4.2.1 DNA Methylation

DNA methylation refers to the addition of a methyl group to cytosine residues, especially in CpG islands, which inhibits gene expression [76]. It plays a meaningful role in silencing tumor suppressor genes and in the early stages of progression from viral hepatitis to HCC.

HBV infection induces abnormal DNA methylation, primarily through the action of the HBx protein. The HBx protein upregulates DNA methyltransferase 1 (DNMT1), causing hypermethylation and inactivation of several tumor suppressor genes [77]. Studies have shown that Ras association domain family 1 isoform A (RASSF1A), a tumor suppressor involved in cell cycle regulation, is significantly hypermethylated in the livers of over 50% of HBV-infected individuals, a pattern seen early in HCC [78]. The p16 (INK4A), a cell cycle inhibitor, also undergoes hypermethylation in HBV-infected hepatocytes, disrupting cell cycle regulation and promoting tumor development [79]. E-cadherin (CDH1) methylation is another critical event in HBV-related HCC. E-cadherin is vital for epithelial–mesenchymal transition (EMT), and its silencing via hypermethylation increases tumor cell invasiveness [80]. G Protein Subunit Alpha 14 (GNA14) expression is significantly downregulated in HBV-related HCC, with DNA methylation being the primary cause. HBx protein regulates GNA14 methylation, affecting the notch homologue protein 1 (Notch1) signaling pathway, a key regulator of tumor proliferation and metastasis [81].

HCV does not integrate into the host genome like HBV, but it promotes liver cancer through epigenetic mechanisms. HCV infection upregulates DNMT1, inducing hypermethylation and silencing of several tumor suppressor genes, including suppressor of cytokine signaling-1 (SOCS-1), RASSF1A, and GSH S-transferase P1 (GSTP1) [82, 83]. SOCS-1, a negative regulator of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, has strong antitumor activity in HCC progression [84]. In HCV-infected HCC patients, SOCS-1 methylation is significantly elevated, indicating that HCV suppresses SOCS-1 expression through methylation, promoting liver malignancy [85]. Furthermore, HCV core protein overexpression inhibits STAT1 activation via methylation, interfering with IFN-α signaling and hindering the antiviral response, facilitating viral replication [86]. The transcription factor cyclic AMP-responsive element-binding protein 3-like protein 1 (CREB3L1) is silenced by methylation in HCV-infected cells, impacting antiviral gene expression and potentially contributing to treatment resistance [86].

DNA methylation not only plays a crucial role in viral hepatitis infections but also dominates the early stages of HCC. Tumor suppressor gene hypermethylation is a hallmark of early HCC development. In early HCC, DNA methylation inhibits the binding of p53 to the tumor suppressor Zinc-finger protein 334 (ZNF334), thereby reducing p53 expression, further emphasizing the importance of DNA methylation in the pathogenesis of HCC [87]. Secreted frizzled-related protein 2 (SFRP2), another potential tumor suppressor, frequently shows promoter hypermethylation in HBV-related HCC. SFRP2 methylation levels exceed normal and have higher diagnostic value than alpha-fetoprotein (AFP), indicating its potential for early HCC diagnosis [88].

In contrast to localized hypermethylation, HBV and HCV infections also trigger global hypomethylation. Genome-wide hypomethylation causes chromosomal instability, increasing gene mutation and rearrangement rates, thus promoting HCC malignancy [89]. HBx protein downregulates DNMT3B, inducing genome-wide hypomethylation, leading to genomic instability and driving tumor progression [78]. The combined effects of localized tumor suppressor hypermethylation and genome-wide hypomethylation caused by viral hepatitis drive the development and progression of HCC.

4.2.2 RNA Methylation

RNA methylation refers to the process of adding methyl groups to RNA molecules, where methyl groups are transferred from methyl donors to RNA bases under the catalysis of RNA methyltransferases [90]. Hepatitis viruses promote HCC progression by regulating RNA methylation, especially N⁶-methyladenosine (m6A). Recent studies have highlighted that m6A modifications are crucial for viral replication, immune evasion, and oncogenesis.

In HBV-related HCC, m6A modifications drive tumor initiation and progression by regulating prominent genes. Methyltransferase-like 3 (METTL3), a core component of the m6A methyltransferase complex, is pivotal in m6A modification after HBV infection [91]. HBV infection induces m6A modification in the 3′-UTR of phosphatase and tensin homolog (PTEN) mRNA by upregulating METTL3, reducing its stability, and activating the PI3K/AKT pathway, thus promoting HCC development [92]. HBV also influences m6A modification via its HBx protein. HBx, an important regulator of viral transcription, promotes m6A modification of viral RNA and induces METTL3 nuclear localization, facilitating m6A addition to viral and host RNA. HBx interacts with the METTL3/14 complex to promote m6A modification of host genes, accelerating tumor progression [93]. YTH domain family 2 (YTHDF2), an “m6A reader,” is another important regulatory protein in HBV-related HCC. Studies have shown that YTHDF2 undergoes O-GlcNAcylation after HBV infection, increasing its stability and oncogenic activity, which promotes the cell cycle by stabilizing m6A-modified transcripts like minichromosome maintenance 2 (MCM2) and MCM5, further driving HBV-related HCC [94]. The m6A demethylase AlkB homolog 5 (ALKBH5) also contributes to HBV-related HCC. ALKBH5 overexpression removes m6A from HBx mRNA, increasing its stability and promoting tumor cell proliferation and migration. Knocking down ALKBH5 inhibits HBV-related HCC progression, indicating it as a potential therapeutic target [95].

Like HBV, HCV also utilizes m6A modification to promote HCC development. M6A modifications are prevalent in the RNA genome of HCV and regulate its life cycle and impact on host cells [96]. Studies have shown that m6A-modified HCV RNA is recognized by the YTHDF protein family, which regulates HCV translation and RNA stability [97]. YTHDC2 recognizes m6A modifications on HCV RNA to promote IRES-dependent translation initiation, while YTHDF2 binds to the 3′-UTR of HCV RNA, inhibiting retinoic acid-inducible gene I (RIG-I)-mediated immune recognition and facilitating immune evasion [98]. HCV also drives liver cancer by modulating m6A modifications in host cells. HCV infection increases m6A levels in genes like RIO kinase 3 (RIOK3) and cold-inducible RNA-binding protein (CIRBP), enhancing their translation, promoting immune evasion, and boosting tumor cell proliferation [99].

In summary, m6A modifications are vital in HBV- and HCV-related HCC transformation through various mechanisms. HBV and HCV exploit m6A regulation of viral RNA and host genes to evade immune surveillance while promoting tumor proliferation and metastasis.

4.2.3 Histone Modifications

Histone modification refers to the process in which histones undergo methylation, acetylation, ubiquitination, and phosphorylation, catalyzed by specific enzymes, to regulate cellular processes [100]. Hepatitis viruses manipulate chromatin structure by altering histone modifications, influencing host–cell transcription and potentially leading to tumorigenesis.

4.2.4 Noncoding RNA

NcRNAs are RNA molecules transcribed from the genome that do not encode proteins, playing critical roles in regulating gene expression and various cellular processes [137]. The abnormal expression of ncRNAs is closely associated with the metastasis, invasion, spread, and recurrence of virus-associated HCC, such as that caused by HBV and HCV infections (Tables 1 and 2).

4.5.1 Hepatocytes

Following hepatitis virus infection, PAMPs, such as viral nucleic acids and proteins, are detected by pattern recognition receptors (PRRs) on the surface of liver cells, including TLRs and RIG-I [307]. Activation of receptors such as TLR4, TLR3, and RIG-I triggers proinflammatory signaling pathways like NF-κB and STAT3, increasing the secretion of inflammatory cytokines, including IL-6, TNF-α, and IFN-γ, which sustain chronic inflammation by activating immune cells and draw macrophages, Natural killer (NK) cells, and T cells to liver [308, 309]. Persistent inflammation damages hepatocytes and worsens the inflammatory response, thus leading to a “cytokine storm” and continuous liver tissue damage and fibrosis.

Remarkably, in early HBV-infected hepatocytes, TLR2 expression is significantly downregulated, suppressing inflammatory signaling pathways and reducing cytokine production [310]. Additionally, HBsAg and HBx interfere with the activation of critical proinflammatory transcription factors, including IFN regulatory factor 3 (IRF-3) and NF-κB, allowing the virus to evade immune detection [311]. HBV further diminishes antiviral activity by suppressing IFN-β and IFN-stimulated genes (ISGs), safeguarding its replication and promoting persistent infection and tumorigenesis [312].

During early HCV infection, activation of TLR3 and RIG-I induces hepatocytes to secrete large amounts of chemokine C-X-C ligand 10 (CXCL10), a process that can occur via nonparenchymal cell (NPC) immune responses independent of type I or type III IFNs, which suggests that HCV regulates hepatocyte–NPC interactions through multiple mechanisms, causing sustained inflammation [313]. Additionally, HCV employs unique mechanisms to evade host immune responses. HCV-infected hepatocytes modify the expression of immune-suppressive molecules, including programmed death-ligand 1 (PD-L1) and TGF-β, inhibiting T-cell activity. While T cells are essential for controlling HCV infection, particularly those of CD4⁺ and CD8⁺ T cells, are significantly impaired in chronic cases, allowing HCV to sustain chronic infection by suppressing T-cell responses [314]. Moreover, HCV-infected hepatocytes secrete exosomes and TGF-β, inducing the expansion of regulatory T cells (Tregs) and further suppressing antiviral responses. Exosomes also carry specific miRNAs that regulate the tumor microenvironment and promote HCC development [315, 316]. In summary, HBV and HCV manipulate hepatocytes through various mechanisms, inducing chronic inflammation and immune evasion, which ultimately promotes HCC development.

4.5.2 Hepatic Stellate Cells

Chronic viral hepatitis induces persistent inflammation and drives HCC progression by activating HSCs, which results in fibrosis. In chronic hepatitis, HSCs transition from a quiescent to an activated state, and produce large amounts of extracellular matrix (ECM) that lead to fibrosis and cirrhosis, creating a microenvironment conducive to HCC development [317].

Activated HSCs are central to fibrosis development. Chronic inflammation from HBV and HCV infections continuously stimulates HSCs, leading to excessive collagen buildup and worsening fibrosis. Studies have shown that activated HSCs secrete cytokines such as TGF-β through autocrine and paracrine effects, which further drive HSCs activation and fibrosis, creating a vicious cycle [318]. Additionally, HSCs secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), promoting tumor angiogenesis and the growth of precancerous and cancerous hepatocytes, thereby creating a tumor-promoting microenvironment conducive to HCC development [319]. HSCs also play a critical role in immune evasion.

HSCs, by secreting immunosuppressive cytokines like TGF-β and IL-10, suppress the activity of immune cells, including NK cells and cytotoxic T lymphocytes (CTLs). In this case, infected hepatocytes can evade immune surveillance and maintain persistent infection [320]. Moreover, research has revealed that HBV and HCV infections upregulate specific genes in HSCs, such as nicotinamide N-methyltransferase (NNMT) and CD44, which enhance cancer cell migration and immune evasion, accelerating HCC progression [321]. HSC-secreted TGF-β induces EMT in cancer cells, enhancing their invasiveness and metastatic potential, thereby driving both tumor progression and fibrosis severity [322]. In summary, HBV and HCV promote chronic inflammation, fibrosis, and HCC progression by activation of HSCs.

