MicroRNA Binding Site Polymorphisms in Hepatocellular Carcinoma: Implications for Pathogenesis, Prognosis, and Therapeutic Response.

1. Introduction

1.1. Hepatocellular Carcinoma: Global Burden and Pathogenesis
The global burden of liver cancer is significant, as it is the fifth most common malignancy and responsible for the fourth highest number of cancer‐related deaths. The highest rates of this disease are found in East Asia and sub‐Saharan Africa [1]. Among men, it is the fourth most diagnosed cancer and second deadliest [2]. Hepatocellular carcinoma (HCC), arising from hepatocytes, accounts for over 80% of primary liver cancers, while intrahepatic cholangiocarcinoma (ICC) originates in the bile ducts [3]. Secondary liver metastases, often from colorectal cancer, further complicate the disease burden [4].
HCC develops through a multistep process driven by genetic and environmental factors, with cirrhosis as a common precursor [5]. Chronic liver injury, triggered by hepatitis B/C viruses (HBV/HCV), alcohol, aflatoxins, obesity, type 2 diabetes, or smoking, creates a pro‐carcinogenic microenvironment [2]. Molecular alterations in a cell can include mutations in tumor suppressor genes (such as TP53, PTEN) and proto‐oncogenes (such as RAS, MYC), chromosomal instability, and activation of inflammatory pathways (including, IL‐6/STAT3, NF‐κB) [6–8]. Inflammatory cytokines, particularly IL‐6 and TNF‐α, activate critical signaling pathways including STAT3 and NF‐κB, promoting hepatocyte proliferation, fibrogenesis, and neoplastic transformation. HCC pathogenesis involves dysregulation of key molecular pathways such as JAK/STAT, Ras/Raf/MAPK, PI3K/AKT/mTOR, and ubiquitin–proteasome systems, which control cellular proliferation, survival, and apoptotic processes [7, 9]. Furthermore, VEGF‐mediated angiogenesis is crucial in HCC progression and metastatic dissemination, with elevated VEGF expression correlating strongly with adverse clinical outcomes [7, 10]. Furthermore, the pathogenesis of HCC can be influenced by factors such as viral infections, conditions like nonalcoholic fatty liver disease (NAFLD), and oxidative stress [11]. These chronic insults are known to induce specific changes in the cellular miRNome, directly linking environmental factors to post‐transcriptional dysregulation.

1.2. Diagnostic Landscape and the Crucial Role of MicroRNAs
Current diagnostic approaches for HCC focus on early detection, crucial for improving outcomes [12, 13]. Diagnosis using conventional methods relies on imaging techniques, including ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) [14]. Ultrasound is the standard HCC screening tool due to its accessibility and low cost, though less sensitive for small tumors. Advanced imaging (CT/MRI) improves detection but is prohibitively expensive for routine use [12, 15, 16].
Serological biomarkers, including alpha‐fetoprotein (AFP), are still the main diagnostic tools for HCC, though their sensitivity and specificity are inadequate, especially for early stages [14, 17]. Several alternatives, such as AFP‐L3 and des‐γ‐carboxy prothrombin (DCP), have been presented, but their diagnostic accuracy is still insufficient [14, 18]. Recent studies also introduced potential diagnostic markers, including Glypican‐3 (GPC3), Golgi protein‐73 (GP73) [13], and microRNAs (including, miR‐122, miR‐21) [19]. Emerging molecular technologies, including liquid biopsies analyzing cell‐free DNA (cfDNA), circulating tumor cells (CTCs), and extracellular vehicles (EVs), offer noninvasive options with high promise for early detection, by capturing tumor‐specific genetic and epigenetic changes [14, 20]. In particular, cfDNA methylation patterns and miRNA signatures in EVs are considered highly promising for improving early detection [21–23]. Despite these advances, challenges in cost, standardization, and validation limit their integration into routine practice [12, 15, 16, 20]. These gaps highlight the importance of developing more specific biomarkers and utilizing molecular advances to enhance HCC early diagnostic and its accuracy [18, 23–25].
MicroRNAs (miRNAs) are short, noncoding RNAs crucial for post‐transcriptional regulation. By binding to specific mRNAs, often within the 3’‐untranslated regions, miRNAs control vital cell processes such as differentiation, proliferation, and apoptosis. There is a complex regulatory network between miRNAs and mRNAs. One miRNA can control the expression of numerous mRNAs, and in turn, a single mRNA can be a target for multiple miRNAs [26]. This interaction typically results in translational inhibition or mRNA degradation, leading to reduced protein expression [27]. miRNAs are categorized as either oncomiRs, which promote tumor growth, or tumor suppressors, which inhibit tumor development and progression [26]. In HCC, dysregulation of miRNAs significantly contributes to tumorigenesis, progression [28, 29], and response to treatment [30]. For example, miR‐122, a liver‐specific miRNA, is downregulated in HCC and associated with tumor growth and poor prognosis. HCC shows an increase in the oncogenic miRNA miR‐21, which drives cell proliferation and invasion. In contrast, miR‐199a‐3p is downregulated in HCC and is linked to angiogenesis, proliferation, and apoptosis [29]. Furthermore, exosomal miRNAs are being explored as noninvasive biomarkers for HCC detection [31]. Given their role in regulating gene expression and their involvement in various stages of HCC progression [19, 32], miRNAs and their binding sites are potential noninvasive biomarkers for the early detection, prognosis, and monitoring of treatment response in HCC [29, 32, 33]. Specifically, they offer a less invasive alternative to tissue biopsies by enabling the identification of genetic alterations in circulating exosomes or cell‐free RNA. Figure 1 demonstrates the schematic representation of microRNA‐SNP interaction and their involvement in HCC pathogenesis.
Recognizing the crucial role of miRNAs in HCC and the potential impact of SNPs on their function, this review aims to provide a first‐of‐its‐kind, comprehensive synthesis of miRNA‐SNP interactions across these four pathogenic pillars, thereby establishing a new molecular framework for HCC risk stratification, prognosis, and the design of targeted therapeutic strategies.

