promotion of IL-11, SERPINE2 and VEGFA mRNA translation [74, 75]. In addition, RBM15 and IGF2BP3 can also exacerbate angiogenesis in HCC by promoting VEGFA mRNA translation [75]. METTL3 and ALKBH4 affect the growth, metastasis, and angiogenesis of HCC by regulating the expression of circ-CCT3 [76]. Furthermore, METTL3 facilitates the translation of YAP1, thereby enhancing vasculogenic mimicry (VM) formation and HCC progression via the Hippo pathway [77]. The latest studies, WTAP facilitates the translation of ATG5 and triggers autophagic iron death via a m6A-YTHDC2-dependent mechanism [25]. KIAA1429 additionally safeguards HCC cells from ferroptosis through m6A-dependent post-transcriptional modification of SLC7A11 [78]. The latest studies indicate that METTL16 extensively interacts with the translation initiation machinery, enhancing ribosome biogenesis and directly engaging with eIF3a/b to facilitate mRNA translation initiation. This process contributes to the initiation and progression of HCC and the self-renewal of liver cancer stem cells (LCSCs) [79]. METTL3 mediates m6A to increase NOVA2 expression and promotes LCSCs self-renewal through the Wnt pathway [80]. BMP9-mediated inhibition of m6A methylation within the 5′-UTR of CyclinD1 mRNA promotes its expression leading to HCC cell cycle progression [81]. YTHDF3 reduces ZFP41 mRNA stability via m6A modification, whereas high expression of ZFP41 inhibits Snail expression, and the EMT pathway suppresses HCC cell proliferation and invasion [82].

5-methylcytosine
5-Methylcytosine (m5C) is a chemical modification located at the fifth carbon atom of cytosine within RNA molecules. This modification occurs across various RNA types, including tRNAs, mRNAs, rRNAs, and ncRNAs. m5C is crucial in influencing RNA fate, encompassing aspects such as RNA stability, translation, transcription, and nuclear export [83]. Not only m6A involved in regulating HBV transcripts, but NSUN2 also mediates m5C modifications to promote HBV replication, and HBV promotes NSUN2 expression [84]. The latest studies indicate that YBX1 enhances HCV replication by mediating m5C modification [85]. Abnormal levels of m5C play a crucial role in the development and progression of HCC [86]. NSUN2 is the writers of m5C. It affects Ras and Wnt/β-catenin signaling pathway by regulating H19 RNA methylation modification to promote its binding to G3BP1, thereby facilitating the progression of HCC cells [40]. Furthermore, NSUN7 enhances the stability of CCDC9B mRNA in a m5C-dependent manner, thereby facilitating tumor progression [43]. ALYREF functions as a m5C reader, and its expression in HCC influences immune cell infiltration levels and correlates with unfavorable patient prognosis [87].

N7-methylguanosine
N7-methylguanosine (m7G) is a modification of RNA methylation that occurs at the seventh nitrogen of guanosine, introducing positively charged or amphiphilic ions into the nucleobase. This modification represents approximately 0.4% of all guanosine residues [88]. This modification is frequently observed in the 5′ cap and internal regions of mRNAs, as well as in tRNAs, rRNAs, and ncRNAs [89]. Numerous studies indicate that m7G methylation correlates with multiple facets of tumor biology, including stress response, cancer initiation, progression, and prognosis [89]. The enzyme primarily involved in this modification is METTL1, which associates with WDR4 complex to facilitate the increase of m7G methylation in the malignant potential of HCC cells [90]. Additionally, sublethal heat exposure following inadequate radiofrequency ablation results in METTL1 facilitating SLUG/SNAIL translation through m7G methylation modification, thereby increasing the malignant potential of HCC cells [91]. The latest studies indicate that METTL1 promotes circIPP2A2 expression by mediating m7G and promotes HCC malignant progression through the Hornerin/PI2K/AKT/GSK2β axis [46].

N1-methyladenosine
N1-methyladenosine (m1A), first discovered in the 1960s, results from the methylation of adenosine at the 1-position and is found in various RNA types, including tRNA, rRNA, mRNA, and lncRNA [92]. The m1A, as a post-transcriptional modification, significantly impacts RNA stability through its influence on base pairing [93]. The m1A methylation process is catalyzed by methyltransferases, including TRMT6/61A, TRMT10A, TRMT61B, and TRMT10C. Conversely, demethylases including ALKBH1, ALKBH3, ALKBH5, ALKBH7, and FTO facilitate the process of demethylation. Currently, proteins that specifically recognize m1A modifications in RNA have not been identified. Several m6A readers, including YTHDF1, YTHDF2, YTHDF3, and YTHDC1, have been demonstrated to recognize m1A modifications and interact with them [92]. The TRMT6/TRMT61A complex mediates m1A modifications that enhance PPARδ translation and facilitate HCC progression [50]. The latest research indicates that the dysregulation of m1A regulators correlates with the occurrence and progression of HCC, leading to effects such as altered tumor cell death, proliferation, invasion, and modifications in the tumor microenvironment. The inhibition of ALKBH3 leads to a reduction in the proliferation and migration of HCC [36].

3-methylcytidine
3-Methylcytidine (m3C) is a modification that occurs exclusively in eukaryotic tRNAs. This modification takes place at position 32 within the anticodon loop of certain tRNAs [94]. Recent research indicates that m3C may be catalyzed by particular methyltransferases, including METTL2A, METTL6, and METTL8 [95, 96]. METTL6 is involved in stabilizing cell adhesion genes, such as ITGA1 and CLDN14, thereby enhancing their expression and facilitating HCC colony formation, cell proliferation, and cell migration [51]. METTL6 facilitates the addition of 3-methylcytidine at C32 of particular serine tRNA heteroreceptors, influencing ribosome occupancy and RNA levels, thus contributing to HCC progression [95].