4.5.3 Hepatoma Cells

Chronic viral infections induce gradual malignant transformation of hepatocytes, eventually forming tumors. Hepatoma cells, in turn, use various mechanisms to evade immune surveillance, enabling their survival and spread in the immune environment.

Normally, cells present antigens to CTLs via major histocompatibility complex class I (MHC I) molecules, triggering an immune response [323, 324]. However, during HBV and HCV infections, the expression of MHC I in HCC cells is downregulated. Hence, the likelihood of antigen recognition by T cells is reduced, allowing HCC cells to evade CTL-mediated attacks and promoting their proliferation and spread [325]. Another important immune evasion strategy involves the overexpression of immune checkpoint molecules like PD-L1 by hepatoma cells, which suppresses T-cell activity. HBV-DNA polymerase interacts with poly (ADP-ribose) polymerase 1 (PARP1), preventing its nuclear translocation and upregulating PD-L1 expression [326]. PD-L1 binds to cell death protein 1 (PD-1) receptors on T cells, inhibiting their function and preventing effective tumor cell attacks. Meanwhile, activation of the PD-L1/PD-1 axis reduces T-cell killing capacity while leading to T-cell exhaustion, further promoting the survival and spread of tumor [326, 327]. Hepatoma cells also secrete immunosuppressive cytokines such as IL-10 and TGF-β, which further dampen the host's antitumor immune response. These cytokines directly suppress effector T cells and promote the activation of Tregs. Increased Treg activity further inhibits CTLs and NK cells, while the accumulation of these immunosuppressive factors in the tumor microenvironment weakens immune defenses, facilitating tumor growth and metastasis [328]. In summary, HBV and HCV can effectively drive immune evasion in liver cancer through multiple pathways, facilitating the malignant transformation of HCC.

4.5.4 CD8⁺ T Cells

CD8⁺ T cells are essential for host antiviral immunity and tumor suppression. They curb viral spread by killing infected hepatocytes directly, secreting proinflammatory cytokines like IFN-γ, IL-2, and TNF-α, and releasing cytotoxic molecules such as granzyme and perforin [329]. Besides, they also contribute to malignant transformation by inducing chronic inflammation in cases of HBV and HCV infections, which promotes aggravation of liver disease. Over time, they gradually become exhausted, hindering the effective clearance of virus-infected cells, which is closely related to the occurrence of HCC [330].

In chronic HBV infection, CD8⁺ T cells persist in circulation and the liver, yet their function is significantly impaired. Studies have shown that HBV-specific CD8⁺ T cells express high levels of inhibitory receptors like CTL-associated antigen 4 (CTLA-4), PD-1, T cell immunoreceptor with Ig and ITIM domains (TIGIT), lymphocyte activation gene 3 (LAG-3) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) [331]. The continued expression of these inhibitory receptors suppresses CD8⁺ T cell functions, limiting their production of antiviral cytokines and ability to eliminate virus-infected hepatocytes, which hinders viral clearance and perpetuates chronic inflammation, raising the risk of HCC [330]. Furthermore, although HBV-specific CD8⁺ T cells exhibit functional exhaustion, they still retain some inflammatory activity. They secrete low levels of IFN-γ and recruit other immune cells, such as macrophages, into the liver, exacerbating chronic inflammation [332]. Macrophages, in turn, secrete proinflammatory cytokines like TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1), which accelerate liver damage and fibrosis, ultimately contributing to HCC progression [333].

In chronic HCV infection, CD8⁺ T cell exhaustion follows a similar mechanism. Despite sufficient numbers of HCV-specific CD8⁺ T cells, their function is severely impaired, limiting effective viral clearance [334]. As in HBV infection, chronic inflammation from HCV infection related to CD8⁺ T cells promotes tumorigenesis and progression via enhanced oxidative stress and proinflammatory pathways [335].

In addition to virus-specific CD8⁺ T cells, nonspecific CD8⁺ T cells also play a crucial role in chronic hepatitis and HCC progression. Activated during chronic inflammation, these cells secrete proinflammatory cytokines such as IFN-γ, driving the inflammatory response, though they cannot directly eliminate viruses [336]. In summary, despite functional suppression from high inhibitory receptor expression, CD8⁺ T cells still recruit inflammatory cells and secrete proinflammatory factors, causing repeated cycles of hepatocyte injury, regeneration, and mutation accumulation. The long-term cycle of inflammation and regeneration fosters liver cancer development.

4.5.5 CD4⁺ T Cells

CD4⁺ T cells hold much significance in the immune system, particularly in antiviral and antitumor responses. In chronic HBV and HCV infections, CD4⁺ T cells produce cytokines that regulate immune responses by differentiating into various effector subgroups, which promote HCC development through chronic inflammation [337].

Th1 cells, a subset of CD4⁺ T cells, mediate immune responses by secreting proinflammatory cytokines like IFN-γ, TNF-α, and IL-2, activating CD8⁺ T cells [338]. In HBV-related HCC, Th1 cell frequency is significantly reduced, especially in tumor tissues, which weakens antiviral and antitumor immunity, facilitating HCC progression [339]. In contrast, Th17 cells secrete cytokines such as IL-17, IL-21, and IL-22, and sustain inflammation. In chronic HBV infection, an imbalance in the Th1/Th17 ratio, particularly an increase in Th17 cells, correlates with liver damage, fibrosis, and late-stage liver disease [340]. Increased tumor-infiltrating Th17 cells are poor prognostic indicators in HBV-related HCC, where a high Th17/Th1 ratio predicts poor survival, especially in patients undergoing liver resection [341, 342].

T follicular helper cells (Tfh cells), another CD4⁺ T cell subset, support B cell differentiation and antibody production, playing a key role in antiviral immunity [343]. In HBV-related HCC, circulating Tfh cell frequency is significantly reduced, and their function deteriorates as the disease progresses. Tumor tissue infiltration of Tfh cells is also markedly decreased, which weakens B cell-mediated adaptive immunity and promotes chronic liver inflammation and HCC development [344].

CD4⁺ CTLs, which can directly kill virus-infected cells by secreting granzyme and perforin, are also impaired in HBV-related HCC. They gradually disappear or lose function as HBV-related HCC develops, correlating with poor outcomes [345]. CD4⁺ CTL dysfunction is linked to increased Tregs, further weakening tumor immune surveillance [346]. In HCV infection, CD4⁺ T cells are similarly impaired. Chronic HCV patients exhibit reduced CD4⁺ T cell numbers and diminished function, with lower levels of IL-2 and IFN-γ secretion, indicating a decline in activation and antiviral efficacy [347].

Tregs maintain immune tolerance by suppressing effector T cell functions, but in chronic HBV and HCV infections, Treg frequency is significantly increased, leading to immune suppression and persistent viral infection [348]. Tregs inhibit CD8⁺ T cell proliferation and cytotoxicity by secreting immunosuppressive cytokines like IL-10 and TGF-β, promoting immune evasion in HCC [348]. In the HCC tumor microenvironment, Tregs suppress both antiviral and antitumor immunity by exhibiting higher levels of PD-1 expression, enhancing their suppressive effects and allowing HCC to progress under immune surveillance, closely associated with poor prognosis and increased tumor aggressiveness [349].

In conclusion, hepatitis viruses promote chronic inflammation and HCC development by impairing CD4⁺ T cell function. In HBV and HCV infections, CD4⁺ T cell function becomes exhausted, with reduced Th1 cells, elevated Th17 and Treg cells, and a cycle of chronic inflammation and immune suppression, which hinders viral clearance and creates an environment conducive to HCC progression.

4.5.6 B Cells

B cells, a key component of adaptive immunity, produce antibodies and regulate immune responses to help eliminate viral infections [350]. However, during chronic HBV and HCV infections, B cell dysfunction contributes to viral persistence and promotes HCC development by fostering a chronic inflammatory environment [351].

In the early stages of HBV infection, B cells exert antiviral effects by producing anti-HBs and anti-HBc antibodies that neutralize the virus and prevent its spread [352]. However, in chronic HBV infection, B cells become functionally impaired and fail to produce sufficient antibodies, particularly HBsAg-specific antibodies. B cells often exhibit an atypical memory phenotype (CD21⁻CD27⁻) and increased PD-1 expression in patients suffering from chronic HBV, leading to reduced functionality and antibody production [352]. The B cell dysfunction enables HBV persistence, activates the host immune system, and triggers chronic inflammation. Furthermore, HCV impairs B cell-mediated humoral responses, limiting the production of neutralizing antibodies against the virus, which creates a chronic inflammatory environment [353].

The inhibition of B cell function not only hampers HBV and HCV clearance but also promotes HCC development through persistent chronic inflammation. One mechanism by which hepatitis viruses induce B cell dysfunction is through increased regulatory B (Breg) cells, which secrete IL-10 and TGF-β, suppressing effector T cells and other immune responses [354, 355]. Breg cells also inhibit CD4⁺ T cell production of granzyme and perforin through TIM-1 expression, weakening T cell cytotoxicity, which promotes immune evasion and contributes to HCC progression [356]. Furthermore, in postoperative HCC patients, Breg cell frequency is significantly increased and correlates with higher HBeAg levels and HBV-DNA copies. It suggests that Breg cells are crucial for tumor progression, as they facilitate the evasion of immune attacks by tumor cells and enhance HCC expansion [352].

Briefly, while B cells are essential for antiviral immunity, their dysfunction in chronic HBV and HCV infections hinders viral clearance and leads to chronic inflammation. The increased presence of Breg cells further intensifies an immunosuppressive environment that facilitates HCC progression through mechanisms of immune evasion.

4.5.7 NK Cells

NK cells are essential components of the innate immune system, capable of directly killing virus-infected cells and playing a key role in early infection control [357]. However, during chronic HBV and HCV infections, NK cell function is modulated by the virus, leading to immune dysfunction that promotes chronic inflammation and the progression to HCC.

In the early stages of HBV infection, NK cells inhibit viral replication and spread by secreting IFN-γ and TNF-α, as well as through cytotoxic activity [358]. However, prolonged NK cell activation exacerbates hepatocyte damage and contributes to chronic liver inflammation [359]. Studies regarding HBV transgenic mouse models have shown that NK cells promote EMT pathways through IFN-γ secretion, leading to hepatocyte injury and facilitating HCC development [360]. As HBV infection persists, the virus regulates NK cell function through various mechanisms, suppressing their antiviral capabilities. For instance, HBsAg inhibits JAK–STAT3 signaling pathway in NK cells, impeding viral clearance and promoting the progression from chronic hepatitis to HCC [361]. Additionally, chronic HBV infection upregulates inhibitory receptors on NK cells, such as PD-1, TIM-3, and NK group 2 member A (NKG2A), reducing their ability to secrete IFN-γ and TNF-α, which not only hinders the clearance of infected hepatocytes but also perpetuates chronic inflammation [362, 363]. NK cells also contribute to fibrosis by secreting proinflammatory cytokines, such as IL-4 and IL-13, which activate HSCs [364].

During HCV infection, NK cells play antiviral and immune regulatory roles in the early stages. In acute HCV infection, NK cells exhibit high cytotoxicity and IFN-γ secretion, effectively suppressing viral replication [365]. However, as HCV infection becomes chronic, the virus diminishes NK cell antiviral activity by downregulating activating receptors, such as NKp46 and NKp30 [366]. Similar to HBV infection, HCV upregulates inhibitory receptors, such as NKG2A, reducing NK cell cytolytic capacity and IFN-γ secretion, facilitating viral persistence and chronic liver inflammation [367, 368]. Additionally, the HCV E2 protein binds to CD81 on NK cells, further suppressing their function, which prevents viral clearance and creates conditions that support HCC development [369, 370].