2. miRNA‐Mediated Regulation of Apoptosis in HCC and the Impact of SNPs
Apoptosis, a programmed cell death process, can function as a protective mechanism in cells, but it also paradoxically occurs alongside the progression and proliferation of tumor cells [26]. Recent research has revealed that miRNAs are involved in both the extrinsic and intrinsic pathways of apoptosis [34]. miRNAs are classified into two groups: pro‐apoptotic, which promote cell death, and anti‐apoptotic, which inhibit it. Pro‐apoptotic miRNAs can suppress cancer development, whereas anti‐apoptotic miRNAs can facilitate it [26]. Furthermore, transcripts related to autophagy and apoptosis can indirectly influence each other by competing for common miRNA binding sites [35]. According to recent research, the presence of SNPs within the miRNA binding sites of specific genes, including BCL-xl, H19, PIK3CD, and FOXO1, impacts the process of apoptosis [6, 19, 22, 30] (Table 1).

miR-146b is located at 10q24.32 [36, 37], is involved in the innate immune response to oncogenic transformation, and is implicated in cancer [38]. Specifically, miR‐146b‐5p promotes apoptosis [39], and its expression levels have been linked to HCC [40]. According to research by Yemei Song et al., miR‐146b‐3p is linked to poor survival in HCC patients. The study demonstrated that this miRNA significantly decreases the expression of H19 at the rs3741219 site [41]. H19 expression may induce cell apoptosis via the p53 protein [42]. Song et al. also found a significant association between the H19 SNP rs3741219 and overall survival in HCC [42].

miR-1539, consisting of 21 nucleotides, has been shown to induce angiogenesis and promote endothelial cell survival during ontogenesis. The functional rs3741219 SNP, located in the 3’‐region of H19, causes allelic downregulation of the H19 gene by creating a binding site for both miR‐146b‐3p and miR‐1539 [40]. The H19 gene is involved in a number of biological functions, including cell proliferation, apoptosis, differentiation, and autophagy. It also plays a role in the development of various diseases, including tumors [43]. The rs3741219 SNP in the H19 gene may increase target sites for miR‐1539. Song et al. suggested that miR‐1539 downregulation of H19 expression significantly contributes to HCC survival [44].

miR-7 is a powerful miRNA involved in numerous signaling pathways [45] and is encoded by three separate genes [46]. In mammals, miR‐7 primarily acts as a tumor suppressor and helping to regulate important cellular functions, including proliferation, differentiation, and apoptosis [45]. Fang et al. showed that mTOR and p70S6K, key downstream signals of PI3K, possess miR‐7 target sites in their 3’UTR [19, 47]. PI3K signaling is important for survival, apoptosis, proliferation, migration, and invasion in various cancer types [48]. Fang et al. reported that miR‐7 prevents tumorigenesis and metastasis in HCC by targeting a new site. Overexpression of PIK3CD and miR‐7 disrupts cancer cell migration through cell cycle arrest [19, 47].

miR-let7b is among the first microRNAs discovered and is located on chromosomes 9, 22, and X [49]. Most let‐7 regulatory proteins have been extensively studied in relation to development, proliferation, differentiation, and cancer. Previous research has shown that this microRNA can regulate several key oncogenes, including RAS, HMGA, c-Myc, and cyclin-D, leading to the inhibition of cancer development, maturation, and progression [50]. The 3’‐UTR of the Bcl-xl gene may contain a potential miRNA binding site for let‐7b [51]. The Bcl‐2 family of proteins is crucial for controlling apoptosis [52], and changes in the expression of BCL-2 family members have been recognized as vital in cancer development and progression [53]. The rs3208684 SNP disrupts the binding of miR‐let‐7b to the 3’UTR of Bcl-xl, leading to the increased expression of this gene, effectively suppressing or disrupting the binding of let‐7b to its target and subsequently improving sensitivity to 5‐FU through downregulation of Bcl-xl in HCC [52, 54].

miR-137 is placed on chromosome 1p21.3 [55] and impacts crucial cellular functions such as apoptosis, playing a major role as a tumor suppressor in various cancer types [56]. The 2015 study by Tan and Chao et al. showed that the rs17592236 SNP can decrease the risk of HCC by modifying the binding affinity of miR‐137 to the FOXO1 3’UTR [57]. The transcription factor FOXO1 regulates cell cycle, apoptosis, and oxidative stress [58], and FoxOs control the proteins that stimulate apoptosis [59]. Tan and Chao et al.’s research showed that miR‐137 can influence FOXO1 expression by interacting with the rs17592236 polymorphic site. This interaction influences the development of HCC and apoptosis through the PI3K‐Akt‐FOXO1 pathway [57].
While the accumulating data robustly link specific miRNA‐SNPs to altered apoptotic pathways in HCC, a critical assessment of the literature reveals several common methodological limitations. Many published association studies suffer from small sample sizes, lack of functional validation, or significant population heterogeneity, which limits the generalizability and mechanistic certainty of their findings. For instance, SNP–outcome associations often vary across different ethnic cohorts, necessitating large‐scale, multi‐ethnic meta‐analyses. Furthermore, the mechanistic interpretations derived solely from in silico predictions should be rigorously validated. Future research should prioritize robust experimental validation using techniques such as luciferase reporter assays to confirm the direct impact of the SNP on miRNA–mRNA binding affinity, alongside CRISPR/Cas9‐mediated gene editing and functional knockdown models in HCC cell lines to precisely define the downstream biological consequences.

