As a crucial reversible epigenetic modification, m⁵C methylation is extensively present in various RNA types, encompassing mRNA, rRNA, tRNA, long non-coding RNA, circular RNA, microRNA, and vtRNA. Methylation at diverse sites assumes a pivotal role in mediating the fate of RNA, including the stability, extranuclear transport, and transcription of RNA (13, 17). Intriguingly, m⁵C methylation sites on RNA can be detected and localized more precisely with the advent of cutting-edge high-throughput sequencing technologies, such as RNA bisulfite sequencing (RNA-BisSeq) (18, 19), 5-azacytidine-mediated RNA immunoprecipitation sequencing (AZA-IP-seq) (20), methylation individual nucleotide resolution crosslinking (miCLIP-seq) (21, 22), and immunoprecipitation sequencing (m⁵C-RIP-seq) (18, 23). For instance, m⁵C-RIP-seq can enrich m⁵C-containing RNA fragments for sequencing via antibodies specifically targeting m⁵C methylation sites¹⁵, (23) ( Figure 1A ). Although m⁵C-RIP-seq can effectively identify highly methylated RNA fragments, it has limited resolution of peaks and much lower rates and fails to obtain the precise location and absolute level of m⁵C in the transcriptome compared with other sequencing methods with single-nucleotide resolution since the RNA fragments enriched by antibodies are about 100−150 nt in length. In addition, m⁵C-RIP-seq cannot identify methylation on low-abundance mRNAs because the statistical analysis is relatively insensitive to low-coverage regions (24). miCLIP-seq can capture NOL1/NOP2/SUN domain 2 (NSUN2)-targeted RNA fragments through antibodies against NSUN2. Hussain et al. utilized miCLIP-seq technology to localize the m⁵C methylation site. Specifically, through ultraviolet light, overexpressed mutant NSUN2 (C271A) was cross-linked with RNA fragments to form RNA-protein complexes containing robust covalent bonds, and truncation or mutations were induced during reverse transcription polymerase chain reaction (RT-PCR) and then identified with sequencing data to map the m⁵C methylation site (21, 22) ( Figure 1B ). However, it has been also reported that because NSUN2 catalyzes m⁵C methylation in only a subset of RNAs, a comprehensive transcriptomic profile cannot be obtained with antibodies against a single RNA m⁵C methyltransferase. Furthermore, miCLIP-seq requires overexpression of NSUN2 with mutations in the m⁵C release domain, which creates an abnormal intracellular environment and limits the application of this technology to cells cultured in vitro ( 25). Furthermore, AZA-IP-seq can detect m⁵C sites in RNA by replacing cytosine with 5-azacytidine (20) ( Figure 1C ). Similar to miCLIP-seq, AZA-IP-seq also can only detect m⁵C sites in a subset of RNAs that are catalyzed by a single RNA m⁵C methyltransferase, with an incomplete transcriptomic map. Additionally, the detection results of AZA-IP-seq depend on the efficiency of 5-azaC introduction, which results in reduced sensitivity to m⁵C sites in low-abundance RNA (20). Although the use of AZA-IP-seq is limited to the detection of toxic and specific RNA subpopulations, this technology remains valuable for research on m⁵C methylation. RNA-BisSeq, additionally, is a widely applied detection method for RNA m⁵C methylation (18, 19) ( Figure 1D ), which converts cytosine to uracil through bisulfite treatment, followed by PCR detection of unconverted cytosine to determine the m⁵C site. Despite the advantages of single-nucleotide resolution and methylation level analysis, which avoids some of the drawbacks of other techniques, RNA-BisSeq fails to distinguish between unconverted cytosines derived from m⁵C and 5-hydroxymethylcytosine (hm⁵C) and may encounter recognition errors owing to the double-stranded structure. Conclusively, the evolution of these technologies tremendously advances the understanding of the pivotal role of RNA modifications in gene expression regulation, disease progression, and cellular functions.

First discovered in 1925, m⁵C methylation is currently known to be present on various RNA molecules in multiple organelles, including nuclei ( Figure 2A ), mitochondria ( Figure 2B ), and ribosomes ( Figure 2C ) (26–28). There are over 90,000 m⁵C sites detected in the human genome, and m⁵C methylation is preferentially deposited at the translation start site, near the 3’-untranslated region, and near the Argonaute binding region of mRNAs (29).

Mechanistically, RNA m⁵C methylation primarily involves three categories of effectors, namely methyltransferases, demethyltransferases, and m⁵C readers.