Notably, miR-146a expression is significantly elevated in NK cells from patients with HBV- and HCV-related HCC, downregulating NK cell activity and reducing IFN-γ and TNF-α production, which renders them unable to effectively eliminate cancerous hepatocytes and promotes tumor growth and metastasis [371]. Furthermore, HBV and HCV secrete exosomes that directly affect NK cell function and survival, contributing to viral transmission and the creation of the tumor microenvironment, thereby driving HCC progression [372, 373].

In summary, while NK cells are crucial for antiviral immunity, their function is suppressed by viral mechanisms in chronic HBV and HCV infections. Downregulation of activated receptors and upregulation of inhibitory receptors on NK cells not only hampers viral clearance but also promotes HCC development by inducing chronic inflammation and creating an immunosuppressive environment.

4.5.8 Macrophages

Macrophages, particularly Kupffer cells, the liver-resident macrophages, play a critical role in hepatitis virus infections and in chronic inflammation-induced malignant transformation, leading to HCC [374]. As primary liver immune cells, Kupffer cells are persistently activated by viral infections, releasing proinflammatory cytokines and contributing to both chronic hepatitis and fibrosis [375].

In chronic HBV and HCV infections, liver macrophages present two primary functional states: proinflammatory M1 and immunosuppressive M2 types. M1 macrophages are activated during the acute infection phase, releasing cytokines like TNF-α, IL-6, and IL-1β to control infection [334]. In chronic infection, macrophages gradually shift toward the immunosuppressive M2 phenotype, with M2 macrophages, especially tumor-associated macrophages (TAMs), promoting immune evasion in the tumor microenvironment by secreting inhibitory cytokines such as IL-10 and TGF-β, thereby fostering immune escape and tumor growth [376]. In HBV and HCV infections, persistent viral stimulation induces M2 macrophage polarization, suppressing T cell function to support tumor growth, and releasing ECM remodeling factors, such as MMP-9, thereby enhancing cancer cell invasion and metastasis [377].

In HBV infection, viral factors such as HBx protein and HBV DNA polymerase regulate macrophage activity through multiple mechanisms. HBx protein induces high IL-8 levels in macrophages via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway, activating TGF-β in liver sinusoidal endothelial cells and promoting immunosuppressive Treg cell accumulation via the IL-8/C-X-C motif chemokine receptor 1 (CXCR1) axis [378]. The process strengthens immunosuppressive effects in the tumor microenvironment, enhancing tumor angiogenesis and invasiveness [379]. Additionally, HBV DNA polymerase interacts with PARP1 in macrophages, blocking its nuclear translocation and increasing PD-L1 expression. Elevated PD-L1 weakens T cell-mediated tumor cell killing in macrophages, enabling tumor cells to evade immune surveillance through checkpoint pathways [380]. Furthermore, Kupffer cells engage with HBcAg via TLR2, leading to CD8⁺ T cell exhaustion and reduced antiviral immunity. This interaction creates an environment that not only fails to eliminate the virus but also encourages chronic inflammation and cancer development [381].

In HCV infection, chronic inflammation is primarily driven by NF-κB and STAT3 pathway activation in macrophages [382]. HCV nonstructural protein NS5A activates the NF-κB pathway via TNF receptor (TNFR)-associated factor 2 (TRAF2), inducing the release of IL-6 and TNF-α, which damages hepatocytes and promotes uncontrollable chronic inflammation [383]. Additionally, macrophages detect viral RNA via TLR2, triggering the NLRP3 inflammasome and inducing IL-1β and IL-18 release, which amplify local inflammation and activate HSCs, thereby promoting fibrosis [384, 385]. Furthermore, HCV modulates host miRNA expression to enhance immunosuppressive function of macrophages. HCV infection upregulates miR-155 expression, promoting M2 macrophage polarization and accelerating tumor cell proliferation and migration by inhibiting src homology 2-containing inositol-5′-phosphatase 1 (SHIP1) expression. The miR-155/SHIP1 axis establishes a potent protumor pathway in HCV-infected livers, maintaining immunosuppressive conditions in the tumor microenvironment [386].

Significantly, within the tumor microenvironment of virus-associated HCC, interactions between macrophages and CSCs further drive tumor malignancy. Single-cell sequencing analyses reveal that stem-associated HCC cell subclones such as CD24⁺, CD47⁺, and intercellular adhesion molecule 1 (ICAM1)⁺ subsets, are more likely to establish ligand–receptor-based communication with macrophages [387]. The interaction drives further M2 TAM polarization and release of tumor-promoting factors like S100 calcium-binding protein A11 (S100A11), enhancing the stemness and invasiveness of CSCs, which promotes tumor stemness and drug resistance [387].

In conclusion, HBV and HCV modulate macrophages to achieve immune evasion, induce sustained chronic inflammation, and create an immunosuppressive tumor microenvironment, ultimately promoting the malignant transformation of HCC. Through various molecular pathways, such as MEK–ERK, NF-κB, and STAT3, macrophages maintain inflammatory responses and immune evasion effects, accelerating HCC progression through interactions with tumor cells.

4.5.9 Dendritic Cells

Dendritic cells (DCs) are crucial antigen-presenting cells that initiate host immune responses [388]. However, HBV and HCV modulate DC function to induce chronic inflammation and hamper immune system's ability to clear infections, promoting the malignant transformation into HCC.

In chronic HBV infection, DC function is significantly impaired. Studies have indicated that plasmacytoid DCs in CHB patients, affected by IL-6 receptor signaling blockage, exhibit marked functional decline, further leading to DC dysfunction [389]. HBV also disrupts interaction between NK cells and DCs, weakening the overall antiviral response [390]. HBV impairs DC maturation without affecting their expression of human leukocyte antigen class I (HLA-I), enabling DCs to evade NK cell lysis and remain immature, while the increased expression of IL-10 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in these immature DCs reduces NK cell proliferation [390]. Normally, mature DCs secrete IL-12, a key cytokine for Th1 polarization, which activates CD8⁺ T cells and strengthens antiviral and antitumor immunity. However, this function is severely suppressed in HBV infection, leading to failure in antiviral responses [391].

Similarly, HCV infection significantly affects DC function. Research has found that monocyte-derived DCs in chronic HCV patients fail to mature properly, suppressing the ability to stimulate allogeneic T cells and diminishing IFN-γ production [392]. Mechanistically, the HCV core protein interacts with the globular C1q (gC1q) receptor on DCs, inhibiting IL-12 production and suppressing DC maturation, which skews CD4⁺ T cell differentiation toward a Th2 phenotype, weakening Th1-mediated antiviral responses. This Th2-biased response not only reduces antiviral efficacy but also promotes chronic inflammation, driving HCC development [393]. Additionally, HCV binds to CD81 receptor on DCs, further suppressing cytokine production, particularly IFN-γ, impairing NK cell and CD8⁺ T cell function. This facilitates viral persistence and exacerbates chronic liver inflammation, contributing to fibrosis and HCC progression [394, 395].

In brief, hepatitis viruses manipulate DC function to maintain chronic inflammation, accelerating malignant transformation into HCC. Impaired DC function leads to weakened antiviral immunity, viral persistence, and chronic inflammation, being important drivers of liver cancer development.

4.5.10 Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) play a significant immunoregulatory role in chronic hepatitis virus infections, particularly in HBV infection [396]. They are immunosuppressive cells accumulated in tissues such as liver, where they suppress the function of effector T cells, especially the antiviral activity of CD8⁺ T cells, which contribute to maintenance of chronic inflammation and promotes tumor progression [397]. During chronic HBV infection, MDSCs induce immune tolerance and facilitate immune evasion through various mechanisms. Studies have revealed that MDSCs accumulating in the liver during HBV infection inhibit the proliferation of HBsAg-specific T cells, weakening the host antiviral immune response [398]. Additionally, MDSCs secrete high levels of immunosuppressive cytokines, such as TGF-β and IL-10, which further impair CD8⁺ T cell function, leading to their gradual exhaustion and reduced ability to effectively clear the virus [399]. In summary, MDSCs modulate the immune microenvironment by suppressing antiviral immunity and sustaining chronic inflammation, thereby acting as key drivers of hepatitis virus-induced malignant transformation into HCC.

4.6.1 Nuclear Factor-κB

NF-κB is a transcription factor complex composed of multiple subunits, including p105/p50, p100/p52, RelA (p65), cRel, and RelB, which plays a critical role in cellular processes such as growth, differentiation, proliferation, apoptosis, angiogenesis, and immune responses [400]. In hepatitis virus infections, aberrant activation of the NF-κB pathway is closely linked to inflammation, fibrosis, and tumor progression.

HBV activates the NF-κB pathway through various mechanisms, with its encoded protein HBx playing a central role. HBx induces oxidative stress and promotes proinflammatory cytokine expression, thereby activating NF-κB pathway and driving the malignant transformation of hepatocytes [401, 402]. Studies have shown that HBx decreases cytoplasmic levels of p105 and p50 while promoting the phosphorylation and degradation of NF-κB inhibitor alpha (IKBα), which releases RelA and prolongs NF-κB activation [403]. Additionally, HBx interacts with TANK-binding kinase 1 (TBK1), further enhancing NF-κB activity, which is strongly associated with HCC invasiveness and metastasis [404]. HBx also indirectly activates NF-κB by modulating multiple signaling pathways, including PIK3C, Ras/rapidly accelerated fibrosarcoma (Raf) /MAPK, proto-oncogene tyrosine-protein kinase Src (Src), and JAK–STAT, all of which contribute to IKBα degradation and persistent NF-κB activation, triggering procarcinogenic signals [405]. NF-κB activation driven by HBx promotes hepatocyte proliferation, dysregulated apoptosis, and accelerates HCC progression by increasing the expression of proinflammatory cytokines such as IL-6, IL-8, and CXCL2, which are crucial in the inflammatory response, and fostering liver fibrosis, thereby creating a microenvironment conducive to HCC transformation [406, 407].

HCV also promotes HCC development by activating the NF-κB pathway, both directly and indirectly, enhancing inflammation and tumor progression. The HCV core protein induces proinflammatory cytokines such as TNF-α, thereby activating the NF-κB pathway through TNFR activation. On the other hand, the core protein also binds to the cytoplasmic domain of TNFR1, mimicking TNF-α signaling and amplifying NF-κB activation [408]. Additionally, HCV promotes Th17 cell activation, leading to secretion of IL-17 and IL-22, which further stimulate NF-κB activity [409]. Beyond the core protein, other HCV proteins, including NS4A, and NS5A, activate NF-κB pathway by inducing oxidative stress. Once activated, NF-κB translocates to the nucleus and initiates the expression of genes like cyclooxygenase-2 (COX-2) and IL-8, which are crucial for inflammation and tumor microenvironment formation [410]. While HCV activates NF-κB through multiple mechanisms, some studies identify that certain HCV proteins (such as NS3 and NS5B) can inhibit TNF-α-induced NF-κB activation, potentially allowing HCV to modulate immune responses and evade immune surveillance, thus contributing to viral persistence [411]. Overall, hepatitis viruses promote a tumorigenic microenvironment and drive HCC development by modulating NF-κB pathway through oxidative stress and proinflammatory signaling.

4.6.2 Janus Kinase/Signal Transducer and Activator of Transcription

The JAK–STAT signaling pathway, mediated by cytokines such as interleukins and IFNs, plays a critical role in controlling gene expression involved in cell proliferation, survival, and immune regulation [412]. In viral hepatitis, abnormal activation of the JAK–STAT pathway is a significant mechanism contributing to hepatocyte malignant transformation and HCC development.