3. MicroRNAs and Their SNPs in DNA Damage Response (DDR) Genes
The human genome constantly faces a barrage of endogenous and exogenous genotoxic insults, leading to numerous DNA damage events daily [60]. This constant assault leads to numerous DNA damage events every day. To protect against these threats and maintain the integrity of the genome, a complex DNA damage response (DDR) network is activated. microRNAs have emerged as crucial regulators of the DDR, influencing key cellular events such as cell cycle control and DNA repair mechanisms [61]. SNPs located within the microRNA target regions of genes involved in DNA repair can modulate miRNA–mRNA interactions. These alterations can affect the expression levels of these critical DNA repair genes, potentially impacting an individual’s capacity for DNA repair and consequently influencing disease susceptibility, including the risk of developing various cancers [62]. Several studies have indicated the relationship between SNPs found within miRNA binding sites of genes such as BTRC, DDB2, RAD51, and RAD52, and their effects on DNA damage repair pathways [63–66] (Table 1).

MicroRNA-920 (miR‐920) is a gene located on chromosome 12 [67] that has a predicted binding site within the 3’ untranslated region (3’UTR) of the BTRC gene. The BTRC gene produces β‐transducin repeat containing E3 ubiquitin protein ligase. This protein is a component of the Skp1‐Cullin‐F‐box (SCF) ubiquitin ligase complex that tags specific targets for proteasomal destruction, including Polo‐like kinase 1 (PLK1) [68]. PLK1 is a key regulator of cell cycle progression and has indirect involvement in DNA repair processes [64]. The SCFβTrCP complex ubiquitinates PLK1 during the G1 and S phases of the cell cycle, leading to its degradation [69]. A study by Chen et al. investigated the functional impact of the rs16405 polymorphism within the BTRC 3’UTR, which lies in the miR‐920 binding site [64]. Their findings indicated that the rs16405 alleles disrupted the binding affinity of miR‐920 to the BTRC mRNA. This impaired interaction led to an upregulation of BTRC expression and, through the modulation of WNT signaling, contributed to HCC development.

MicroRNA-197 (miR‐197), transcribed from chromosome 1p13.3, exhibits significant dysregulation across a spectrum of diseases, notably various cancers [70]. Functionally, miR‐197 can promote apoptosis by activating pro‐apoptotic proteins such as BAD, BAX, BID, and BIM, which are central to the apoptotic pathway and influence a cell’s response to DNA damage [71]. Qiu et al. [65] explored the interplay between the DDB2 SNP rs1050244 and the binding of miR‐133a and miR‐197. The DDB2 gene encodes the damage‐specific DNA binding protein 2, a crucial component of the nucleotide excision repair (NER) pathway, responsible for recognizing and removing bulky DNA lesions [65]. Their study suggested that the rs1050244 variant disrupted the interaction of both miR‐133a and miR‐197 with the DDB2 mRNA, leading to an upregulation of DDB2 expression and a potentially reduced risk of HCC [65]. This observation highlights a scenario where a SNP in a miRNA target site can have a protective effect against cancer development by enhancing the expression of a key DNA repair gene. miR‐133a, located on chromosome 11q13.3, is a tumor suppressor miRNA involved in tumor initiation and progression and positively regulates the p53/p21 signaling pathway, further underscoring the complex interplay between miRNAs and DNA repair [72, 73]. Mutations in the DDB2 gene are known to impair DDB2–DNA or DDB2–DDB1 complex formation, consequently compromising NER activity [74].

MicroRNA-129-3p (miR‐129‐3p), located on chromosome 11p11.2 [47], is often dysregulated in many cancer types [75]. It has been linked to the regulation of DNA damage and intracellular calcium signaling pathways [76]. The rs12593359 polymorphism is in the 3’UTR of the RAD51 gene, within a predicted binding site for miR‐129 [66]. The RAD51 gene is a central player in maintaining DNA fidelity by facilitating homologous recombination repair of DNA double‐strand breaks (DSBs) [77]. A more recent study by Qiu et al. [66] hypothesized that the rs12593359 SNP in the RAD51 3’UTR could interfere with miR‐129‐3p binding [77]. Their findings suggested that this disruption could lead to increased RAD51 expression, potentially hindering the suppression of HCC invasion and metastasis normally mediated by miR‐129‐3p. This illustrates how a SNP can modulate miRNA‐mediated regulation of a critical DNA repair gene, influencing cancer progression.