2′-omethylation
2′-O-methylation (Nm) represents a prevalent category of RNA modifications, found in rRNA, tRNA, mRNA, snRNA and lncRNA [97]. Nm modifications occur in the 2′-OH position of the RNA ribose and comprise 2′-O-methyl adenosine (Am), 2′-O-methyl guanosine (Gm), 2′-O-methyl cytidine (Cm), and 2′-O-methyl uridine (Um). Nm modifications enhance the hydrophobicity of nucleotides, subsequently influencing RNA structure, stability, and interactions, thereby regulating various biological processes [97]. Research indicates that Nm is found not only in the 5′ cap of mRNAs but also within the interior of certain RNAs [98]. SNORA23 influences ribosome biogenesis by interfering with Nm methylation of 28S rRNA, subsequently inhibiting HCC progression through the modulation of the PI3K/Akt/mTOR signaling pathway [99]. LncRNA MIR4435-2HG inhibits the degradation of NOP58, thereby facilitating the occurrence and progress sion of HCC through the enhancement of rRNA 2′-O-methylation [34]. In addition, CMTR1 catalyzes the Nm of the first transcribed nucleotide. Multi-omics studies have demonstrated that CMTR1 is overexpressed in a wide range of tumor types, including HCC, and regulates key aspects of RNA metabolism and ribosomal biogenesis by affecting ribosomal proteins and SnoRNAs [100]. Nm maintains ribosomal structural integrity and promotes accurate translation. The Box C/D snoRNP complex (containing FBL methyltransferase) directs rRNA-specific Nm modification. Overexpression of FBL leads to increased levels of rRNA Nm modification and promotes IRES-dependent oncogene translation [101].

Pseudouridine
Pseudouridine (Ψ) is the C5-glycosidic isomer of uridine. Pyrimidine nucleosides feature heterocyclic N-1 atoms linked to the C-1′ atom of pentose, resulting in a glycosidic bond. In contrast, the pseudouridine nucleoside is the C-5 atom of the heterocycle bonded to the C-1′ atom of the pentose sugar to form a glycosidic bond [102]. Uridine is converted to Ψ through the action of Ψ synthases, commonly referred to as pseudouridine synthases (PUSs), in eukaryotic organisms. As of now, six Ψ synthase families have been identified: TruA/B/D, RsuA, RluA, and Pus10p [103]. PUS1 enhances the protein levels of several oncogenes, such as IRS1 and c-MYC, through the incorporation of pseudouridine into mRNA [56]. HCC cells deficient in SNORA24-mediated Ψ-modification exhibited an increased incidence of miscoding and stop codon read-throughs, correlating with decreased patient survival [104]. Overexpression of DKC1 serves as a marker for the proliferative potential of HCC cells. Increased levels of reactive oxygen species (ROS) modulate cytoplasmic PDIA3 levels, contributing to the survival of HCC cells through the promotion of DKC1 [58].
In summary, several forms of RNA methylation are ubiquitous in HCC and influence tumor formation by modulating RNAs, either promoting or preventing it (Fig. 2). Nonetheless, the comprehension of m3C, Nm, and Ψ methylation in HCC is currently insufficient, which is essential for clarifying the underlying mechanisms and pinpointing pertinent regulatory proteins.

The role of RNA methylation in the tumor microenvironment
The tumor microenvironment (TME) is a structured ecosystem comprising cancer cells and various non-malignant cells, all co-embedded within an altered vascularized extracellular matrix. The tumor microenvironment comprises various immune cell types, cancer-associated fibroblasts, endothelial cells, pericytes, and numerous other tissue-resident cell types [105]. The tumor microenvironment plays a critical role in coordinating tumor immunity and significantly influences tumorigenesis, invasion, migration, progression, and therapeutic response [106]. TME is characterized by three primary features: hypoxia, metabolic reprogramming, and immune evasion. These features collectively foster an immune-suppressive microenvironment and modulate tumor immune evasion through diverse mechanisms [107]. Furthermore, in progressive cancers, both the tumor and the tumor immune microenvironment (TIME) exhibit dynamic characteristics. The growth of the tumor leads to evolving interactions between the tumor and the associated immune cells and stromal cell types [106]. Recent studies have emphasized the strong correlation between RNA methylation and various immunobiological processes, particularly tumor immunity [64]. RAN methylation is essential for maintaining tumor microenvironment homeostasis and metabolic reprogramming, thereby affecting immune cell function. It may also facilitate tumor immune evasion by influencing oncogenic and metastatic abilities, disrupting the tumor microenvironment’s equilibrium, and impairing immune cell functionality. For example, the m6A writer METTL3 sustains elevated glycolysis levels and promotes metabolic reprogramming in HCC [108]. The enzyme influences macrophage polarization, effector T cell differentiation and proliferation, dendritic cell activation, and immune checkpoint expression [109–111]. These studies elucidate the connection between RNA methylation, the tumor microenvironment, immune cells, and mechanisms of tumor immune evasion.
Thus, we explored the composition of the tumor microenvironment and clarified its role in mediating metabolic reprogramming and tumor immune evasion in HCC (Fig. 3).

Hypoxia
Hypoxia is a significant characteristic of the tumor microenvironment and is closely linked to tumor progression and immune response [112]. Hypoxia-inducible factor (HIF) is crucial for the regulation of cellular oxygen homeostasis. HIF modulates the equilibrium between oxidative and glycolytic metabolism and promotes enhanced oxygen supply through the activation of genes associated with erythropoiesis and angiogenesis [113]. These alterations foster an environment conducive to the proliferation of tumor cells. HIF is closely associated with tumor metabolism and significantly contributes to immune evasion.
Research indicates that hypoxia induces epigenetic remodeling of m6A in tumor cells. Dynamic alterations in RNA m6A modifications throughout cancer progression facilitate swift adaptation to changes in the microenvironment [37]. For example, hypoxia decreases YTHDF2 expression in an HIF-2α-dependent manner, which intensifies inflammation and remodeling of the HCC vasculature system [74]. HIF-1α-induced YTHDF1 enhances autophagy in HCC by promoting the translation of autophagy-related genes ATG2A and ATG14 in a m6A-dependent manner [37]. Under normoxic conditions, METTL14 facilitates the m6A modification of SLC7A11 mRNA. m6A-modified SLC7A11 mRNA is identified by YTHDF2 and transported to P-bodies for degradation. Reduction of SLC7A11 results in diminished cystine uptake and an increase in cysteine and GSH levels, subsequently promoting ROS generation and triggering ferroptosis. Hypoxia, in an HIF-1α-dependent manner, inhibits METTL14, which subsequently obstructs ferroptosis through the METTL14/YTHDF2/SLC7A11/ROS axis, thereby facilitating the progression of HCC [114]. Reoxygenation strategies may enhance the efficacy of m6A-targeted therapies in reactivating tumor-suppressive ferroptosis. In a hypoxic microenvironment, the overexpression of METTL16 facilitates the degradation of CSMD1-7 through m6A methylation modification, subsequently enhancing HCC metastasis [115]. Furthermore, under hypoxic conditions, HBx interacting protein (HBXIP) upregulates METTL3 expression, resulting in elevated levels of HIP-1α and sustained high glycolytic activity, which contributes to the progression of HCC [108]. The application of METTL3 inhibitors has the potential to reverse this outcome; however, further extensive experiments are required to validate this hypothesis. Hypoxia upregulates RNA polymerase I activity, which in turn alters Nm modification of rRNA and promotes VEGF- and IRES-dependent translation [116].