In HBV infection, the viral protein HBx enhances JAK–STAT activity by interacting with JAK1, increasing its kinase activity, and upregulating STAT3 phosphorylation, leading to sustained activation. This, in turn, activates genes related to cell proliferation and survival, such as cyclin D1 (CCND1) and myeloid cell leukemia 1 (MCL1), promoting uncontrolled hepatocyte proliferation [413]. Additionally, HBx activates STAT3 to induce Twist, a gene promoting EMT, increasing tumor invasiveness and metastatic potential [414]. In HBV-related HCC, IL-6 is a key activator of the JAK–STAT pathway. It binds to its receptor, glycoprotein 130 (GP130), initiating JAK1 phosphorylation and STAT3 signaling, which further drive hepatocyte malignant transformation [415]. Elevated IL-6 levels are strongly linked to HCC development, particularly in men. Mechanistically, estrogen can inhibit IL-6 secretion from macrophages, reducing HCC risk in women [416].

HCV also accelerates liver cancer progression by modulating the JAK–STAT pathway. HCV infection increases IL-6 and other proinflammatory cytokines production, leading to STAT3 activation, which drives uncontrolled hepatocyte proliferation and upregulation of antiapoptotic genes such as B-cell lymphoma-extra-large (Bcl-xL) and CCND1, promoting HCC development [417]. In HCV-related HCC patients, overactivation of STAT3 is closely linked to tumor progression and poor prognosis, underscoring the pivotal role of JAK–STAT pathway in HCV-induced HCC [415]. Notably, as viral load increases in the later stages of infection, HCV upregulates SOCS-1, a negative regulator of the JAK–STAT pathway. HCV infection increases SOCS protein expression through various mechanisms specific to different HCV proteins. Specifically, the HCV protein p7 induces SOCS-3 via STAT3- and ERK-mediated pathways. Conversely, SOCS-7 expression induced by HCV core protein genotype 3a operates independently of STAT3 and may be regulated by PPAR-γ activity. Elevated SOCS-1 levels inhibit the antiviral effects of type I and type III IFN signaling, creating conditions favorable for viral replication and tumorigenesis [418].

Both HBV and HCV promote HCC by modulating the JAK–STAT pathway, enhancing cell proliferation, EMT, and accelerating tumor growth and metastasis. Dysregulation of the pathway is critical for HCC development and progression in chronic viral hepatitis.

4.6.3 Phosphoinositide 3-Kinase/Protein Kinase B

The PI3K/AKT signaling pathway plays a crucial role in regulating cellular processes such as proliferation, survival, metabolism, and migration, and its dysregulation is closely linked to HCC development [419]. Hepatitis viruses, particularly HBV and HCV, activate the pathway through multiple mechanisms, promoting HCC onset and progression.

HBV interacts with the PI3K/AKT pathway primarily through HBx, driving HCC malignancy. HBx regulates the PI3K/AKT pathway by activating AKT and inhibiting GSK-3β, stabilizing β-catenin and upregulating CCND1 expression, which leads to uncontrolled cell proliferation and carcinogenesis [123, 420]. Different HBx isoforms exhibit distinct roles in cell survival. The HBx–S31 isoform, phosphorylated at Ser31, activates AKT, promoting antiapoptotic effects and tumor growth, while the HBx–L31 isoform, lacking the phosphorylation site, induces apoptosis and inhibits tumor formation [421]. Persistent AKT activation supports HCC progression by inhibiting apoptosis, enhancing cell survival through phosphorylation of transcription factors such as c-Myc and NF-κB, which accelerates cell cycle and tumor growth [422, 423].

In a similar manner, HCV promotes HCC development by activating the PI3K/AKT pathway. HCV NS3 protein interacts with key regulators such as p21 and p53, disrupting cell cycle control and activating epidermal growth factor receptor (EGFR) signaling, which further enhances PI3K/AKT activation. This signaling promotes downstream proliferation and survival pathways, driving malignant transformation [424]. Additionally, the HCV NS4A protease activates the PI3K/AKT signaling pathway, promoting infected cell growth and contributing to HCC progression [425]. The PI3K/AKT pathway also interacts with other oncogenic pathways, such as the Ras/ERK signaling pathway, forming a complex network that coregulates HCC development in hepatitis virus infection [426].

In summary, hepatitis virus promotes HCC by activating the PI3K/AKT pathway, which enhances cell proliferation and survival, driving further cancer progression.

4.6.4 Mitogen-Activated Protein Kinase

The MAPK pathway, comprising Ras, Raf, MEK, and ERK, is a notable signaling cascade activated by external growth factors or cytokines. Ras, a critical protein in the pathway, binds to guanosine triphosphate (GTP), activating Raf, which in turn activates MEK and ERK [427]. Hepatitis viruses, including HBV and HCV, hijack the pathway, activating the Ras–ERK signaling cascade to upregulate cell cycle proteins, as well as oncogenes, ultimately driving abnormal cell proliferation and malignant transformation.

In HBV infection, the viral protein HBx directly impacts MAPK pathway activation. HBx interferes with Ras–GTP turnover, sustaining Ras and MAPK pathway activation, which also enhances EGFR expression, further promoting the Ras/MEK/ERK cascade [428]. The amplified signaling drives abnormal hepatocyte proliferation and inhibits apoptosis, allowing the virus to persist and replicate [429]. Moreover, HBx activates the ERK pathway, upregulating the cell cycle inhibitor cyclin-dependent kinase inhibitor 1 (p21Cip1) and inducing G2-phase cell cycle arrest, which benefits HBV replication as the virus thrives during the G1 and G2 phases [430]. Additionally, HBV middle surface antigen activates the Ras/MEK/ERK pathway through a protein kinase C (PKC)-dependent mechanism, further enhancing the survival of HBV-infected cells and contributing to tumor formation [431].

Persistent EGFR activation by HCV triggers the MAPK/ERK signaling pathway, enhancing multiple oncogene expression. Microarray analysis has shown that HCV infection significantly upregulates genes related to inflammation and angiogenesis, including amphiregulin (AREG), IL-8, and C-C motif chemokine ligand 20 (CCL20), which accelerate HCC progression by promoting inflammation and angiogenesis in the tumor microenvironment [432]. The HCV core protein, key factor in HCC development, affects the expression of transcription factors, such as polyoma enhancer-activator 3 (PEA3), serum response factor (SRF), and c-Fos by activating the MAPK/ERK pathway [433]. Specifically, the core protein activates the Raf/MEK/ERK cascade, driving abnormal proliferation and antiapoptotic responses in liver cells. Further research has shown that core protein acts directly upstream of MEK or promote oncogenic gene expression by enhancing downstream transcription factor ETS-like transcription factor (ELK1) activity [434]. Activation of these pathways increases liver cell proliferation and enhances migration and invasion by upregulating critical molecules like matrix metalloproteinases MMP-2 and MMP-9 [3].

In conclusion, aberrant activation of the MAPK pathway plays a pivotal role in HCC transformation from viral hepatitis infection. MAPK activation disrupts cell cycle regulators, enabling viral replication while preventing apoptosis. Sustained MAPK activation also triggers transcription factors like STAT3, enhancing tumor cell survival, invasiveness, and metastatic potential, exacerbating HCC malignancy and worsening patient prognosis.

4.6.5 Nuclear Factor Erythroid 2-Related Factor 2

NRF2 is a notable transcription factor involved in the cellular response to oxidative stress, regulating antioxidant defense mechanisms by controlling AREs [435]. Under normal conditions, NRF2 protects cells from ROS damage. However, during viral infections, viruses such as HBV and HCV hijack NRF2 activation to evade the host immune system, promoting tumorigenesis and disease progression [436].

In HBV infection, viral proteins like HBx and the large surface proteins (LHBs) activate the NRF2 pathway. HBx, in particular, activates NRF2 through c-Raf and MEK pathways, leading to the expression of antioxidant genes that enhance the antioxidant capacity of infected liver cells, helping them resist ROS-induced damage and survive [437]. However, HBV-driven NRF2 activation also serves as an immune evasion strategy. Studies have shown that upregulation of NRF2-regulated genes, such as NADPH quinone oxidoreductase 1 (NQO1), GSH-Px, and glutamate cysteine ligase (GCLC), strengthens antioxidant defenses, modulates proteasomal activity, reduces viral antigen presentation, and diminishes T-cell recognition, allowing HBV-infected cells to survive immune surveillance, thereby promoting chronic infection and worsening liver disease [438, 439].

Similarly, HCV activates the NRF2/ARE pathway through viral proteins, including the core protein, NS3, NS4B, and NS5A, which activate signaling molecules such as PKC, casein kinase 2 (CK2), and PI3K. It induces NRF2 phosphorylation and dissociation from KEAP1, allowing NRF2 to translocate to the nucleus and promote antioxidant gene expression [287, 440]. However, HCV employs a nuanced strategy to regulate NRF2. While NRF2 is activated, the HCV core protein binds to small Maf (sMaf) proteins, preventing NRF2 from entering the nucleus, which limits excessive antioxidant protection. This controlled oxidative stress environment is beneficial for HCV replication and viral release [440].

In summary, NRF2 activation following HBV and HCV infection promotes liver cell proliferation and tumorigenesis by inhibiting apoptosis and enhancing survival signaling. The upregulation of NRF2-regulated genes not only protects cells from oxidative stress but also improves metabolism and reduces ROS production, creating a tumor-friendly environment. Additionally, viral proteins like HBx and NS5A further enhance NRF2 activity through PI3K/AKT and MAPK pathways, promoting cell proliferation and inhibiting apoptosis, which accelerates chronic liver inflammation and cell damage, ultimately leading to HCC.

4.6.6 Hippo

The Hippo signaling pathway is an important pathway that regulates cell proliferation, apoptosis, and organ size [441]. In viral hepatitis, inactivation of the Hippo pathway leads to the nuclear translocation and activation of transcription factors, disrupting the balance between cell proliferation and apoptosis, contributing to malignant transformation, and activating tumorigenic genes that promote cell proliferation, migration, and survival [442].

HBV influences the Hippo/Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway through multiple mechanisms, driving the malignant transformation of HCC. Studies have shown that HBx is closely associated with YAP activation in liver cancer. HBx activates YAP by upregulating the transcription factor forkhead box A1 (FOXA1) and the ubiquitin E3 ligase MSL2, which regulate histone H2B ubiquitination, enhancing YAP transcriptional activity [125]. Additionally, HBx interacts with the E3 ligase human double minute 2 (HDM2), preventing YAP ubiquitination and degradation, further stabilizing YAP in the nucleus [443]. Activated YAP induces oncogene expression, promoting hepatocyte proliferation and tumor progression [444]. Furthermore, in some HBV-related HCC cases, HBV integrates into the host genome, particularly in the C-terminal truncation of HBV surface proteins, which inhibits miR-338-3p expression, activates TAZ and increases tumor invasiveness and metastatic potential [445].

Likewise, HCV modulates the Hippo pathway to drive HCC malignancy. HCV NS5B protein inhibits Hippo signaling, activating YAP and upregulating Snail, which promotes EMT [446]. HCV E2 protein also regulates the Hippo pathway by mimicking the function of CD81, which is a significant regulator of the Hippo pathway and a significant ligand for glypican-3 (GPC3). By reducing Hippo pathway activation, E2 decreases YAP inhibition, promoting cell proliferation and facilitating early tumor cell expansion, making HCV-related HCC more aggressive [447, 448].