(1) Methyltransferases: m⁵C methylation is typically catalyzed by methyltransferases, such as NSUN family members (NSUN1-7) and DNA methyltransferase homolog 2/tRNA-aspartic acid methyltransferase 1 (DNMT2/TRDMT1). During m⁵C methylation, the cysteine residue in the methyltransferase forms a covalent intermediate with the cytosine in the target RNA, which enables the C atom at position C5 to become nucleophilic and to bind to the methyl group of S-adenosyl methionine, contributing to the transfer of the methyl group (27). (2) Demethyltransferases: m⁵C demethylation is primarily achieved by the ten-eleven translocation (TET) family (TET1-3) and ALKBH1. Specifically, TET enzymes, as α-ketoglutarate- and Fe²⁺-dependent dioxygenases, are responsible for catalyzing m⁵C demethylation to generate hm⁵C. Recent studies have unveiled that TET overexpression substantially enhances RNA hm⁵C levels, highlighting that members in the TET family may also function as RNA demethylases to remove m⁵C methylation. Unlike TETs, ALKBH1 initially converts m⁵C to the intermediate hm⁵C and further oxidizes hm⁵C to produce 5-formylcytosine, thus catalyzing m⁵C demethylation (30). (3) m⁵C readers: m⁵C-modified RNA within cells is predominately recognized and regulated by ALYREF and YBX1. ALYREF encourages the nuclear export of mRNAs, whilst YBX1 stabilizes mRNAs by binding to m⁵C-modified mRNAs via its cold shock domain. Overall, the m⁵C regulatory network entails sophisticated molecular mechanisms and is essential for the regulation of cell functions and gene expression (28, 31).

TLRs, as key PRRs within the innate immune system, are responsible for recognizing invading pathogens, double-stranded RNA, single-stranded RNA, and CpG-DNA. Upon recognition, TLRs stimulate immune responses by activating intracellular pathways and transcription factors such as IRF7 and NF-κB via adapter proteins including MyD88 (7). There are 10 types of TLRs in the human liver, which are distributed on the cell surface and within intracellular compartments (52). Given the constant exposure of the liver to gut microbes, TLRs assume crucial roles in repressing inappropriate pro-inflammatory responses. On the contrary, prolonged TLR activation may elicit chronic liver damage and elevate the risk of HCC (53). Notably, diverse biological functions vary across TLRs. For instance, TLR4 activation can obviously promote the development of HCC by up-regulating pro-inflammatory factors and malignant tumor-related molecules and mediating immune cell infiltration (54–59). In contrast, TLR3 activation can protect against HCC cell growth by inducing reactive oxygen species generation and activating apoptotic pathways (60–62). Additionally, TLR2 (63), TLR5 (64), TLR7 (65, 66), TLR8 (66), and TLR9 (67) are also implicated in the development of HCC, whose activities may affect various processes such as cell proliferation, migration, and apoptosis.

Through comparisons between m⁵C-modified RNA and unmodified RNA, Kariko et al. observed that m⁵C-modified RNA inhibited the expression of cytokines and activation markers in DCs and also suppressed the signaling activity activated by TLR3, TLR7, and TLR8, thereby affecting the immune activity of DC cells (16) ( Figure 3 ). Furthermore, because base modification in mRNAs changes their immune activity, researchers used m⁵C in mRNA therapy alone or in combination with other modifications, which successfully inhibited the immune effects of TLR-mediated mRNAs (16). This breakthrough signifies a major advancement in the realms of RNA and immunotherapy. The research by Andries et al. further highlighted m⁵C-modified mRNAs as a novel mRNA therapy, partly because of their ability to boost evasion from the innate immune response by inhibiting endosomal TLR3 (68). In summary, m⁵C methylation may exert complex and critical effects on TLRs and the pathogenesis of HCC, offering essential insights into the immunological mechanisms of HCC and the development of treatment strategies for HCC.

As a cytoplasmic PRR and a DNA sensor, cGAS activates the IFN pathway by recognizing cytoplasmic DNA, including DNA released from viruses, bacteria, mitochondria, tumor cells, or dead cells. The C-terminus of cGAS contains a conserved zinc ion-binding region, which favors DNA binding and dimerization of cGAS. DNA binding changes the structure of cGAS, leading to cGAS activation. STING, a small protein in the endoplasmic reticulum, is retained in the endoplasmic reticulum through interactions with the Ca²⁺ sensor STIM1. When binding to DNA, cGAS is activated by ATP and GTP, catalyzing the formation of cGAMP, which further activates STING. Activated STING is palmitoylated and phosphorylated to recruit TBK1 and IKK, among which TBK1 triggers the release of IFN (a pivotal pathway in cancers, particularly HCC) by activating IRF3, while IKK induces inflammation by activating NF-κB (69, 70).