Metabolic reprogramming
Metabolic reprogramming serves as a significant mechanism for tumor immune evasion [117]. Numerous studies indicate that RNA methylation plays a significant role in the metabolic processes of glucose, amino acids, and lipids, subsequently regulating the tumor microenvironment balance that influences tumor immune evasion [118].

Glucose metabolism
Cancer cells exhibit glucose consumption via Warburg metabolism. The Warburg effect denotes the phenomenon whereby cancer cells preferentially utilize aerobic glycolysis for energy production, even in the presence of adequate oxygen, rather than relying on oxidative phosphorylation (OXPHOS) [119]. Cancer cells suppress anti-tumor immune responses by competing for essential nutrients and diminishing the metabolism of tumor-infiltrating immune cells. This metabolic approach influences various immune cells within the tumor immune microenvironment (TIME), such as dendritic cells (DCs), activated T cells, natural killer (NK) cells, neutrophils, and M1 macrophages [120]. The latest research indicates that glucose deprivation conditions result in decreased m6A modification, which subsequently reduces necrosis in HCC cells. Under conditions of glucose deprivation, safeguarding HCC cells from necrosis produced by glucose deprivation through the reduction of m6A modification of FOSL1 and the enhancement of its mRNA stability in a YTHDF2-dependent way [121]. METTL14 enhances SIRT6 mRNA stability via m6A modification, consequently diminishing HCC glycolysis and malignancy [122]. The latest studies indicate that IGF2BP1 increases c-Myc stability by mediating m6A, which in turn enhances aerobic glycolysis in HCC [123]. Furthermore, NOP2 facilitates the increase of m5C in c-Myc mRNA expression and the stimulation of glycolytic gene expression to augment glycolysis in HCC [39]. HNRNPA2B1 facilitates tumor proliferation via regulating the methylation modification of PCK1 mRNA, and thereby limiting gluconeogenesis [30]. Inhibition of FTO leads to a reduction in tumor cell glycolysis through the downregulation of glycolytic enzymes, including PFKP, PGAM1, and HK1 [124]. In addition, silencing of METTL6 leads to reduced energy turnover and altered substrate utilization [95]. PUS1 can mediate mRNA translation through Ψ-modification enhances protein levels of c-Myc and promotes HCC progression through the MYC and mTOR pathways [56, 125]. The role of RNA methylation in HCC gluconeogenesis requires additional investigation.

Amino acid metabolism
Aberrant amino acid metabolism inhibits the anti-tumor immunity of immune cells and may facilitate tumor immune evasion [126]. Glutamine catabolism is a significant aspect of metabolic remodeling in tumors [127]. Furthermore, inhibiting glutamine metabolism reduces the immunosuppressive tumor microenvironment and counteracts tumor immune evasion, thereby leading to a decrease in tumor growth [128].
Previous studies have established the regulatory function of m6A modifications in glutamine metabolism. FTO-mediated m6A demethylation enhances the expression of the glutamine transporter protein SLC1A5 [129]. Furthermore, FTO exhibited increased expression following the inhibition of glutamine catabolism, which enhanced ATF4 mRNA stability in a YTHDF2-dependent manner. This process subsequently up-regulated DDIT4 expression and inhibited the mTOR signaling pathway, thereby activating cellular autophagy [130]. Furthermore, ALKBH5, acting as a m6A eraser, reduces glutathione (GSH) via the ROS-YAP-AXL-ALKBH5-GLS2 loop, leading to metabolic remodeling and subsequently inhibiting the metastasis of GC cells [131]. The latest research indicates that TARBP1 facilitates the translation of ASCT2 mRNA through Nm-modified tRNA, subsequently enhancing glutamine metabolism in HCC cells [54].

Lipid metabolism
Lipids serve as essential constituents of biological membranes, play a critical role in biosynthesis, and are involved in energy storage mechanisms. Enhanced lipid anabolism is regarded as a significant contributor to tumorigenesis [132]. Additionally, the heightened affinity of tumor cells for lipids and cholesterol will intensify the malignant transformation of these cells and contribute to the abnormal accumulation of lipids in the tumor microenvironment [133]. Dysregulated lipid metabolism inhibits the anti-tumor capacity of immune cells and promotes immune evasion by tumor cells, thereby impairing the immune response and remodeling the immunosuppressed tumor microenvironment.
Numerous studies indicate that RNA methylation is essential for lipid metabolism in HCC. In HCC cells, m6A modification enhances the fatty acid oxidation (FAO) process and contributes to chemoresistance by facilitating shearing and promoting the expression of ERRγ precursor mRNA [134]. METTL5 facilitates lipogenesis and the progression of HCC via ACSL4-mediated fatty acid oxidation [135]. The overexpression of FTO increased adipogenesis and enlarged lipid droplets in the liver while inhibiting CPT1-mediated fatty acid oxidation via the SREBP1c pathway [136]. Additionally, the silencing of FTO facilitates the decay of FASN mRNA through a YTHDF2-dependent mechanism, subsequently leading to decreased levels of ACC1 and ACLY proteins, which inhibit adipogenesis in HCC cells [137]. Furthermore, the overexpression of METTL14 resulted in elevated protein levels of ACLY and SCD1, which in turn enhanced triglyceride and cholesterol production, along with the accumulation of lipid droplets [138]. METTL3 increases the stability of PPARα mRNA through a YTHDF2-dependent mechanism, subsequently influencing the circadian rhythm of hepatic lipid metabolism [139]. The m1A writer TRMT6/TRMT61A complex enhances cholesterol synthesis by activating Hedgehog signaling, which facilitates PPARδ translation, thereby promoting LCSCs self-renewal and HCC progression [50]. The m5C writer NSUN2 promotes CDKN1A translation through an ALYREF-m5C-dependent mechanism, which in turn inhibits MCE processes and adipogenesis [140]. The latest studies indicate that LINC00618 promotes NSUN2 stabilization by inhibiting ubiquitin–proteasome-mediated degradation, which in turn mediates m5C to promote SREBP2 translation. And SREBP2 induces HCC cell proliferation, migration and EMT by mediating the cholesterol synthesis pathway [141].
In summary, RNA methylation plays a role in abnormal metabolic processes and the formation of a hypoxic microenvironment in HCC. It influences various downstream pathways, resulting in immune system dysfunction, HCC development, chemotherapy resistance, and immune evasion. Aberrant metabolisms inhibit immune cell functions and enhance tumor biological behaviors. RNA methylation modulates the tumor microenvironment, a phenomenon observed in various tumors, including HCC. FTO decreases APOE mRNA-m6A levels and modulates the IL-6/JAK2/STAT3 signaling pathway, which inhibits glycolysis and suppresses tumor growth in thyroid cancer [142]. FTO enhances lipid droplet formation in esophageal cancer by promoting HSD17B11 expression via a YTHDF1-dependent mechanism [143]. NSUN2 facilitates the modification of lncRNA-m5C, which increases the stability of GLS mRNA, thus advancing the reprogramming of glutamine metabolism and expediting the progression of gastric cancer [144].