The let-7 family of microRNAs participates in the regulation of diverse cell signaling pathways [78], and is generally considered to function as tumor suppressors. Li et al. [63] investigated the rs7963551 polymorphism located within a let-7 target site in the 3’UTR of the RAD52 gene. The RAD52 gene encodes a protein involved in DNA repair, particularly in single‐strand annealing and potentially in alternative double‐strand break repair pathways. Rad52 can establish physical connections with replication protein A (RPA), a single‐stranded DNA binding complex It has also been implicated in RNA bridging to facilitate DNA break synapsis and ligation, potentially utilizing RNA as a template for reverse transcription‐dependent DNA repair [79]. Li et al. concluded that the rs7963551 allele reduced the binding affinity of let-7 miRNAs to the RAD52 3’UTR, resulting in increased RAD52 gene expression. Notably, this SNP was markedly linked with an increased risk of hepatitis B virus (HBV)‐related HCC [63].

4. Modulation of Immune Homeostasis in HCC by MicroRNAs and Related SNPs
The interaction between the host immune system and cancer development is a critical determinant of disease progression. While efficient anti‐tumor immunity can effectively control and eliminate malignant cells, tumor‐associated inflammation can paradoxically foster cancer growth, invasion, and metastasis [80]. MicroRNAs are determinant in modulating host immune homeostasis [81]. Certain miRNAs can facilitate immune evasion by cancer cells through mechanisms such as reducing cancer cell immunogenicity and dampening anti‐tumor immune responses [82]. The significant role of miRNAs in regulating gene expression, particularly of inflammatory mediators, is well‐established. Consequently, SNPs within the 3’UTRs of immune‐related genes can disrupt miRNA binding, altering their regulatory efficacy and potentially influencing cancer susceptibility [83]. Previous investigations have implicated SNPs within the miRNA binding sites of genes such as IFNAR1, TLR4, PDCD1 (PD-1), and IL1A in the context of HCC [84–87] (Table 2).

MicroRNA-1231 (miR‐1231) exhibits downregulation in several cancer types [88]. Emerging evidence suggests an association between miR‐1231 and liver cancer risk, with a demonstrated role in inhibiting HBV replication by targeting its core mRNA [89]. A 2012 study by Zhou et al. identified the rs17875871 polymorphism within a putative miR‐1231 target sequence in the 3’UTR of the IFNAR1 gene [86]. Type I interferons (IFNs), including IFNα and IFNβ, exert anti‐tumor effects primarily indirectly by activating immune cells to mediate the elimination of cancerous cells [90]. Proteins induced by IFN signaling possess antiviral, antiproliferative, and immunomodulatory properties, acting through the interferon alpha/beta receptor (IFNAR) complex [91]. In their study, Zhou et al. proposed that the rs17875871 SNP resides within a functional binding site for miR‐1231. The presence of this SNP could potentially alter the binding affinity of miR‐1231 to the IFNAR1 mRNA transcript, leading to dysregulation of IFNAR1 expression. Negative regulation of IFNAR1 by enhanced miR‐1231 binding, influenced by the SNP, could potentially impair downstream IFN signaling and increase susceptibility to chronic HBV infection, a major etiological factor for HCC development [86].

MicroRNA-34a (miR‐34a) is a well‐established tumor suppressor miRNA and is expressed in immune cells, where it regulates their function, development, and survival [92]. This miRNA exerts significant immunomodulatory effects by targeting over 30 genes involved in diverse cellular pathways [92]. A 2014 study by Jiang et al. studied the effects of the rs1057317 polymorphism on miR‐34a binding to TLR4 mRNA and its association with HCC risk [84]. Toll‐like receptor 4 (TLR4) is a very important pattern recognition receptor of the innate immune system, and its hyperactivation can trigger the release of various pro‐inflammatory cytokines in the pathogenesis of several diseases [92]. TLRs, in general, are essential for the activation of both innate and adaptive immune responses [93]. The findings from Jiang et al.’s research indicated that the rs1057317 SNP, within the miR‐34a binding site in the TLR4 gene, could significantly influence the progression of hepatocellular carcinoma. The SNP‐mediated alteration in miR‐34a binding and subsequent TLR4 expression could disrupt the delicate balance of immune signaling, contributing to chronic inflammation and promoting HCC development.

MicroRNA-4717 (miR‐4717) is engaged in the pathogenesis of chronic HBV infection, as one of the most important causes of HCC [94]. Functionally, miR‐4717 can downregulate the production of PD‐1 mRNA by directly binding to its 3’UTR [95], subsequently leading to increased production of IFN‐γ and TNF‐α [96] . A 2015 study by Zhang et al. predicted that the rs10204525 polymorphism influences the binding of miR‐4717 to the PDCD1 3’UTR and consequently affects PD‐1 expression [87]. Their research suggested that miR‐4717 modulates PD‐1 expression by interacting with the 3’UTR of PDCD1 mRNA, resulting in the modification of immune regulation and influencing vulnerability to chronic HBV infection and HCC [96]. The SNP‐mediated alteration in this interaction could disrupt the fine‐tuning of T cell exhaustion and inflammatory responses in the liver [87].

MicroRNA-122 (miR‐122) is an abundant miRNA found primarily in the liver. It plays a significant role in various liver diseases, including infections caused by hepatitis C and hepatitis B viruses. [97]. Accordingly, miR‐122 can modulate host immune responses by affecting the production of inflammatory cytokines [98]. A 2009 study by Gao et al. identified the rs3783553 polymorphism in the 3’UTR of the IL1A gene, demonstrating its impact on miR‐122 binding and the subsequent regulation of interleukin 1 alpha (IL‐1α), and its association with HCC risk [85]. IL‐1α is a pleiotropic cytokine with both pro‐ and anti‐tumorigenic roles in different cancer contexts and has been shown to be upregulated in several tumor types [99]. Gao et al.’s research indicated that the rs3783553 SNP disrupts a binding site for miR‐122, leading to increased transcription of IL1A. Consequently, this polymorphism in IL1A may play a role in susceptibility to HCC by altering the local inflammatory milieu within the liver [85].