In conclusion, hepatitis viruses significantly contribute to the malignant transformation of HCC by modulating the Hippo–YAP/TAZ pathway, enhancing the aggressive nature of liver cancer.

4.6.7 Wingless-int/β-Catenin

The Wnt/β-catenin signaling pathway is essential for cell proliferation, differentiation, and survival, abnormal activation of which is closely linked to HCC development [449]. Both HBV and HCV modulate the Wnt/β-catenin pathway through distinct mechanisms, promoting HCC onset and progression.

In HBV-related HCC, the abnormal activation of the Wnt/β-catenin pathway is a prominent driver of tumorigenesis. Studies have shown that mutations in the catenin beta1 (CTNNB1) gene, which encodes β-catenin, are present in about 12% of HBV–HCC patients and directly cause abnormal activation of the Wnt/β-catenin pathway [450]. In HBV-infected livers, several regulators of the Wnt/β-catenin pathway exhibit abnormal expression patterns. For example, Wnt2, Wnt7, and disheveled segment polarity protein 3 (DVL-3) are upregulated in tumors, while negative regulators like naked cuticle homolog 1 (NKD1) and NKD2 are significantly downregulated, underscoring the crucial role of the Wnt pathway in HBV-related tumor development [451]. Furthermore, HBx activates the Wnt/β-catenin pathway via multiple mechanisms. HBx first suppresses E-cadherin expression, disrupting cell adhesion, and promotes Wnt pathway activity via promoter hypermethylation and Src signaling activation [452]. HBx also directly binds to the tumor suppressor gene adenomatous polyposis coli (APC), preventing β-catenin degradation, which causes β-catenin accumulation in the cytoplasm and its translocation to the nucleus, where it activates Wnt target genes [453]. Inhibition of GSK-3β degradation complex further accelerates the process, owing to persistent Wnt pathway activation [454].

HCV similarly promotes HCC by modulating the Wnt/β-catenin pathway. In HCV-related HCC patients, CTNNB1 mutations are found in approximately 26% of cases, significantly higher than the 12% observed in HBV–HCC [455]. HCV proteins like NS3 disrupt DNA repair mechanisms, increasing CTNNB1 mutation frequency and promoting Wnt/β-catenin activation [456]. The HCV core protein, a major regulator of the Wnt/β-catenin pathway, activates the pathway through modulation of nuclear transcription factors [457]. The core protein also enhances Wnt signaling by upregulating Frizzled (FZD) receptors and LDLR-related proteins 5/6 (LRP5/6) while inhibiting antagonists like FZD-related protein 2 and Dickkopf (DKK) [458]. In early HCV infection, DDK1 recruits DNA methyltransferases and HDACs, causing epigenetic silencing of gene promoters, which further drives β-catenin activation [459]. Additionally, HCV NS5A protein interacts directly to GSK-3β, preventing β-catenin degradation and further enhancing Wnt pathway activation [460].

In conclusion, hepatitis viruses modulate the Wnt/β-catenin pathway via multiple mechanisms, significantly promoting HCC malignant transformation. Abnormal Wnt/β-catenin activation drives hepatocyte proliferation and metastasis, while fostering fibrosis and the tumor microenvironment, ultimately accelerating HCC onset and progression.

4.6.8 p53

p53, often called the “guardian of the genome,” is a critical tumor suppressor protein activated in response to DNA damage or cellular stress, which regulates downstream genes to induce cell cycle arrest or apoptosis, preventing the proliferation of abnormal cells [461]. However, hepatitis viruses like HBV and HCV inhibit p53 activity through various mechanisms to evade immune surveillance, promote viral persistence, and contribute to HCC development.

In HBV infection, the viral HBx protein directly interferes with p53 function. HBx forms a complex with p53, preventing it from binding to DNA, thus blocking its role in DNA repair and apoptosis [462]. This inhibition mimics the behavior of mutated p53 in cancers, allowing the accumulation of DNA damage and promoting hepatocyte transformation into tumor cells [463, 464]. Murine double minute 2 (MDM2), a noteworthy negative regulator of p53, binds to p53 and facilitates its ubiquitination and degradation, inhibiting p53 tumor-suppressing functions [465]. During HBV infection, HBx interacts with MDM2, promoting its nuclear translocation and further suppressing p53 transcriptional activity, which not only diminishes p53 anticancer functions but also stabilizes HBx, enhancing its carcinogenic effects [466]. Furthermore, MDM2 undergoes NEDDylation, increasing HBx stability and accelerating HBV-induced liver fibrosis [443]. By disrupting the MDM2–p53 axis, HBV evades immune surveillance and promotes hepatocyte malignancy. MDM2 also interacts with the TGF-β1 signaling pathway, contributing to liver fibrosis and tumorigenesis [467]. Beyond HBx, HBeAg suppresses p53-dependent apoptosis pathways, such as the Fas/Fas Ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) pathways, enabling infected hepatocytes to evade immune clearance [468]. HBeAg interacts with NUMB endocytic adaptor protein (NUMB), weakening p53 transcriptional activity, making p53-dependent apoptosis ineffective. Additionally, HBeAg accelerates p53 degradation via MDM2-mediated ubiquitination, contributing to p53 inactivation. This dual mechanism allows HBV-infected hepatocytes to survive longer, accumulate genetic damage, and eventually progress to liver cancer [469].

In like manner, HCV disrupts the p53 pathway to promote HCC development. HCV infection increases oxidative stress, inhibiting p53 acetylation and enhancing MDM2-mediated p53 degradation [470]. The HCV core protein suppresses p53 transcriptional activity, reducing p53-dependent apoptosis and helping infected hepatocytes evade cell death [471]. Furthermore, HCV activates the Nrf2 signaling pathway, promoting MDM2 expression and further disrupting the p53 pathway, enabling long-term survival of infected hepatocytes and accumulation of damage that drives tumorigenesis [472].

Through these modes, both HBV and HCV disrupt p53 function, allowing infected hepatocytes to evade immune surveillance, accumulate mutations, and progress to HCC. The inactivation of p53 is a crucial step in the carcinogenesis associated with viral hepatitis.

4.6.9 Vascular Endothelial Growth Factor

Angiogenesis is a critical process for growth and metastasis of HCC and is closely associated with VEGF [473]. Hepatitis viruses, such as HBV and HCV, promote HCC development by activating the VEGF signaling pathway, thereby enhancing angiogenesis.

In HBV-related HCC, VEGF serves as a significant angiogenic factor. Studies have shown that VEGF and COX-2 expression is significantly increased in HBV–HCC mice and patient tumor tissues, promoting an increase in microvascular density (MVD) [474]. MVD, a crucial marker of tumor angiogenesis, is strongly driven by the overexpression of VEGF, which stimulates the formation of new tumor blood vessels [474]. HBx, the HBV-encoded protein, contributes to the process by stabilizing HIF-α and activating mechanistic target of rapamycin (mTOR) and IκB kinase beta (IKKβ) signaling pathways, thereby enhancing VEGF expression [475]. Additionally, HBx activates the MAPK signaling pathway, increasing the secretion of angiopoietin-2 (Ang-2), further promoting angiogenesis in liver tissues [476]. Beyond VEGF stimulation, HBx upregulates MMPs, such as MMP-2, MMP-9, and MMP-14, which degrade the ECM, facilitating endothelial cell migration and new blood vessel formation, thereby enhancing HCC angiogenic capacity [477].

In a comparable way, HCV promotes HCC progression by regulating angiogenesis pathways. The HCV core protein is closely linked to angiogenesis, as it activates various growth factor signaling pathways, including p38 MAPK, PI3K, and JNK, leading to increased VEGF and Ang-2 expression, significantly enhancing angiogenesis in liver cancer [478, 479]. HCV core protein also activates the STAT3 signaling pathway, promoting VEGF expression, and interacts with androgen receptors to increase VEGF transcription [480]. Additionally, the proangiogenic effect of HCV core protein is dose-dependent, with activation of the activator protein-1 (AP-1) pathway being another important mechanism driving increased VEGF expression [481].

By regulating the VEGF pathway, hepatitis viruses enhance angiogenesis, providing tumor with essential nutrients and oxygen, while also creating pathways for tumor cell dissemination and metastasis, which accelerates HCC progression.

4.6.10 Insulin-Like Growth Factor

The IGF signaling pathway, involving IGF-1, IGF-2, and their receptors (IGF-1R and IGF-2R), plays a crucial role in regulating cell proliferation, metabolism, and antiapoptotic processes [482]. Aberrant activation of the IGF signaling pathway is a key factor in the progression from viral hepatitis to HCC.

In HBV infection, which is strongly associated with HCC development, the IGF/IGF-1R signaling pathway is activated. HBx upregulates IGF-1R expression and increases IGF-2 transcription by interacting with the Sp1 binding site on IGF-2 promoter, which collectively lead to IGF-2 overexpression and aberrant IGF signaling, accelerating liver cancer progression [483]. Additionally, HBV enhances IGF-1R expression through the activation of HIF-1α, further promoting HCC development. Notably, HIF-1α activation under hypoxic conditions exacerbates IGF signaling, facilitating tumor growth and invasion [484].

Dysregulation of the IGF signaling pathway in chronic HCV infection, mediated by insulin resistance and chronic inflammation, drives HCC progression [485]. Insulin resistance and impaired growth hormone function, common in HCV-infected patients, are associated with reduced IGF-1 levels [486]. Low levels of IGF-1 not only reduce proliferative capacity of liver cells but also heighten their sensitivity to inflammatory and tumor growth factors, thereby facilitating cancer cell formation and proliferation. IGF-1 activates STAT5 signaling and induces EMT in HCC cells by downregulating E-cadherin and upregulating N-cadherin and vimentin [487].

The IGF signaling pathway not only promotes liver cancer cell proliferation but is also linked to CSCs characteristics. IGF-1 treatment activates IGF/IGF-1R signaling in HBV-positive HCC cells, increasing expression of cancer stemness markers like nanog homeobox (NANOG) and octamer-binding transcription factor 4 (OCT4). This suggests that IGF pathway activation maintains CSCs properties, promoting HCC recurrence and progression [488].

In conclusion, hepatitis viruses, particularly HBV and HCV, promote the malignant transformation of hepatocytes by regulating the IGF signaling pathway, particularly through upregulation of IGF-2 and IGF-1R. Aberrant activation of the IGF pathway not only exacerbates liver inflammation and metabolic dysregulation but also enhances CSCs self-renewal, contributing to tumor invasiveness and recurrence risk.

5.2.1 Antiviral Therapies

Chronic infections with HBV or HCV are well-recognized primary risk factors for HCC development through multiple mechanisms. Consequently, achieving sustained viral suppression or eradication has proven effective strategy in preventing HCC. Over the last decade, advancements in antiviral agents, including IFN, NAs, and DAAs, have enabled sustained viral suppression or cure in most chronic HBV/HCV patients, significantly impacting HCC prevention (Table 3).

5.2.2 Antiliver Fibrosis/Cirrhosis Therapies

Chronic liver injury caused by HBV and HCV leads to disease progression, from hepatocyte injury to fibrosis, cirrhosis, and ultimately HCC. Although no therapy approved by the US FDA or EMA directly target liver fibrosis or cirrhosis, several promising agents have shown success in reversing liver fibrosis in clinical/preclinical trials (Table 3) [554].