The role of RNA methylation in modulating innate immune responses
The liver exhibits a distinctive immune landscape characterized by a high density of immune cells, including specific types such as Kupffer cells (KCs), which are absent in other body regions [145]. These cells are regulated by RNA methylation. In the KCs, METTL3 enhances STING activation and up-regulation, thus contributing to radiation-induced liver disease (RILD) through the TEAD1-STING-NLRP3 signaling pathway [146]. KCs are implicated in the pathogenesis and progression of HCC due to their capacity for phenotypic changes and cytokine secretion [147]. Altering RNA methylation levels in KCs to restructure their function may serve as a therapeutic strategy for HCC. The system contains a significant quantity of innate immune cells, such as NK cells, macrophages, monocytes, dendritic cells, and myeloid-derived suppressor cells (MDSCs) [145]. The characteristics of the tumor microenvironment, such as hypoxia and metabolic abnormalities, equally impact these immune cells [148]. We investigated immune cells where RNA methylation is significantly linked to innate immunity in various tumors (Fig. 4).

Macrophage
Macrophages are crucial in both the innate and adaptive immune responses, functioning through the phagocytosis of foreign substances, the presentation of antigens, and intercellular communication with other immune cells [149]. Tumor-associated macrophages (TAMs) constitute a significant infiltrating cell population in tumors and are crucial in shaping the tumor immune microenvironment [150]. Tumor-associated macrophages are typically categorized into two primary subtypes: M1 macrophages and M2 macrophages. M1 macrophages are typically regarded as anti-tumorigenic, while M2 macrophages are viewed as pro-tumorigenic [151]. TAMs exhibit significant plasticity, enabling the polarization of M2-type macrophages into M1-type macrophages and modification of their functions, thereby contributing to the inhibition of tumor progression [109].
Research indicates that RNA methylation regulates macrophage polarization through the modulation of various signaling pathways and the reprogramming of the tumor microenvironment. The latest research indicates that silencing YTHDF2 promoted the polarization of anti-tumor macrophages and enhanced their antigen cross-presentation capacity by increasing the stability of CX3CL1 mRNA [152]. METTL3 is essential for macrophage polarization. In models with METTL3 silenced, the therapeutic efficacy of PD-1 blockade diminished, resulting in accelerated tumor cell progression and metastasis [153]. In a similar manner, the silencing of METTL3 reduced M2 polarization of KCs by lowering RBM14 expression via YTHDF1-dependent m6A modification [154]. The m6A modification facilitated by METTL3 promotes M1 polarization of macrophages and induces pyroptosis, thereby accelerating liver fibrosis [153, 155]. Research indicates that METTL3 regulates M2 macrophage polarization, while M2 macrophages also influence METTL3 expression. The latest research indicates that M2 macrophages enhance METTL3 expression through the IL-6/STAT3 signaling pathway. In turn, METTL3 facilitates SLC16A1-AS1 expression via IGF2BP3-dependent m6A modification, subsequently promoting M2 macrophage polarization [156]. The latest studies indicate that IGF2BP3 promotes the malignant phenotype of HCC cells and macrophage M2 polarization by mediating m6A for RRM2 translation [157]. Furthermore, the knockdown of FTO suppressed the polarization of M1-type and M2-type macrophages by inhibiting the NF-κB signaling pathway and silencing YTHDF2 [158]. ALKBH5 enhances CCL5 production via various pathways, facilitating monocyte infiltration and M2 polarization [159]. Moreover, various modified RNA types influence TAM polarization. For example, FBL functions as a 2′-O-methyltransferase, inhibiting innate immune responses by reducing the expression of IFN-Ⅰ and ISGs in macrophages [160].
These findings indicate that RNA methylation affects TAM infiltration in HCC by catalyzing and modulating the polarization capacity of TAMs, thereby influencing their infiltration. The findings underscore the significant role of RNA methylation in immunotherapy, potentially offering new avenues and targets for therapeutic intervention.

Natural killer cells
Natural killer (NK) cells, integral to the innate immune system, are crucial in tumor immunity [161]. Research indicates that RNA methylation plays a role in the regulation of anti-tumor immunity and natural killer cell function. METTL3-deficient NK cells demonstrate disrupted homeostasis within the tumor immune microenvironment and display an abnormal infiltration state, which impairs anti-tumor immunity [162]. The latest research indicates that METTL3 and METTL14 play complementary roles in the regulation of NK cell development and function [163]. Moreover, the defective m6A reader YTHDF2 negatively affects the antitumor and antiviral functions of NK cells in vivo, which are essential for sustaining NK cell homeostasis and terminal maturation [164]. Furthermore, IL-2 and IL-15 are essential for the functionality of NK cells, playing significant roles in their proliferation, activation, and survival. FTO functions as a significant negative regulator of IL2/15-mediated JAK/STAT signaling in NK cells. FTO-deficient NK cells demonstrate increased tumor cytotoxicity and improved cell viability [165].