5. Modulation of Tumor Migration, Invasion, and Angiogenesis by MicroRNAs and SNPs
Angiogenesis, the natural process of forming new blood vessels, is controlled by a delicate balance between factors that promote it and those that inhibit it. In diseases like cancer, this process becomes dysregulated, with an increase in pro‐angiogenic factors that are essential for a tumor to grow, invade surrounding tissues, and spread [100, 101]. Cancer cells exhibit disruptions in the normal regulatory mechanisms governing cell migration, facilitating local invasion and the establishment of distant metastases [102]. By controlling the expression of genes linked to angiogenesis, endothelial cell proliferation, migration, and tube formation, miRNAs are key regulators of these processes [103]. They can influence both pro‐ and anti‐angiogenic signaling pathways [104]. Several studies highlighted the critical involvement of miRNA deregulation in promoting tumor cell invasion, migration, and subsequent angiogenesis [105]. For instance, recent studies have identified miRNA binding site polymorphisms in genes implicated in migration, invasion, and angiogenesis, such as RASA1, COL1A2, CA9, CD147, VHL, and RYR3, which can influence susceptibility to hepatocellular carcinoma (HCC) [101, 106–110] (Table 3).

MicroRNA-130a (miR‐130a) and miR-19b have been linked to the development of several diseases, including HCC [111]. MiR‐130a has been shown to dampen the NF‐κB signaling pathway and its target gene VEGFA by reducing TNF‐α expression [112], contributing to accelerated invasion and migration in HCC [111]. Similarly, the miR‐19 family modulates angiogenic activity and the process of angiogenesis in endothelial cells, promoting proliferation or migration in various cancers [113, 114]. In a 2011 study by Ding et al., a screen of SNPs in the microRNA‐binding sites of 50 genes related to HCC identified six significant SNPs, including rs10474257 located within the miR‐19b and miR‐130a binding sites of RASA1 [115]. RASA1 encodes RAS P21 protein activator 1, whose levels are associated with liver cancer cells [116], and which regulates cell migration and angiogenesis. Defects in RASA1 function can lead to irregular angiogenic remodeling of the capillary network, potentially contributing to HCC development [115].

MicroRNA let-7g
plays crucial roles in regulating diverse biological processes [117] and is frequently dysregulated in various cancer types [117]. Research has demonstrated that let‐7 g influences cell growth, migration, and invasion when studied in HCC cell lines [115], as well as angiogenesis, vascular remodeling, and EMT [118]. Research by Zhu et al. demonstrated that the rs3917 polymorphism in the 3’UTR of COL1A2 acts as a direct target site for let-7 g [117]. COL1A2 is responsible for encoding the alpha 2 chain of type I collagen, and its elevated expression impacts the invasion, migration, and growth of cancer cell lines [119]. Collagen I can promote the malignant properties of tumors by stimulating JNK1 signaling, which, through the upregulation of N‐cadherin levels, enhances invasion and metastasis [117]. Zhu et al.’s findings indicated that the rs3917 SNP in COL1A2 could affect HCC risk, likely through modulation of let-7g binding and subsequent COL1A2 expression [117].

MicroRNA-34a has been shown to influence almost all stages of cancer progression [120], including angiogenesis, cell migration, and tumorigenesis [120, 121]. Previous studies have highlighted the role of the miR‐34a/NF‐κB/HMGB1 axis in angiogenesis in primary liver cancer [122], and upregulation of miR‐34a‐5p expression has been shown to decrease the invasive capacity of HCC cells [123]. A 2017 study by Hua et al. revealed that the rs1048638 polymorphism influences HCC risk and development by altering the expression of Carbonic Anhydrase IX (CA9) targeted by miR‐34a [107]. CA9 prevents hypoxia‐induced upregulation of genes involved in angiogenesis, and knockdown of the DDX11‐AS1 axis, which is regulated by CA9, can significantly suppress cell proliferation, migration, and invasion [124]. Hua et al.’s research suggested that the CA9 rs1048638 SNP disrupts the binding of miR‐34a to the CA9 3’UTR, thereby influencing HCC risk [107].
A microRNA-3976 binding site SNP (rs6757) has been shown to significantly impact the differentiation, proliferation, metastasis, prognosis, and invasion of hepatocellular carcinoma by changing the quality of miR‐3976 binding to the CD147 3’UTR [108]. CD147 encodes a glycoprotein that is significantly overexpressed on the surface of most malignant cancer cell types [125], and participates in tumor cell invasion, metastasis, and angiogenesis through various mechanisms. It is overexpressed in human cancers such as CNS, head and neck, breast, and gastrointestinal carcinomas [126, 127]. A study by Guo et al. demonstrated that the rs6757 SNP affects the efficiency of miR‐3976 binding to the CD147 3’UTR and affects HCC risk in the South Chinese population [108].