Antiviral therapy targeting the underlying infection has made significant progress in preventing and even reversing liver fibrosis progression. In CHB patients with hepatic steatosis, antiviral therapy was independently associated with a lower risk of fibrosis progression [555]. Additionally, a prospective study of HCV patients treated with DAAs found that HCV clearance improved liver function and fibrosis [556]. Further research has underscored the significance of chemokine-related proinflammatory pathways in the immunopathology of liver fibrosis. Antiviral treatment has been shown to reduce CXCL9, CXCL10, and CXCL11 levels, thereby mitigating the severity of liver cirrhosis [557].

Furthermore, anti-inflammatory, hepatoprotective, and antifibrotic agents address hepatitis virus-related-fibrosis symptoms and play distinct roles at different disease stages. Silymarin, a flavonoid compound, protects healthy and partially damaged liver cells by reducing oxidative stress and mitigating cytotoxicity. It has been approved in several countries for treating liver conditions [558]. In a small clinical trial, silymarin adjunct therapy enhanced DAA efficacy, normalizing hepatological, serological, hormonal, and antioxidant markers in HCV patients [507]. Berberine has also been investigated as a potential antifibrotic agent, with mechanisms including the activation of ROS-mediated HSC ferroptosis [511], inhibition of inflammation via a sirtuin 3-dependent mechanism [559], and regulation of lipid metabolism and gut microbiota to improve liver health [512].

Beyond traditional drug therapies, mesenchymal stem cells (MSCs) have emerged as a novel treatment for liver fibrosis and cirrhosis. They can migrate to injury sites, inhibit HSC activation, differentiate into hepatocytes, and participate in immune regulation, thereby reducing ECM deposition and ameliorating hepatic fibrosis [560]. In a randomized controlled trial, umbilical cord-derived MSC treatment significantly improved long-term survival rates and liver function in patients with decompensated cirrhosis induced by HBV, introducing a novel therapeutic approach [518]. Additionally, autologous MSC infusion via peripheral veins has shown promise in treating patients with HCV and end-stage liver disease, demonstrating satisfactory effects on liver synthetic function and fibrosis [519]. However, due to the complex mechanisms involved in liver fibrosis formation and significant individual variations, antifibrotic drug development continues to face considerable challenges.

5.2.3 Anti-HCC Therapies

Surgical resection, liver transplantation (LT), and local ablative therapy are the primary treatments for HCC [561]. LT is the preferred treatment for early-stage HCC patients who are not candidates for resection due to hepatic dysfunction or multifocal neoplasms, as it addresses both HCC and the underlying liver pathophysiology. For patients with minimal tumor load and no vascular invasion or metastatic spread, transcatheter arterial chemoembolization (TACE) is the frontline therapy [561]. Due to its aggressive nature and potential for asymptomatic progression, many patients are diagnosed at intermediate or advanced stages, limiting the opportunity for optimal intervention. Conventional therapeutic approaches have demonstrated suboptimal efficacy in advanced HCC, prompting a paradigm shift toward the utilization of systemic therapeutics.

Targeted therapies have introduced new treatment approaches for hepatitis virus-associated HCC, particularly in advanced-stage patients. Sorafenib remains the primary first-line therapy, followed by lenvatinib, bevacizumab (BVZ), and donafenib as additional first-line options. Sorafenib, a multikinase inhibitor, exerts antineoplastic effects by inhibiting VEGF receptor (VEGFR), platelet-derived growth factor receptor-beta (PDGFR-β), and Raf/MEK/ERK signaling cascades, thereby inhibiting tumor proliferation and angiogenesis [525]. In 2018, lenvatinib, a multikinase inhibitor with a spectrum of activity against VEGFR, FGF receptor (FGFR), PDGFR, rearranged during transfection (RET), and stem cell factor receptor (KIT), was approved by the US FDA and China's NMPA as a frontline therapeutic option for patients afflicted with unresectable HCC [526]. BVZ, a recombinant IgG1 monoclonal antibody, demonstrates high-affinity binding to VEGF and thereby preventing VEGF–VEGFR interaction [562]. The combination regimen of atezolizumab with BVZ has been established as the standard first-line treatment protocol for patients with unresectable or metastatic HCC [563]. Donafenib, an independently developed molecular-targeted drug in China and a derivative of chemically modified sorafenib, targets receptor tyrosine kinases (RTKs), including VEGFR and PDGFR, and directly inhibits Raf kinases and Raf/MEK/ERK pathways. The mechanism of anti-HCC is to inhibit tumor cell proliferation and neovascularization, exerting antitumor effects through multitarget blockade (Table 3) [528].

Resistance to first-line targeted therapy has necessitated the expansion of second-line treatment options for HCC. Regorafenib, a multitarget kinase inhibitor structurally sharing structural homology with sorafenib, exerts its therapeutic effects by targeting VEGFR, FGFR-1, PDGFR, RAF, KIT, and RET, thereby inhibiting angiogenesis and modulating tumor microenvironment, which was approved by the US FDA in 2017 for HCC patients who have demonstrated refractoriness to sorafenib [529]. Cabozantinib is also a multitarget small molecule tyrosine kinase inhibitor (TKI) that inhibits mesenchymal-epithelial transition (MET), VEGFR, ROS proto-oncogene 1 (ROS1), AXL receptor tyrosine kinase (AXL), neurotrophic tyrosine receptor kinase (NTRK), KIT, RET, and was approved by the US FDA in 2019 for advanced HCC [530]. Ramucirumab, an IgG1 monoclonal antibody, binds to VEGFR-2 thereby inhibiting angiogenesis. The US FDA approved ramucirumab in 2019 for advanced HCC patients with AFP levels ≥400 ng/mL [531]. Apatinib, a TKI selectively inhibiting of VEGFR-2 and blocking of VEGF signaling, has also been approved for advanced HCC (Table 3) [532].

In summary, antiviral treatments, including IFN, NAs and DAAs, have proven effective strategies in preventing HCC by achieving sustained viral suppression and eradication. Several promising agents such as silymarin and berberine, as well as MSCs therapy, have demonstrated potential in ameliorating liver fibrosis caused by HBV and HCV. At the HCC stage, targeted therapies such as sorafenib have successfully inhibited tumor proliferation and angiogenesis. However, side effects and patient tolerance may limit the clinical use of these drugs, highlighting the need for new targets and therapies to improve efficacy.

5.3.1 Immune Checkpoint Inhibitors

Virus-infected liver cells and HCC cells evade immunosurveillance by upregulating PD-L1 expression on their surface, which interacts with T-cell immune checkpoint receptors, including B- and T-lymphocyte attenuator (BTLA), PD-1, and CTLA-4, inhibiting T-cell activation and immune responses, enabling malignant cells to escape immune detection, proliferate uncontrollably, and accelerating the progression of viral hepatitis to HCC [588]. Immune checkpoint inhibitors (ICIs) reshape the tumor microenvironment by activating suppressed immune cells to attack the tumor. US FDA-approved ICIs include agents targeting PD-L1 (atezolizumab, durvalumab), PD-1 (pembrolizumab, nivolumab), and CTLA-4 (ipilimumab, tremelimumab). By disrupting immune checkpoints, ICIs relieve tumor-induced T-cell inhibition, restoring their activation and cytotoxic function.

Two pivotal clinical trials, IMbrave150 and HIMALAYA, have demonstrated a significant enhancement in overall and progression-free survival for patients with unresectable HCC treated with ICIs compared with sorafenib [589, 590]. In the IMbrave150 trial, the combination of atezolizumab and BVZ exhibited superior efficacy over sorafenib, with improved median OS (19.2 vs. 13.4 months), median progression-free survival (6.8 vs. 4.3 months), and objective response rates (30 vs. 11%) [589]. The HIMALAYA trial investigated the synergistic effect of two ICIs: durvalumab and tremelimumab. The STRIDE (single tremelimumab regular interval durvalumab) treatment, based on phase II data, demonstrated superior OS compared with sorafenib (median 16.4 vs. 13.8 months) and a higher objective response rate (20.1 vs. 5.1%) [590].

Meta-analyses have indicated that HCC patients with viral hepatitis achieve favorable survival outcomes with ICIs treatments [591]. Notably, ICIs have the potential to rejuvenate exhausted T-cell immunity, which may facilitate not only the treatment of cancer but also potentially lead to the cure of CHB. In HCC patients receiving ICIs, a functional cure of hepatitis B has been observed, with a higher cumulative incidence of HBsAg loss, particularly in patients with baseline HBsAg levels <100 IU/mL [592].

While ICIs have emerged as valuable treatment options for solid tumors, further research is warranted to confirm their suitability as a primary treatment for HCC. However, not all patients benefit from immunotherapies. As more data become available, personalized treatment for HCC is likely to become the prevailing approach.

5.3.2 Chimeric Antigen Receptor-T Cell Therapy

Chimeric antigen receptor (CAR)-T cell therapy is a novel immunotherapeutic modality that targets and eliminates virus infected liver cells and HCC cells. The CAR is a recombinant protein engineered to bind specific tumor-associated antigens, independent of MHC restriction, enabling T cells to recognize and eliminate malignant cells expressing the designated antigen [593]. The application of CAR-T cell therapy to HCC necessitates the identification of antigens that are selectively expressed on the surface of cancer cells [594]. GPC3, a membrane-anchored member of the HSPG family, is specifically upregulated in HBV/HCV-associated HCC, making it a compelling therapeutic target. Experimental evidence suggests that GPC3-targeted CAR-T cells are capable of inducing sustained tumor regression in murine models of HCC [595]. Phase I clinical trials have documented the antitumor activity of CAR–GPC3 T cells in HCC patients, thereby establishing the feasibility and preliminary safety of treatment [565]. Recently, a dual-target CAR-T cell therapy that targets both GPC3 and PD-1 has been developed, demonstrating the ability to eliminate PD-L1-positive HCC tumor cells and modulate the immunosuppressive tumor microenvironment, thereby exhibiting enhanced tumor inhibition compared with single-target CAR-T cells [568].

Recent studies have also concentrated on engineered T cells targeting the hepatitis virus genome or proteins for treating viral hepatitis-induced HCC. Tan et al. identified that HBV-related HCC tumor cells express antigenic epitopes translated from integrated HBV mRNAs, which can activate T cells. Therefore, autologous T cells engineered to express T-cell receptors (TCRs) specific for HBV-DNA epitopes were adoptively transferred to two HCC patients with posttransplant recurrence [596]. For chronic HCV infection, a CAR-T therapy targeting the HCV/E2 glycoprotein has been developed to secrete antiviral and proinflammatory cytokines while lysing HCV-infected hepatocytes, thereby controlling HCV infection [597]. However, no clinical trials have been conducted to date for HCV-targeting CAR-T therapy, and its efficacy in treating HCV-related HCC remains undetermined.

5.3.3 Vaccinations

Vaccines are essential for preventing and treating hepatitis virus-induced HCC, functioning in two primary categories: preventive and therapeutic. Prophylactic hepatitis B vaccines induce neutralizing antibodies against HBV envelope proteins, preventing hepatitis B and reducing both HBV infection and HCC incidence [598]. However, the development of effective prophylactic hepatitis B vaccines is complicated by viral hypervariability and immune evasion mechanisms [599]. Therapeutic vaccines are essential for HCC treatment as they aim to elicit a robust T-cell response that enhances the liver's immune microenvironment and inhibits the progression from viral hepatitis to HCC.