Dendritic cells
Dendritic cells (DCs) serve as the primary antigen-presenting cells (APCs) in the organism, facilitating the connection between innate and adaptive immunity [166]. A significant quantity of aberrant m6A mRNA modifications has been identified in cancerous dendritic cells [167]. METTL3 enhances dendritic cell activation and dendritic cell-mediated T-cell responses by facilitating the translation of CD40 and CD80 through m6A modification in dendritic cells [110]. YTHDF1 enhances the expression of various DC lysosomal proteases by interacting with m6A modifications in their transcripts, thereby facilitating their translation and influencing DC cell function. Inducing checkpoint blockade through YTHDF1 depletion in dendritic cells may represent a potential immunotherapy strategy [168]. CCR7 enhances the expression of lnc-Dpf3 by eliminating the m6A modification, thereby facilitating the metastasis of dendritic cells to lymph nodes and leading to an exaggerated adaptive response [169]. Furthermore, YTHDF2 decreases the expression level of lnc-Dpf3 in resting mature dendritic cells in a m6A-dependent manner [169]. Collectively, m6A modifications are essential for the activation and functionality of dendritic cells, as well as their migration.

Myeloid-derived suppressor cells
Myeloid-derived suppressor cells (MDSCs) constitute a significant element of the tumor microenvironment, exhibiting strong immunosuppressive properties [170]. These cells demonstrate immunosuppressive properties and affect tumor outcomes by markedly inhibiting T-cell function [171]. The inhibition of YTHDF2 expression has been demonstrated to enhance the expansion and suppression of MDSCs [172]. Silencing of ALKBH5 expression resulted in a reduction of MDSC accumulation and an increase in DC abundance [173]. Moreover, METTL1 enhances the expression of CSCL5/8 in a m7A-dependent manner, subsequently resulting in the accumulation of MDSCs and immunosuppression in HCC and intrahepatic cholangiocarcinoma [174].

The role of RNA methylation in the modulation of adaptive immunity
RNA methylation serves as a crucial regulator in the modulation of adaptive immune processes within the host [167]. T and B lymphocytes are the primary immune cells engaged in adaptive immune processes. These cells play a critical role in the immune microenvironment of the liver and affect the initiation and progression of HCC [145]. RNA methylation may affect the development, differentiation, maturation, and depletion of immune cells by regulating RNA expression and protein translation (Fig. 4).

T-lymphocytes
T-cells are produced in the bone marrow and subsequently migrate to the thymus for development and maturation. T-cells in the thymic cortex mature and differentiate into CD4 or CD8 positive cells [175]. Research indicates that RNA methylation plays a critical role in T-cell functions such as proliferation, activation, and apoptosis, mediated by various regulatory factors [176].
Silencing METTL3 has been demonstrated to reduce c-Myc protein expression, resulting in diminished value-addition of allogeneic CD4+ T-cells, heightened apoptosis, and a halt in the cell cycle at the G0 phase [111]. Defective METTL3 in naïve T-cells inhibits IL-7-mediated STAT5 activation as well as T-cell proliferation and differentiation, thereby disrupting T-cell homeostasis and differentiation [177]. Furthermore, METTL3 and FTO facilitate the regulation of CD4L mRNA expression through a YTHDF2-dependent mechanism, subsequently influencing CD4+ T-cell activation [178]. Besides METTL3, WTAP also influences CD4+ T-cell activation, apoptosis, and various other processes. The silencing of WTAP resulted in reduced CD4+ T-cell proliferation and increased apoptosis following TCR signaling activation [179]. Furthermore, TRMT61A facilitated the m1A modification of tRNA, enhanced the translation of several critical proteins during CD4+ T-cell activation, and contributed to the swift immune response of CD4+ T-cells [180].
Furthermore, RNA methylation not only affects the function of CD4+ T-cells, but also CD8+ T-cells. For example, YTHDF2 deficiency enhances CD8+ T-cell effector function by inhibiting glycolysis and promoting mitochondrial respiration in CD8+ T-cells [152]. In mice lacking YTHDF1, there is an increase in antigen-specific CD8+ T-cell antitumor responses [168]. IGF2BP3 enhances macrophage infiltration and M2 polarization while inhibiting CD8+ T-cell activation through the facilitation of CCL5 and TGF-β1 secretion and direct binding [181]. Furthermore, WTAP enhances the stability of circCCAR1 in a m6A-dependent manner and facilitates its packaging into exosomes in a hnRNPA2B1-dependent manner, leading to accelerated depletion of CD8+ T-cells [182]. FTO-mediated modification of m6A demethylation enhances the expression of the bZIP family, including JUNB transcription factors, while JUNB overexpression suppresses CD8+ T-cell activation [124]. The latest research indicates that the silencing of FTO increased the expression of CD8+ T-cell markers CD45 and CD8, subsequently promoting the infiltration, activation, and recruitment of tumor-infiltrating CD8+ T-cells. This also led to an elevated expression of M1 markers such as CD68, CD80, and CD86, thereby enhancing the migration of anti-tumor M1 macrophages within the HCC tumor microenvironment [183]. The latest research indicates that silencing of FTSJ3 has been demonstrated to increase cellular infiltration and enhance cytotoxic activity in CD8+ T-cells, thereby improving the efficacy of PD-1 blockade therapy [53]. Silencing of RPUSD3, a writer of Ψ, resulted in a significant increase in CD8+ T cell infiltration [59].

B-lymphocytes
B cell maturation and development is a systematic process involving the progressive differentiation of B cells from pro-B cells to pre-B cells and ultimately to immature B cells. The spleen is the site for the further maturation of immature B cells [184]. Pre-B cells can be categorized into two primary subpopulations: large, immature, actively dividing large-pre-B cells and small, more mature, quiescent small-pre-B cells [185]. Research indicates that RNA methylation plays a role in the maturation and differentiation of B cells. The m6A modification, facilitated by METTL14, enhances the decay of mRNA encoding negative immunomodulatory factors in a YTHDF2-dependent manner while also supporting the positive selection and proliferation of GC B cells [186]. METTL14 deletion hindered interleukin-7 (IL-7)-induced pro-B cell proliferation and the transition from large-pre-B to small-pre-B cells in a m6A-dependent manner, subsequently affecting B cell developmental maturation [187]. Furthermore, the differentiation of YTHDF2-deficient B cells into germinal center B cells is inhibited, while B cell proliferation and activation remain unaffected [188]. WTAP negatively regulates the expression of CD40 mRNA, thereby influencing B cell development and activation [189].
These findings indicate that RNA methylation may be a therapeutic target for improving innate and adaptive immune responses and plays a role in various biological processes within immune cells. RNA methylation plays a role in regulating physiological processes, including macrophage infiltration and differentiation, the accumulation of MDSC, T-cell activation, and B-cell differentiation.