MicroRNA-367 is influential in a variety of disease states, including cancer [128]. In HCC, miR‐367 acts as an oncogenic factor by activating the PI3K/AKT signaling pathway. Research has shown that when miR‐367 is inhibited, it can suppress cell proliferation, migration, and invasion. Peng et al. found that the rs1044129 polymorphism located in the 3’UTR of RYR3 could alter the binding affinity between this SNP and miR‐367, which was linked to survival outcomes in HCC [109]. RYR3 encodes the ryanodine receptor 3, a calcium channel that regulates cytosolic calcium concentrations [109]. Aberrant Ca2+ signaling is a hallmark of numerous cancers, and patients carrying RYR mutations have shown significantly higher tumor mutational burden (TMB) across most cancer types compared to those without these mutations [129]. Peng et al.’s work demonstrated that the rs1044129 SNP in the microRNA binding region of RYR3 can serve as a potential biomarker for predicting outcomes in HCC [109].

MicroRNA-300 and miR-381 have been shown to regulate a variety of genes in cancer, and their expression levels often correlate with tumor stage and survival rates [130]. MiR‐300 is upregulated in HCC tissues [131], while miR‐381 is frequently downregulated in various cancers [132], and modulates multiple cellular functions including growth, cell cycle progression, migration, and invasion [133]. In a 2021 study by Chen et al., the rs1642742 polymorphism, located in the 3’UTR of the VHL gene, was shown to reduce VHL expression by enhancing its interaction with miR‐300 and miR‐381 [110]. The VHL gene produces the Von Hippel–Lindau tumor suppressor protein, and its mutations lead to the stabilization of HIF1α and HIF2α, which subsequently activate various oncogenic signaling pathways. Consequently, individuals with VHL mutations have an increased risk of developing various neoplasms [134]. Chen et al.’s study demonstrated that the VHL rs1642742 SNP plays a role in susceptibility to HCC and is associated with tumor growth and metastasis through its interaction with miR‐300 and miR‐381 [110].

6. MicroRNA Biogenesis and the Influence of SNPs
The process of miRNA biogenesis, which encompasses miRNA processing and maturation, exerts a significant influence on various aspects of cancer development [135, 136]. This multi‐step process is managed by a cohort of proteins collectively known as proteins involved in miRNA biogenesis [137]. Aberrant miRNA expression in malignancy is evidenced by its association with cancer‐related changes and by alterations in the expression or function of proteins crucial for miRNA production [138]. Prior research has demonstrated that SNPs within miRNA biogenesis genes, such as RAN and DICER1, can modulate the binding affinity of miRNAs to the 3’UTR of these genes, thereby affecting susceptibility to HCC (Table 2).

MicroRNA-199-3p (miR‐199‐3p) is involved in the initiation and progression of various cancers and modulates multiple signaling pathways [139]. The exportin 5 (XPO5)/RAN‐GTP complex is essential for the nuclear export of precursor miRNAs (pre‐miRNAs), and alterations in the expression or function of its components have been linked to cancer risk [136]. A 2013 study by Liu et al. indicated that the SNP rs3803012 could alter the binding capacity of miRNAs to the 3’UTR of the RAN gene [140]. The RAN gene encodes a small GTPase protein crucial for nuclear transport, including the translocation of pre‐miRNAs. Mutations in RAN can disrupt normal miRNA processing and expression, thereby contributing to the development and progression of tumor cells [141]. Specifically, the RAN rs3803012 variant was associated with the elevated risk of persistent HBV infection and subsequent HBV‐related HCC [140], suggesting a link between impaired pre‐miRNA transport and hepatocarcinogenesis.

MicroRNA-574-3p (miR‐574‐3p) has been identified as a crucial miRNA mediating various cellular processes [142]. Liu et al. also demonstrated that the DICER1 SNP rs1057035 might alter the affinity of miR‐574‐3p to this gene and contribute to the risk of HBV‐related HCC [140]. DICER1 encodes the DICER enzyme, a type III ribonuclease that plays a pivotal role in cleaving precursor miRNAs (pre‐miRNAs) into mature miRNAs. Consequently, DICER function is critical in the hepatocarcinogenesis process [141]. DICER is recognized as an essential component in the biogenesis of both miRNAs and small interfering RNAs (siRNAs) [143]. While DICER upregulation has been reported in several cancers [144], alterations in its activity due to genetic polymorphisms can disrupt the normal repertoire of mature miRNAs. In their study, Liu et al. hypothesized and provided evidence suggesting that the DICER1 rs1057035 SNP is associated with an increased risk of HCC [140], potentially by affecting the levels or processing efficiency of specific miRNAs, including miR‐574‐3p.

7. MicroRNAs and the Impact of Single Nucleotide Polymorphisms on Tumor Growth and Progression
MicroRNAs exert significant influence on the intracellular signaling pathways activated by growth factors, which are critical regulators of tumor advancement, playing remarkable roles in this process and ultimately in tumor development [145]. Aberrations in the quantity and function of miRNAs are consistently associated with cancer initiation, progression, and metastasis [146]. MiRNAs can control not only tumor cells themselves but also the homeostasis of the tumor microenvironment, a critical factor underpinning tumor growth and dissemination [147]. During liver carcinogenesis, miRNAs are implicated in all facets of tumor cell behavior, including proliferation, survival, and migration [148]. Competitive endogenous RNAs (ceRNAs), which carry binding sites for microRNAs and compete with target mRNAs for miRNA binding, often underlie processes associated with cell differentiation and development [149]. Previous studies have shown that SNPs within miRNA binding sites of genes involved in tumor growth and progression, such as ERBB4, EGFR, STAT3, ERBB2, JAK1, SOX6, SGSM3, and SET8, can influence the risk and outcome of HCC [150–157] (Table 3).