In a phase II trial with GPC3 peptide vaccination for HCC, the vaccine reduced 1-year recurrence by 15% and improved 5- and 8-year survival rates by 10 and 30%, respectively. The vaccine demonstrated greater efficacy in HCC patients with elevated GPC3 expression, highlighting the effectiveness of cancer vaccination in specific HCC subgroups [575]. In another phase II study focused on HBV infection, the GS-4774 vaccine was shown to enhance the production of IFN, TNF, and IL-2 by CD8⁺ T cells exposed to antigenic peptides, thereby disrupting immune tolerance in HBV-infected individuals [600]. In a phase I/II multicenter trial for HCC, HepaVac-101, which combines multipeptide antigen IMA970A with the TLR7/8 RIG-I agonist CV8102, demonstrated a favorable safety profile and elicited immune responses against tumor-associated antigens (TAAs) [574].

In addition to peptide-based vaccines, RNA-based vaccines have also been developed for therapeutic intervention in HCC. Specifically, poly-ICLC, a synthetic double-stranded RNA molecule designed to emulate viral infection, has demonstrated potential in stimulating immune responses. Studies have indicated that a combinatorial therapeutic approach, involving initial intratumoral administration followed by intramuscular injections of poly-ICLC, significantly suppressed tumor growth and enhanced CD8⁺ T cell infiltration [580]. Furthermore, combining therapeutic vaccines with ICIs has also demonstrated potential in HCC treatment. In a phase I/II trial, a personalized therapeutic cancer vaccine coadministered with plasmid-encoded IL-12 and pembrolizumab, exhibited clinical activity in advanced HCC patients by inducing antitumor T cell responses [571].

Serum levels of AFP are commonly elevated in most HCC tumors and can serve as a predictor of HCC recurrence in patients with viral hepatitis [601]. A study by Lu et al. demonstrated that AFP-based vaccine immunization combined with anti-PD-L1 treatment significantly inhibited HCC progression in AFP-positive tumor models [602]. Additionally, poly(lactic-coglycolic acid (PLGA)) micro/nanoparticle vaccination combined with anti-PD1 antibodies and ovalbumin (OVA) has been shown to enhance the activation and proliferation of OVA-specific CD8⁺ T cells, suggesting its potential to augment antitumor immune responses in HCC [577]. An advanced in situ nanovaccine, utilizing layered double hydroxides as a vehicle for cyclic GMP-AMP (cGAMP), a stimulator of IFN genes (STING) agonist, and adsorbed TAAs, has been reported to significantly modulate tumor immune microenvironment and augment the efficacy of anti-PD-L1 immunotherapies for HCC, offering a promising strategy for in situ cancer vaccination [578].

These vaccination strategies have demonstrated safety profiles, with the majority inducing antigen-specific responses without toxic or autoimmune reactions. However, the immunosuppressive environment of HCC presents challenges for eliciting effective immune responses, and the heterogeneous expression of TAAs in HCC tumors may limit the effectiveness of vaccines and increase the potential for immune evasion [603]. Consequently, enhancing vaccine efficacy against HCC malignancy requires a deeper understanding of TAAs and the strategic integration of vaccination with immune-activating approaches.

5.3.4 Oncolytic Virus

The immunosuppressive tumor microenvironment in viral hepatitis-related HCC may constrain the therapeutic efficacy of ICIs [604]. Oncolytic virus (OV) therapy is a novel immunotherapeutic approach that selectively targets and eliminates tumor cells, inducing tumor lysis and antitumor immune responses [605]. For instance, the oncolytic influenza virus delNS1- granulocyte-macrophage colony-stimulating factor (GM-CSF), when combined with PD-1 blockade therapy, has been shown to effectively target and eliminate HCC cells without impacting normal hepatocytes by activating CD4⁺ and CD8⁺ T cells through the JAK2–STAT3 pathway [584]. Comprehending the immune microenvironment in HCC is essential for elucidating the effects of OV. Spatial transcriptomics and single-cell RNA sequencing revealed that SynOV treatment increases CD8⁺ T cells infiltration, enhances Cxcl9–Cxcr3-mediated communication, and normalizes Kupffer cells in the tumor microenvironment. The tumor-reducing effects of SynOV have been clinically observed in metastatic HCC patients during a phase I clinical trial [606]. Ideally, OVs should selectively replicate in malignant cells to reverse the immunosuppressive microenvironment and be cleared by the host's immune system. The live attenuated vaccine West Nile virus (WNV)-poly(A) has emerged as a promising oncolytic agent. Mechanistically, it selectively targets and kills tumor cells while sparing normal cells due to its high sensitivity to IFN. Furthermore, WNV-poly(A) activates DCs, triggering a tumor antigen-specific response mediated by CD8⁺ T cells, thereby inhibiting the growth of both primary and metastatic tumor cells [587].

To avert host antiviral responses and achieve tumor-selective targeting, a surface engineering strategy has been devised to mask oncolytic herpes simplex virus (oHSV) with a galactose–polyethylene–glycol (PEG) polymer chain. The glycosylated-PEG–oHSV mitigates the immunosuppressive tumor microenvironment by augmenting the infiltration of CD8⁺ T cells and NK cells, stimulating the secretion of antitumor cytokines, ultimately impeding HCC progression [607]. A novel recombinant OV has also been constructed by incorporating NA fragments and GV1001 peptides, which exhibits selective cytotoxicity toward tumor cells. In alignment with in vitro observations, the recombinant OV significantly suppresses liver tumor growth and induces an antitumor immune response characterized by increased CD4⁺ and CD8⁺ T cells, thereby enhancing survival rates [585]. Recently, a recombinant Sindbis virus carrying GM-CSF has been developed for HCC therapy [608]. Studies have demonstrated that Sindbis virus effectively infects HCC cell lines and induces cell death, with the addition of GM-CSF potentiating its tumoricidal effects and augmenting immune cell infiltration in the tumor microenvironment.

Although OVs have demonstrated potential in the therapeutic management of cancer and preliminary research has yielded promising findings, the absence of substantive clinical data creates a significant void in the comprehensive assessment of their efficacy in the context of hepatic malignancies. Addressing this gap between basic research and clinical application is essential for validating OVs as a viable treatment for patients with viral hepatitis-associated HCC.

In conclusion, enhancing immune responses is essential to inhibit hepatitis virus replication and promote tumor cell regression. ICIs targeting PD-1, PD-L1, and CTLA-4 can relieve hepatitis virus-induced inhibition of T-cells, restoring their activation and cytotoxic function, and serving as a systemic treatment option for HCC alongside targeted therapies. However, low response rates limit their clinical application. CAR-T therapy modifies T cells to target specific tumor antigens and induce a strong T cell response, enhancing the liver immune microenvironment. Therapeutic vaccines aim to elicit a robust T-cell response that enhances the liver's immune microenvironment and inhibits the progression from viral hepatitis to HCC. Additionally, OV therapy can selectively replicate in virus-infected or tumor cells, sparing healthy cells, thereby enhancing immunotherapies efficacy and inhibiting the progression of viral hepatitis to HCC.

5.4.1 MicroRNAs

MiRNAs have emerged as promising, novel, noninvasive biomarkers for the early diagnosis, prognosis, and evaluation of HCC. In HCV patients, serum levels of miRNA-122 expression are elevated compared with controls but decreases in chronic cirrhosis and HCC, suggesting its potential as a biomarker for diagnosing the progression of HCV infection [609]. Detection of specific miRNA expression has also revealed that miR-210-3p is uniquely upregulated in HBV-related HCC, enhancing HBx expression [610]. Consequently, miRNA-122 and miR-210-3p may serve as a valuable biomarker of HBV-related HCC, and their inhibition could prevent hepatocarcinogenesis associated with HBV. Additionally, a study examining patients with HCV-related fibrosis, cirrhosis and HCC has demonstrated that miR-484 and miR-524 expression offers promising diagnostic potential: miR-484 could differentiate late-stage fibrosis from mild fibrosis and HCC, while miR-524 could distinguish cirrhosis from fibrosis, suggesting that miRNAs can serve as sensitive biomarkers for staging HCV-related liver disease progression [611].

MiRNA-based therapies for HCC provide a dual therapeutic strategy: miRNA replacement to restore tumor-suppressive miRNAs, thereby inhibiting tumor growth, and miRNA antagonism to target oncogenic miRNAs and mitigate their adverse effects. One promising example is RG101, a GalNAc-conjugated anti-miR-122 oligonucleotide, which has shown promise in reducing viral load in chronic HCV patients [612]. Combining miRNA replacement and antagonism therapies can amplify their benefits for HCC. For instance, cotreatment with a miR-122 mimic and miR-221 inhibitor inhibits cancer cell proliferation and angiogenesis while promoting apoptosis and necrosis by targeting specific peptidase 1 (SENP1) and ADP-ribosylation factor 4 (ARF4) genes, respectively [613]. Innovative delivery systems have also been developed to enhance the therapeutic potential of miRNAs in HCC. A synthetic miRNA-based targeted therapy using ultrasound-targeted microbubble destruction (UTMD) to locally deliver miRNA-loaded nanoparticles has been developed for HCC treatment [614]. This UTMD-mediated delivery of miRNA-122 and anti-miRNA-21 successfully modulates the tumor immune microenvironment at the cytokine level. Another approach involves encapsulating four miRNAs, including miRNA-122, miRNA-100, anti-miRNA-21, and anti-miRNA-10b, within biodegradable PLGA–PEG nanoparticles, and delivering them to HCC models. This significantly enhances the effectiveness of doxorubicin chemotherapies in HCC, achieving marked tumor growth inhibition and an increased apoptotic index [615].

5.4.2 Long Noncoding RNAs

LncRNAs have presented promising avenues for HCC precision medicine by acting as potential biomarkers, prognostic indicators, and therapeutic targets. HOX antisense intergenic RNA (HOTAIR) is significantly upregulated in HBV-infected cells and peripheral blood mononuclear cells (PBMCs) from CHB patients, making it a potential diagnostic and therapeutic biomarker for HBV [165]. The combined serum expression profiling of HULC, UCA1, and HOTAIR lncRNAs shows strong diagnostic performance in distinguishing HCC from liver diseases, including chronic HBV infection, fatty liver and cirrhosis, with potential implications for early diagnosis and personalized HCC treatment [616]. In HCC patients, contrast-enhanced ultrasound (CEUS) grading positively correlates with metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression [617]. MALAT1 is also heavily involved in HBx-related HCC progression, and silencing MALAT1 can block HBx-induced CSCs generation, stemness-related factor activation, and tumorigenicity via the PI3K/AKT signaling [147].

Targeting lncRNAs involved in tumor progression presents a promising strategy for virus-related HCC. For instance, metformin shows an inhibitory effect on HBV-associated HCC by suppressing HBx-induced HULC overexpression [618]. Gallic acid has been observed to reduce MALAT1 expression, thereby inhibiting MALAT1-mediated Wnt/β-catenin signaling and suppressing tumorigenesis [619]. Furthermore, an endosomal pH-responsive nanoparticle platform designed for codelivery of siRNA and a cisplatin prodrug specifically transports siRNA into the nucleus, reversing cisplatin resistance by silencing nuclear-localized MALAT1 and inhibiting HCC tumor growth [620].

5.4.3 M6A Modifications

M6A modifications hold promise as diagnostic, therapeutic, and prognostic tools in HBV/HCV-associated HCC. ALKBH5 exhibits elevated expression in HBV–HCC tissues, moderate expression in paracancerous tissues, and diminished expression in normal liver tissues, suggesting its potential as a biomarker for diagnosing the progression of HBV-related HCC [621]. Additionally, m6A modifications serve as prognostic markers for HCC, with METTL3, YTHDF2, and YTHDF1 highly expressed in high-risk HCC groups, while zinc finger CCCH-type containing 13 (ZC3H13) is highly expressed in low-risk groups [622]. Additionally, m6A-modified lncRNAs may predict OS in HCC patients by revealing differences in tumor-infiltrating immune cells, immune checkpoint expression, and chemotherapy sensitivity, providing new insights into HCC prognosis, immunotherapies and chemotherapies [623].