The involvement of the cGAS-STING pathway in HCC has been increasingly reported. For instance, a prior study revealed that olaparib activated STING chemokine signaling to enhance radiation-induced systemic anti-tumor effects (71). Another study showed that the cGAS-STING pathway was involved in the promoting effects of oncolytic influenza viruses combined with PD-L1 antibodies on CD8+ T cell activation (72). Su et al. found that TAK1 deficiency modulated ferroptosis and macrophage cGAS-STING signaling to foster liver injury and tumorigenesis (73). A study by Li et al. demonstrated that hyperbaric oxygen boosted teniposide-induced cGAS-STING activation, therefore increasing the anti-tumor efficacy of PD-1 antibodies in HCC (74). Gut microbiota was reported to modulate radiotherapy-induced anti-tumor immune responses in HCC via STING signaling (75). Sorafenib combined with STAT3 knockdown was revealed to accelerate HCC cell apoptosis and encourage cGAS-STING-mediated anti-tumor immunity (76). The disruption of the BRCA1-PALB2 interaction was demonstrated to induce tumor immunosuppression and T lymphocyte infiltration in HCC through the cGAS-STING pathway (77). According to the results of Du et al., radiotherapy induced PD-L1 up-regulation via cGAS-STING activation to facilitate immune evasion in HCC (78). Zhao et al. observed that hypoxia-induced RNASEH2A constrained cGAS-STING signaling activation in HCC and might portend the poor prognosis of patients (79). Of note, another study displayed that STING deficiency accelerated tumor growth and reduced autophagy and apoptosis in mice with HCC, which could be nullified by STING agonists.

Furthermore, the research of Chen et al. demonstrated that NSUN5-mediated m⁵C methylation of GPX4 contributed to cGAS-STING signaling activation, which fostered anti-cancer immune responses in colon adenocarcinoma (80). Another study revealed that NSUN2 activation maintained global m⁵C methylation of RNA, including TREX2, in tumor cells and stabilized TREX2 expression, consequently impeding the accumulation of cytoplasmic double-stranded DNA and blocking the activation of the cGAS-STING pathway, which facilitated tumorigenesis and PD-L1 immunotherapy resistance (14). Additionally, NSUN2-mediated m⁵C methylation of IRF3 mRNA may negatively affect type I IFN responses in various viral infections (81). These findings underscore the important effect of m⁵C methylation on the cGAS-STING pathway in HCC and highlight its potential as a hot spot for future research on HCC ( Figure 4 ).

RLRs, a class of intracellular PRRs, are primarily tasked with the surveillance of RNA viruses, DNA viruses, and pathogenic RNA stemming from certain bacterial infections. Upon detection, RLRs lead to the activation of IRF3, a transcription factor, transcriptionally up-regulating cytokines such as IFNs and then bolstering immune responses. Mounting evidence supports the indispensable role of intact IFN signaling in the efficacy of numerous traditional chemotherapeutic drugs and targeted anti-cancer drugs. Accordingly, activation of RIG-I or MDA5 signaling has emerged as a promising therapeutic strategy for cancers (82, 83).

A prior study exhibited that hepatocyte-specific RIG-I deficiency facilitated diethylnitrosamine-induced hepatocarcinogenesis but mitigated non-alcoholic fatty liver disease-induced hepatocarcinogenesis. This study also demonstrated that interleukin-6 (IL-6) diminished RIG-I expression in HCC progenitor cells (HcPCs), driving IL-6 effector signaling and the development of HCC from HcPCs (84). NFATc3 was found to impede HCC and hepatitis B virus replication by positively regulating RIG-I-mediated IFN transcription, illustrating that NFATc3 may exert an effect on the RIG-I pathway, which is vital for maintaining liver health and repressing viral replication (85). In stem cells, RIG-I deficiency led to TGF-β/AKT/Smad2 signaling activation and precipitated the formation of tolerogenic DCs, underscoring the significance of RIG-I in immune regulation (86). Additionally, RIG-I has been identified not only as an anti-oncogene but also as a biomarker for the efficacy of IFN-α treatment for HCC, emphasizing its critical role in modulating the growth and treatment responses of tumors (6, 87).

Moreover, a prior study displayed that deletion of the m⁵C methyltransferase NSUN2 boosted type I IFN responses and substantially depressed the replication of various RNA and DNA viruses and that NSUN2 loss diminished the RNA m⁵C methyl group in the host and elevated the polymerase III transcription of non-coding RNAs recognized by RIG-I, particularly RPPH1 and 7SL RNA, consequently enhancing type I IFN signaling. In addition, this study also showed that this m⁵C-mediated antiviral innate immunity was also conserved in mouse models (15). Overall, this study elucidates the role of m⁵C in regulating innate immunity and illustrates m⁵C as a promising target for the development of broad-spectrum antiviral and anti-tumor therapeutics ( Figure 5 ).