MicroRNA let-7c
is a compelling candidate for its involvement in the development of various diseases, particularly cancer [158]. It participates in organogenesis and fundamental cellular activities [159]. The let-7 family of miRNAs generally functions as tumor suppressors in cancer cells, inhibiting metastasis and tumor growth [158]. Overexpression of let-7c, which can inhibit the Ras/NFκB signaling pathway, has been shown to reduce malignant transformation and cancer stem cell development [158]. A study by Yu et al. indicated that the rs6147150 polymorphism is located within the seed region of miR‐let-7c, potentially targeting a sequence in the 3’UTR of ERBB4 [150]. ERBB4, upon binding with epidermal growth factor (EGF), can activate downstream genes in the nucleus, thereby promoting cell division and proliferation [160]. According to Yu et al.’s research, the rs6147150 SNP, situated in the miR‐let-7c seed region, may contribute to HCC development in the Chinese population by affecting the post‐transcriptional regulation of ERBB4 [150].

MicroRNA-3196 (miR‐3196) is mainly involved in tumor development [161] and is significantly downregulated in HCC tissues, where its reduction promotes HCC cell growth [162]. In a study by Zhang et al., miR‐related SNPs in EGFR were investigated, revealing that the rs884225 polymorphism enhances the binding efficiency of miR‐3196 to the 3’UTR of EGFR, reducing epidermal growth factor receptor (EGFR) expression levels and suppressing cell proliferation [154]. The EGFR is a key component of a complex signaling pathway that regulates cancer cell growth, adhesion, metastasis, and survival [162]. MicroRNAs, including miR‐3196, play critical roles in modulating EGFR signaling [163]. EGFR can also facilitate EMT, promoting liver cancer progression [164]. Zhang et al.’s study suggested that rs884225 probably enhances miR‐3196 binding to EGFR, leading to decreased EGFR levels and consequently inhibiting cell proliferation in HCC [154].

MicroRNA-214 (miR‐214) is linked to the development and proliferation of several tumors [165, 166]. This miRNA is located within the dynamin 3 gene at chromosome 19q13.3 [165] and has been found to exhibit tumor‐suppressing activity in HCC [167]. Fan et al. showed that miR‐214 might regulate the SNP rs111904020, leading to an increase in STAT3 levels [152]. Signal transducer and activator of transcription 3 (STAT3) is crucial in the pathogenesis of liver diseases [168]. STAT3 inhibition can promote apoptosis in cancer cells and prevent tumor growth [168], but it may also enhance the aggressiveness of HCC tumors by increasing the expression of EMT‐related proteins [169]. Fan et al.’s study indicated that the rs111904020 SNP in the STAT3 3’UTR acts as a factor that promotes HCC development by interfering with the regulatory role of miR‐214 on STAT3 expression [152].

MicroRNA-221-5p (miR‐221‐5p) has been extensively investigated for its role in cancer progression and its potential as a valuable biomarker in cancer research [170]. It acts as a regulator of chronic liver injury and inflammation‐associated processes in liver cells [170]. MiR‐221 may promote angiogenesis, and its overexpression can enhance cell growth and invasion [171, 172]. A study by Fan et al. proposed that miR‐221‐5p might regulate the rs113054794 SNP, leading to increased levels of ErbB2 in HCC patients [153]. Nuclear ErbB2 positivity has been strongly associated with histological indicators of hepatocellular injury [173]. ErbB2 has also been shown to inhibit the growth of HBx‐associated HCC cells [174]. Fan et al.’s study demonstrated that the rs113054794 SNP in the 3’UTR of ERBB2 was associated with poor differentiation and larger tumor size in HCC by impeding the regulatory function of miR‐221‐5p on ErbB2 expression [153].

MicroRNA-502 (miR‐502) expression levels have been associated with various HCC cell lines and patient liver tissues [175]. Overexpression of miR‐502 significantly suppresses HCC proliferation, tumor growth, invasion, and metastasis [175]. A study by Guo et al. showed that the polymorphism rs16917496 is located in the miR‐502 seed region within the 3’UTR of SET8 in Chinese patients with hepatocellular carcinoma [151]. SET Domain Containing 8 (SET8) expression can be linked to the poor survival in HCC patients [176], and silencing of SET8 can reduce the proliferation, migration, and invasion of tumor cells in HCC [177]. Guo et al.’s data indicate that SET8 affects HCC outcomes by altering its expression, which depends on its binding affinity with miR‐502 [151].

MicroRNA-431-5p (miR‐431‐5p) can inhibit the development and progression of some cancers, including HCC [178]. Earlier research showed that it is notably reduced in HCC cells, and its upregulation restricted the growth of HCC cells [179]. A study by Yu et al. showed that the rs112395617 SNP in JAK1 disrupted the binding site for miR‐431‐5p [155]. Mutations in JAK1 have been observed in HCC [180]. Janus kinase 1 (JAK1) enhances growth factor and cytokine‐activated STAT signaling pathways and influences cellular growth, immune responses, and differentiation [155]. Yu et al. suggested that the rs112395617 polymorphism could play a role in HCC susceptibility by affecting JAK1 transcriptional activity through interfering with its interaction with miR‐431‐5p [155].