The role of RNA methylation in the modulation of tumor immune evasion
The disruption of immune cell activation and phenotypic switching processes leads to immune depletion and suppression of the tumor immune microenvironment, subsequently facilitating tumor immune escape [118]. The anti-tumor immune response primarily relies on the recognition of tumor antigens by activated CD8+ T-cells [190]. However, tumor cells are extremely cunning, capable of emitting inhibitory signals that compromise T cell immune function and impede effective immune responses [191]. Tumor cells evade immune surveillance and elimination by producing an immunosuppressive tumor microenvironment and upregulating immune checkpoint proteins [190]. Furthermore, the interaction between PD-1 and PD-L1 inhibits the proliferation, survival, and responsiveness of CD8 cytotoxic T lymphocytes (CTLs) while promoting peripheral immune tolerance, thereby facilitating tumor evasion of immune system surveillance [192].
Evidence indicates that RNA methylation regulates tumor immune evasion through the modulation of immunosuppressive factors associated with tumors (Fig. 5). For example, the m6A modulates PD-1/PD-L1 via several physiological processes, including RNA-selective shearing, translation, and stability, thereby facilitating immune evasion [193]. The latest studies, METTL3 has been shown to mediate cyclic RNA stability in an m6A-dependent manner, which in turn is involved in regulating malignant progression and immune escape in HCC [63]. METTL3 enhances CD47 expression through an IGF2BP1-dependent mechanism, subsequently facilitating immune evasion in HCC [194]. m6A modification facilitated the up-regulation of TUG1 expression, which subsequently enhanced PD-L1 to suppress the anti-tumor immune response of CD8+ T-cells while also promoting CD47 to inhibit macrophage phagocytosis, ultimately aiding tumor immune escape [195]. METTL14 enhances MIR155HG expression in a m6A-dependent manner, subsequently promoting PD-L1 expression via the miR-223-STAT1 pathway, which ultimately facilitates immune escape in HCC [196]. LRPPRC, as m6A Writers, can increase PD-L1 stability by m6A modification and thus mediate HCC immune evasion [29]. Besides m6A, modifications like Nm and m5C also play a role in regulating tumor immune evasion. The latest research indicates that SAMD4B decreases the stability of APOA2 in a Nm-dependent manner, subsequently leading to reduced PD-L1 levels and impairing immune escape in HCC [55]. Furthermore, the m5C-reading protein YBX1 enhances PD-L1 expression and markedly reduces CTL proliferation upon interaction with PD-1, thereby inhibiting the immune response of the organism [45]. These studies illustrate the significant function of RNA methylation in the regulation of tumor immune evasion, indicating that targeting this mechanism may serve as a viable therapeutic approach.

Integrated mechanisms of RNA methylation in metabolic-immune crosstalk
RNA methylation regulates metabolic reprogramming and the composition of the immune microenvironment. In addition, RNA methylation appears to modulate the composition of the immune microenvironment by regulating metabolic reprogramming. c-Myc promotes metabolic reprogramming in HCC, enhancing glycolysis and glutamine metabolism to facilitate rapid tumor growth [197]. The latest research indicates that IGF2BP1 promotes PD-L1 transcription and enhances CD8+ T-cell-mediated HCC immune evasion by mediating m6A modification of c-Myc [123]. NUSN2 augmented cholesterol biosynthesis via m5C modification of SOAT2, concurrently impairing CD8+ T cytotoxicity and facilitating HCC immune evasion [198]. Glucose directly promotes oligomerization and activation of NSUN2. Activated NSUN2 mediates TREX2 stability via m5C, which in turn drives anti-PD-L1 immunotherapy resistance by limiting cGAS/STING signaling [199]. TRMT61A-mediated tRNA-m1A modification enhances tumor killing of CD8 + T cells by promoting cholesterol biosynthesis [200].
These researches indicate that RNA methylation regulatory proteins can affect the immune microenvironment through metabolic reprogramming, supporting the idea of integrating metabolic reprogramming inhibitors with immunotherapy.

RNA methylation influences the efficacy of immunotherapy and systemic therapy
Numerous small-molecule inhibitors targeting RNA methylation have been developed, potentially enhancing treatment outcomes in HCC (Table 2). STM2457, a METTL3 inhibitor that decreases m6A levels, has demonstrated a significant enhancement in treatment outcomes for HCC when used in conjunction with lenvatinib [201]. STM2457 can also suppress c-Myc expression [111]. The latest research indicates that the FTO inhibitor CS2, when used in conjunction with anti-PD-1, demonstrated a more pronounced reduction in tumor mass in HCC mice [183]. In parallel, Dac51, an FTO inhibitor, enhanced T-cell infiltration, and the combination of this treatment with PD-L1 blockers further augmented therapeutic effects [124]. A prior investigation identified a potential YTHDF2 inhibitor, DC-Y13-27, which enhances the anti-tumor immune response by targeting YTHDF2 in MDSC [202]. The latest study identified a more effective YTHDF2 inhibitor, DF-A7, which improves anti-tumor activity by targeting m6A modification in tumor cells [152]. A nanoparticle drug, MFMP, consists of hollow mesoporous manganese dioxide (MnO2) nanoparticles, the MAT2A inhibitor FIDAS-5, macrophage membranes, and anti-PD-L1. MFM enhances HCC antigenicity through a number of biological functions including reduction of m6A modifications, inhibition of EGFR expression to promote cytotoxic T cell recognition and cytotoxic killing [203]. Inhibition of YTHDF1, when combined with anti-PD-1 therapy, markedly reduced tumor progression. CircRHBDD1 may function as a cofactor for YTHDF1, facilitating its association with PIK3R1 mRNA, which promotes aerobic glycolysis and plays a role in resistance to anti-PD-1 therapy [204]. LNP-siRNA is a potential option for disease treatment [205]. Research indicates that LNP-siYTHDF1, which targets YTHDF1, decreases tumor burden and enhances the efficacy of anti-PD-1 treatment [206]. In future research, LNP-siRNA may be integrated with additional RNA methylated proteins to investigate the potential of RNA methylation therapy.
RNA methylation has been demonstrated to influence the sensitivity of HCC to single-agent targeted therapies, such as sorafenib and levatinib. For example, METTL3-mediated m6A modification enhances resistance to sorafenib in HCC cells, resulting in a diminished therapeutic outcome of sorafenib [207]. METTL3-mediated recognition of m6A in FZD10 and NOVA2 transcripts by YTHDF2 and YTHDF1, respectively, increases their expression and leads to a worse treatment outcome of levatinib for improving HCC [80, 208]. Moreover, the silencing of METTL14 increases the stability of HNF3γ mRNA via m6A-dependent mechanisms, leading to heightened sensitivity of HCC cells to sorafenib [209]. Besides m6A modifications, m5C and m7G also influence sensitivity to single-agent targeted therapies. NSUN2 inhibits the sensitivity of HCC cells to sorafenib by activating the Ras pathway, leading to reduced apoptosis rates and cell cycle arrest [210]. Moreover, the silencing of WDR4 markedly enhanced the efficacy of sorafenib in treating HCC [47]. The latest studies have shown that lactoylated IGF2BP3 enhances PCK2-mediated gluconeogenesis and maintains high levels of SAM and GSH. Whereas increased SAM concentration enhances RNA m6A methylation via METTL3, subsequent binding of lactoylated IGF2BP3 to m6A sites on PCK2 and NRF2 mRNA promotes their translation, which in turn leads to levatinib resistance [211]. High expression of IGF2BP1 enhances sensitivity to oxaliplatin in HCC patients [123].
In summary, these studies indicate that RNA methylation regulators significantly influence immunotherapy and systemic therapy, highlighting their potential to impede tumor progression. Specifically, modifying the expression of RNA methylation regulatory factors markedly suppressed tumor progression in conjunction with immune checkpoint inhibitors or sorafenib. Inhibitors of RNA methylation regulatory factors are anticipated to yield substantial progress in the future and offer novel combination therapies for patients.