Given the role of m6A dysregulation in the pathophysiology of viral hepatitis-associated HCC, it represents a promising therapeutic target. ISG20, a 3′–5′ exonuclease enzyme, binds to m6A-containing HBV transcripts through YTHDF2, thereby promoting HBV RNA degradation, inhibiting HBV replication, and potentially preventing HBV-driven hepatocarcinogenesis [624]. Another m6A reader YTHDF1 has been shown to promote HCC progression through the YTHDF1–m6A–NOTCH1 epigenetic axis and is a viable therapeutic target for HCC. Lipid nanoparticles targeting YTHDF1 significantly enhance the efficacy of lenvatinib and sorafenib in HCC models [625]. Additionally, a mannose-modified antigen-capturing nanoplatform has been developed to codeliver TAAs and an m6A demethylase inhibitor to tumor-infiltrating DCs. In vivo study has demonstrated that the nanoplatform promotes DCs maturation and enhances effector T cell tumor infiltration, which synergizes with ICIs to inhibit distant tumor growth and lung metastasis [626]. Research on targeting m6A is still in the early stages, with limited therapy available, and further validation of their effectiveness is needed in preclinical and clinical studies.

5.4.4 CRISPR/Cas9

The CRISPR/Cas9 is originally a self-defense mechanism in prokaryotes and an adaptive immune system in bacteria to resist foreign genetic material, has evolved into a powerful gene-editing tool. Characterized by its simplicity, efficiency, low cost, and versatility, CRISPR/Cas9 opens new avenues for treating viral hepatitis-induced HCC [627]. On one hand, CRISPR/Cas9 enables targeted disruption of specific genomic regions in infectious agents or modulation of cellular factors implicated in viral replication through RNA-guided double-strand DNA breaks [628]. On the other hand, it enables rapid and precise gene editing. Integrating CRISPR/Cas9 with other therapeutic approaches may further inhibit tumor growth and enhance survival rates in patients with HBV/HCV-associated HCC [627].

As previously noted, the HBx is a momentous mediator in HBV-induced HCC pathogenesis by promoting EMT, making it an attractive target for CRISPR/Cas9. A novel HBx-specific single-guide RNA (sgRNA) designed with CRISPR/Cas9, termed HBx–CRISPR, has been found to reduce cccDNA levels and HBsAg production in HBV–HCC cells [629]. However, the genetic diversity of the HBV genome limits the applicability of genome editing across all patients. To counteract this, adenovirus vectors expressing multiplex guide RNAs for CRISPR/Cas9 allow simultaneous gene knockouts, addressing target heterogeneity and preventing escape mutants in genome-editing therapy [630]. Additionally, disruption of cyclophilin D with CRISPR/Cas9 impairs mitochondrial membrane integrity, reduces viral replication, and protects against HCV-induced inflammation, cell damage, and the heightened HCC risk [631].

Despite the promise of CRISPR/Cas9, challenges such as off-target effects, off-cell activity, and immunotoxicity should be addressed. To enhance precision and safety, a nanocomplex delivery system enables efficient coloading of gene-drug combinations, allowing for accurate gene editing and synergistic tumor inhibition without affecting normal tissues [632]. For instance, a polyamidoamine-aptamer-coated hollow mesoporous silica nanoparticle codelivering sorafenib and CRISPR/Cas9 enables efficient EGFR gene therapies and tumor inhibition without harming major organs [633]. Growth differentiation factor 15 (GDF15), a cytokine elevated in HCC patients following DAA treatment, promotes immunosuppression in the tumor microenvironment [634]. Targeted delivery of CRISPR/Cas9 against GDF15 using nanocapsules coated with HCC-specific SP94 peptides, enhances immune cell infiltration in HCC and promotes favorable immune microenvironment changes, as evidenced by cytometry by time-of-flight (CyTOF) analysis [635]. Additionally, a biomimetic delivery nanoplatform has been designed for HCC treatment by self-assembling a PD-L1-targeted CRISPR/Cas9 system with ursolic acid (UA). UA activates the natural immune system through the TLR-2-myeloid differentiation primary response 88 (MyD88)–TRAF6 pathway, while PD-L1 blockade promotes cytotoxic T cell infiltration, leading to significant tumor regression by simultaneously engaging innate and adaptive immunity [636].

These gene-targeted strategies highlight the potential to regulate multiple dysregulated genes and pathways in HCC, offering a more holistic approach compared with single-target drugs. However, as most of these studies remain in preclinical stages, further clinical trials are essential to confirm the efficacy and safety of these therapies in patients.

5.5.1 Gut Microbiota

Gut microbiota regulation can repair the intestinal barrier and enhance the antiviral immune defense in the liver, thus inhibiting viral hepatitis-induced HCC [637]. Certain agents with anticancer properties have been identified to modulate the gut microbiota, thereby improving the tumor immune microenvironment. For instance, stigmasterol has been shown to modulate diversity of the intestinal microbiota and significantly enhance populations of Lactobacillus johnsonii, Lactobacillus murinus, and Lactobacillus reuteri, leading to a reduced Treg/CD8⁺ T cell ratio, which enhances immune responses within the tumor microenvironment [638]. Additionally, intervention with Echinacea purpurea polysaccharide in HCC promotes expression of intestinal tight junction proteins, thereby repairing the intestinal barrier, which aids in controlling LPS leakage, subsequently inhibiting the TLR4/NF-κB pathway and reducing inflammatory factor expression [639]. A novel “Trojan-horse” strategy has been proposed, using an oral dextran–carbenoxolone (DEX–CBX) conjugate to combine prebiotics with glycyrrhetinic acid (GA) for targeted GA delivery to HCC via the gut–liver axis [640]. DEX–CBX significantly increases the abundance of Akkermansia, a bacterium known to strengthen systemic immune responses, which enhances NK T cells, CD8⁺ T cells, and M2 macrophages, offering a novel strategy to precisely modulate hepatic inflammation and gut microbiota in HCC.

Probiotics, live organisms that support host health, may also benefit HCC patients by improving the intestinal microenvironment [641]. Lactobacillus brevis SR52-2 and Lactobacillus delbrueckii Q80 have exhibited anti-HBV properties by inhibiting HBeAg and HBsAg expression and supporting gastrointestinal health in HBV-associated HCC patients [642]. Moreover, modulating the gut microbiota has further been shown to enhance the efficacy of anticancer drugs. In a study of advanced HCC patients treated with sorafenib, Enterococcus faecium (Efm) was significantly enriched in those who responded to treatment compared with nonresponders, suggesting that Efm could enhance sorafenib's therapeutic effects [643]. Additional in vivo study has shown that Efm colonization induces IL-12 and IFN-γ production and increases the proportion of IFN-γ and CD8⁺ T cells in the tumor microenvironment. Notably, butyrate, a SCFA produced by gut bacteria from dietary fiber and probiotics, possesses multiple anticancer mechanisms, including epigenetic regulation, immune modulation and metastasis suppression [644]. Che et al. developed nanoparticles coencapsulating butyrate and sorafenib, coated with an anti-GPC3 antibody, which prolonged drug retention time and demonstrated efficient, safe targeting to HCC cells [645].

Fecal microbiota transplantation (FMT), which transfers functional gut bacteria from a healthy donor to the patient, is a novel approach to restore gut microbiota balance and has shown promise in treating CLDs [646]. In a case-controlled, open-label pilot trial, FMT successfully cleared HBeAg in patients with persistent HBeAg positivity after long-term antiviral therapy [647]. In chronic HBV infection, oral capsule-based intestinal microbiota transplantation (IMT) decreased HBsAg and total bile acid (TBA) levels and restored gut microbiota abundance [648]. As a unique biological therapy derived from the human body and not classified as an organ transplant, FMT requires stringent monitoring and preservation of donor specimens for broader application.

5.5.2 Artificial Intelligence

Due to delayed diagnoses and the suboptimal efficacy of current therapeutic interventions, the prognosis for HCC remains poor. Recently, AI has risen as a distinctive opportunity to enhance the entire spectrum of HCC clinical management, encompassing risk prediction, diagnosis, and prognostication [649]. A gradient-boosting machine (GBM)-based model has been developed to generate risk scores for predicting HCC development in a cohort of 13,508 patients with CHB treated with ETV or tenofovir, which has demonstrated superior performance in risk stratification, identifying a subgroup of patients at minimal risk of developing HCC who could benefit from less intensive surveillance [650]. Using data from 5701 observations of 1985 HCC patients at a single center from 2000 to 2021, an AI-based system, PRAID, was constructed and evaluated for its prognostic value, specifically in predicting 1- and 3-year survival. Results have demonstrated that PRAID provided valuable prognostic information for short- and medium-term survival, aiding in therapeutic decision-making for both initial and recurrent HCC cases [651].

Machine learning (ML), a subtype of AI, enables computer programs to “learn” from data and improve through experience. Evidence suggests that ML offers a more accurate risk stratification model for HCC development after SVR compared with conventional methods [649]. Minami et al. developed a novel prediction model, the SMART model, which uses ML algorithms to predict HCC occurrence following HCV eradication. The SMART model incorporates seven accessible parameters: age, platelet count, serum AFP, gamma-glutamyl transferase (GGT), albumin, AST levels, and body mass index (BMI), allowing for the generation of individualized predictive curves, thereby supporting personalized and cost-effective surveillance after SVR [652]. Additionally, the SMART-HCC score, an ML-based HCC risk score incorporating LS measurements, identified LS as one of the top clinical predictors. The SMART-HCC score has been trained to predict HCC in 5,155 adult patients with various CLDs in Korea and subsequently validated in two prospective cohorts from Hong Kong (N = 2732) and Europe (N = 2384), which holds promise for clinicians in stratifying HCC risk among CLD patients [653]. Deep learning (DL) is a more advanced subtype of ML that utilizes multilayered neural networks to process large datasets. When applied to digital histopathologic images, DL can generate recurrence risk scores, potentially enhancing current stratification methods and refining the clinical management of patients undergoing primary surgical resection for HCC [654]. Multi-DL, a neural network comprising encoder, prediction, and segmentation pathways, has been developed using contrast-enhanced computed tomography (CT) images, effectively stratifying patients into low- and high-risk groups with significant survival differences, which offers clinicians valuable insights for tailoring therapeutic regimens [655].

AI is expected to significantly transform the treatment and care of patients with virus-related HCC. Although significant advancements have been made over the past decade, further refinement in risk prediction, diagnosis, and prognostication remains critical. Implementing these technologies into clinical practice presents several challenges, including the need for standardized data collection, sharing, and storage frameworks, as well as further validation to confirm the reliability and robustness of these models.

In conclusion, innovative therapeutic approaches have provided more precise treatment options for HBV/HCV-associated HCC. Gut microbiota-targeted treatments, tailored to the unique intestinal microenvironment of each individual, can repair the intestinal barrier, enhance liver antiviral immunity, and inhibit HCC induced by viral hepatitis. Additionally, AI can help predict disease progression, personalize treatment plans, improve outcomes, and reduce side effects through the analysis of large medical datasets. These strategies offer multiple treatment options to inhibit the progression of viral hepatitis to HCC, with the potential to improve outcomes and patient survival rates.