MicroRNA-1269 (miR‐1269) is crucial in controlling the progression of HCC [156]. Downregulation of miR‐1269 in liver cancer tissues has been associated with reduced proliferation of liver cancer cells [181]. MiR‐1269 holds diagnostic value in HCC and is also linked to a negative prognosis for the cancer [182]. Xiong et al. suggested that overexpression of miR‐1269 with a stronger binding ability to SOX6 promoted cell proliferation in HCC [156]. SRY‐Box Transcription Factor 6 (SOX6) is expressed at low levels and acts as a promising prognostic biomarker for HCC [183], and its overexpression results in decreased cell growth and proliferation [184]. Results from Xiong et al. indicate that the SNP rs73239138 in the miR‐1269 binding site acts as a protective element that prevents binding to the 3’UTR of SOX6, therefore inhibiting tumor growth in patients with HCC [156].
The functional role of microRNA‐151‐5p (miR‐151‐5p) is influenced by the genetic characteristics of cancer tissue, with its expression levels ranging from downregulated to upregulated depending on the cancer type [185]. Expression of miR‐151‐5p is increased in liver cancer cells and is notably linked to their growth and spread [185]. A study showed that the rs56228771 SNP was located within the 3’UTR of SGSM3. Small G protein signaling modulator 3 (SGSM3) participates in the signaling pathway of small G protein‐coupled receptors, influencing susceptibility to hepatocellular carcinoma. Wang et al. indicated that rs56228771 interfered with a binding site for miR‐151‐5p, leading to an increase in SGSM3 levels, thus contributing to HCC risk [186].

8. MicroRNAs Effects on Drug Metabolism in HCC

MicroRNA-128-3p (miR‐128‐3p) is a remarkable factor in the development and pathogenesis of some cancers [187]. MiR‐128‐3p has potential as a noninvasive biomarker for predicting the overall survival of individuals with HCC [188]. Studies have shown an inverse correlation between the expression levels of miR‐128‐3p and Cytochrome P450 2C9 (CYP2C9) in HCC tumor tissues [189]. CYP2C9, a key enzyme predominantly expressed in the liver, plays a significant role in the metabolism of various therapeutic agents used in cancer treatment, as well as exogenous carcinogens [190]. Approximately 18% of the cytochrome P450 (CYP) proteins found in liver microsomes consist of CYP2C9 [191]. Alterations in the expression of CYP genes, such as CYP2C9, mediated by hsa‐miR‐128‐3p, can significantly influence the efficacy of xenobiotic detoxification processes. Furthermore, these changes can affect the generation of signaling molecules that modulate downstream signaling pathways, potentially exerting a paradoxical effect on HCC carcinogenesis (Table 3).

9. Conclusion
This review highlights the complex involvement of miRNAs and their associated SNPs in the pathogenesis, prognosis, and therapeutic response of HCC. The miRNA binding site polymorphisms are critical master modulators linking genetic variations to the dysregulation of key HCC pathogenic pathways (apoptosis, DNA repair, immune evasion, and metastasis). The interaction between miRNAs and SNPs within their binding sites increases the complexity of HCC. These genetic variations can modulate miRNA‐mRNA interactions, altering the expression of key genes and ultimately impacting HCC susceptibility, development, and treatment outcomes. The potential of miRNAs as noninvasive biomarkers could be utilized for timely diagnosis, prognosis, and monitoring therapeutic efficacy, which could improve patient management and outcomes. Future research, including large‐scale, multi‐ethnic cohort studies, functional validation using CRISPR‐Cas9 and in vivo models, and the development of SNP‐tailored combination therapies is crucial to fully understand the regulatory networks modulated by miRNAs and SNPs in HCC.

Nomenclature
HCCHepatocellular carcinoma
miRNAsMicroRNAs
SNPsSingle nucleotide polymorphisms
ICCIntrahepatic cholangiocarcinoma
HBVHepatitis B virus
HCVHepatitis C virus
IL‐6Interleukin‐6
TNF‐α
Tumor necrosis factor‐alpha
VEGFVascular endothelial growth factor
NAFLDNonalcoholic fatty liver disease
CTComputed tomography
MRIMagnetic resonance imaging
AFPAlpha‐fetoprotein
AFP‐L3Alpha‐fetoprotein‐L3
DCPDes‐γ‐carboxy prothrombin
GPC3Glypican‐3
GP73Golgi protein‐73
cfDNACell‐free DNA
CTCsCirculating tumor cells
EVsExtracellular vehicles
3’UTR3’ Untranslated region
BTRC
β‐Transducin repeat containing E3 ubiquitin protein ligase
SCFSkp1‐Cullin‐F‐box
PLK1Polo‐like kinase 1
DDB2Damage‐specific DNA binding protein 2
NERNucleotide excision repair
RPAReplication protein A
IFNARInterferon alpha/beta receptor
TLR4Toll‐like receptor 4
PD‐1Programmed cell death protein 1
IL‐1α
Interleukin 1 alpha
RASA1RAS P21 protein activator 1
CA9Carbonic anhydrase IX
CNSCentral nervous system

Consent
Not applicable.

Conflicts of Interest
The authors declare no conflicts of interest.

Author Contributions
S.A.R. mainly participated in literature search, study design, writing, and critical revision. F.K.M. and M.D. mainly participated in literature search, writing, and critical revision. S.M. and S.A. mainly participated in literature search, writing, critical revision, study design, and supervision. All authors read and approved the final manuscript.

Funding
No funding was received for this manuscript.