Future perspectives
While numerous studies elucidate the mechanism of RNA methylation in HCC, the majority focus exclusively on the m6A methylation modification, with other methylation types in the HCC tumor microenvironment and immune evasion receiving insufficient scrutiny. A significant proportion of RNA methylation-regulated proteins in HCC have undefined functions. For instance, only a limited number of regulatory proteins, including m3C, Nm, and m1A, have been documented in HCC. Comprehensive investigations focusing on RNA methylation beyond m6A in the immune milieu and immune evasion of HCC tumors are essential to clarify the functions of RNA methylation alterations in the development, invasion, migration, and proliferation of HCC. For instance, Ψ can be incorporated into pre-mRNAs during co-transcription, therefore influencing the interaction of pre-mRNAs with the spliceosome [212]. In vitro, Ψ can enhance mRNA stability through base stacking, fortifying secondary structures, and influencing protein binding [213]. The impact of Ψ on mRNA stability in vivo and on pre-RNAs in HCC remains ambiguous and necessitates additional investigation.
Much research has demonstrated that c-Myc serves as a convergent node for various RNA methylations. m6A influences c-Myc via METTL3 and IGF2BP1 [111, 123], m5C regulates c-Myc expression through NOP2 [39], and Ψ elevates c-Myc protein levels through PUS1 [56] (Fig. 6A). This complex amplification effect facilitates metabolic reprogramming and immunological evasion. However, significant gaps persist concerning the separate or synergistic regulation of immune escape by Ψ and m5C via c-Myc. Alongside c-Myc, the VEGF family may act as shared targets, demonstrating synergistic effects across methylations. The M6A regulatory proteins RBM15, YTHDF2 and IGF2BP3 facilitate VEGFA translation [75], while ALKBH5 and METTL3 influence circ-CCT3 through m6A alteration [76], hence enhancing angiogenesis. Under hypoxic conditions, Nm modification reprogramming enhanced VEGF translation [116] (Fig. 6B). In the future, the impact of co-modification on HCC can be substantiated by validating RNA methylation-regulated protein interactions by co-modified transcriptome analyses and both in vitro and in vivo research.
RNA methylation has potential in HCC immunotherapy. Regulatory factors can be utilized to estimate the diagnosis and prognosis of HCC, as well as to influence HCC progression and drug resistance. In summary, RNA methylation-targeted therapies may offer integrated strategies for the treatment of HCC. Nevertheless, current research has concentrated on traditional anti-PD-1 treatments, overlooking alternative immunotherapeutic options. Chimeric antigen receptor (CAR) represents a promising cancer immunotherapy encompassing several modalities, such as CAR-NK cell therapy and CAR-T cell therapy; nonetheless, its utilization in solid tumors, including HCC, presents significant challenges [214, 215]. Recent research indicates that RNA methylation is crucial for regulating the effector function, longevity, and metabolic adaptability of these immune cells, offering novel opportunities to enhance their efficacy. M6A methylation facilitated by METTL3 is crucial for the maturation and cytotoxicity of NK cells [162]. Engineering CAR-NK cells with elevated METTL3 expression may improve the anti-HCC response by preserving NK cell activation and lifespan. FTO-deficient NK cells demonstrate increased tumor cytotoxicity and improved cell viability [165]. FTO deficiency promotes the proliferation of CD8+ T cells and improves tumor cytotoxicity [124]. Developing RNA methylation-targeted treatments alongside CAR may enhance the therapeutic efficacy of CAR in solid malignancies.
Small molecule inhibitors of RNA methylation not only improve therapy in combination with immunosuppressants but also synergize with metabolic inhibitors. PGAM1 is essential in cancer metabolism, serving a pivotal catalytic function in the conversion of 3-PG to 2-PG during aerobic glycolysis. KH3, functioning as a metabolic inhibitor, suppresses PGAM1 expression.KH3 suppresses PGAM1 expression by targeting PGAM1 to enhance CD8 + T cell infiltration and its associated anti-tumor immunity [216]. Inhibition of IGF2BP1 has been demonstrated to similarly downregulate PGAM1 expression [124]. The concurrent application of small molecule inhibitors of RNA methylation may enhance the efficacy of metabolic inhibitors, thereby significantly impeding HCC metabolism.
Crucially, combining RNA methylation-targeted therapies with molecularly targeted agents holds immense promise for improving HCC outcomes, as evidenced by compelling preclinical synergy, such as FTO inhibitors enhancing anti-PD-1 efficacy and METTL3 inhibitors overcoming lenvatinib resistance. This combinatorial approach uniquely co-targets tumor-intrinsic vulnerabilities (proliferation, metabolism, drug resistance) and the immunosuppressive TME, potentially offering superior efficacy compared to monotherapies or existing combinations. To translate this promise into reality, priority should be given to advancing the most robust combinations (such as FTOi/anti-PD-1 or METTL3i/lenvatinib) into early-phase clinical trials. These trials must incorporate biomarker-driven patient selection, such as FTO or METTL3 expression, carefully manage potential on-target/off-tumor toxicities through improved inhibitor selectivity or delivery systems like nanoparticles, and focus on populations refractory to current standards. Concurrently, exploring RNA methylation regulation to enhance CAR-NK/T cells and reduce tumor metabolism for next-generation therapies is also a frontier for long-term innovation. Overcoming these challenges will be pivotal in harnessing the full potential of RNA methylation modulation to revolutionize HCC treatment paradigms.
While m6A dominates current research, emerging evidence implicates non-m6A modifications (m5C, m1A, m7G, Ψ, etc.) in HCC immune evasion and therapy resistance. Future studies should prioritize: Decoding the immune mechanism of non-m6A modifications. Investigate the impact of specific modifications, such as m5C-NSUN2 in SOAT2 mRNA that enhances cholesterol synthesis and Ψ-PUS1 in oncogene translation, on the reprogramming of immune cell function, including T-cell exhaustion and macrophage polarization, through the integration of single-cell epitranscriptomics and immune phenotyping. Investigate crosstalk between non-m6A writers/readers, such as TRMT61A for m1A-tRNA, ALYREF for m5C, and immune checkpoint pathways (PD-L1, CTLA-4) using CRISPR screens in HCC-immune cocultures. In addition, explore if non-m6A modifications drive adaptive resistance to targeted therapies (such as m7G-METTL1-mediated SLUG/SNAIL translation post-RFA and Ψ-DKC1 stabilizing c-MYC under sorafenib). Longitudinal multi-omics of patient biopsies pre-/post-therapy could identify dynamic modification signatures. Finally, making technological innovations will help the RNA methylation regulatory mechanism to be further improved. For example, develop modification-specific antibodies and in vivo mapping tools for low-abundance marks (m3C, Nm) to resolve spatial distribution in HCC tumor-immune niches. Integrate artificial intelligence with ribosome profiling to predict modification-dependent immune gene translation efficiency. Evaluate the interactions of non-m6A inhibitors, such as NSUN2 blockers and PUS1 antagonists, with immune checkpoint inhibitors and metabolic drugs in immunocompetent hepatocellular carcinoma models, utilizing CAR-T and NK cell engineering to maintain effector cell functionality in the context of modification-induced immunosuppression. Addressing these gaps will unveil hidden epitranscriptomic axes controlling HCC immunometabolic plasticity and expand the druggable landscape beyond m6A.
The translational potential of RNA methylation in HCC is hindered by limitations in biomarkers and targeted therapeutics. The dynamic spatiotemporal variability of RNA changes complicates the establishment of homogeneous biomarkers and obstructs the discovery of clinically actionable biomarkers. For instance, METTL3 regulates both YAP1 to promote HCC progression [77] and MTF1 to inhibit HCC progression [18]. Several small-molecule inhibitors that target RNA methylation regulatory proteins have been identified and thoroughly evaluated in animal studies for the treatment of HCC, such as STM2457, CS2 and DC-Y13-27, either as monotherapy or in combination with other agents. Unfortunately, most of the small-molecule inhibitors associated with RNA regulatory proteins have not been discovered, and only a small number of them have been identified. And there are no approved pharmacological agents specifically targeting RNA modification in HCC. Investigations into small molecule inhibitors remain in preliminary phases, necessitating extensive clinical validation to ascertain the true efficacy of RNA methylation-targeted therapy in HCC. The advancement of supplementary small molecule inhibitors aimed at these biological functions related to RNA methylation, particularly in conjunction with immune checkpoint inhibitors or targeted therapeutic agents, presents significant potential for the clinical management of HCC. Abrine has demonstrated the ability to enhance phagocytosis in macrophages, whereas m6A alteration is crucial for the regulation of macrophages. Nonetheless, it remains ambiguous if Abrine modulates macrophages through the influence of m6A alteration; this unexplored domain necessitates additional examination and inquiry, anticipated to be a pivotal accomplishment in elucidating RNA small molecule inhibitors for HCC treatment. Integrating multi-omics to elucidate the characteristics of combination biomarkers and advancing the development of small molecule inhibitors may expedite the transition from mechanistic understanding to clinically applicable RNA methylation targeting strategies, thereby enhancing HCC patient stratification and personalized treatment approaches.

Conclusions
RNA methylation is a crucial epigenetic mechanism that regulates the progression of hepatocellular carcinoma through various interactions within the tumor microenvironment. The seven principal RNA modifications—m6A, m5C, m1A, m7G, m3C, Ψ, and Nm—play a critical role in HCC pathogenesis by influencing metabolic reprogramming, including glucose, amino acid, and lipid metabolism, as well as hypoxic responses and immune evasion. The modifications govern essential oncogenic pathways through changes in RNA stability, translation, and processing, consequently facilitating tumor proliferation, invasion, and metastasis. RNA methylation dysregulation significantly affects both innate and adaptive immunity by altering macrophage polarization, NK cell function, T-cell activation, and immune checkpoint expression, such as PD-L1, thereby promoting immune escape in HCC.
Regulatory proteins involved in RNA methylation, including writers, erasers, and readers, represent potential diagnostic and prognostic biomarkers. Targeting these proteins with small-molecule inhibitors, such as METTL3 inhibitor STM2457, FTO inhibitors CS2/Dac51, and YTHDF2 antagonist DC-Y13-27, synergizes with existing therapies, including lenvatinib and anti-PD-1, to address drug resistance and improve treatment efficacy. Future research must focus on elucidating the mechanisms underlying understudied modifications (m3C, Nm, Ψ) and their interactions in immune-metabolic regulation. Innovative therapies that integrate RNA methylation-targeted agents with immunotherapy, such as CAR-NK and T cells, for the treatment of solid tumors; clinical application of multi-omics-informed biomarkers and inhibitors to enhance personalized therapy for HCC.
In summary, Targeting RNA methylation presents a strategic approach to hinder HCC progression, reshape tumor immunity, and enhance patient outcomes.