“Hallmarks of Cancer" describes a set of functional abilities that human cells acquire upon their transition from normal growth stage to neoplastic growth states, focusing on the abilities that are crucial for the growth of malignant tumors (Hanahan, 2022). The hallmarks include abilities to maintain proliferative signaling, avoid immune attack, avoid cell death, enable replicative immortality, induce/access vasculature, activate invasion and metastasis and reprogram cellular metabolism (Hanahan, 2022, Hanahan and Weinberg, 2000). However, these hallmark traits alone cannot address the complexities of cancer pathogenesis, and therefore, two additional hallmarks were added which were genome instability and tumor-promoting inflammation (Ramzy, 2022, Elemam, 2024). Moreover, the investigation and recognition of the tumor microenvironment (TME) led to the discovery of new hallmarks such as unlocking phenotypic plasticity of cancer cell, non-mutational epigenetic reprogramming, polymorphic microbiomes and senescent cells (Hanahan, 2022).

Almost all malignancies exhibit these aforementioned hallmarks (Abdel-Latif and Youness, 2020, Fahmy, 2022, Elemam, 2023), in this review we will focus on liver cancer, particularly hepatocellular carcinoma (HCC). The incidence of liver cancer is rising globally and it continues to be a concern for global health (Samant et al., 2021). By 2025, liver cancer is expected to impact more than 1 million people annually (Llovet, 2016, Villanueva). The most prevalent type of liver cancer is HCC, which accounts for about 90% of cases (Rahmoon, 2017, Youness, 2016, Youness, 2016). HCC is the sixth most prevalent neoplasm and the third most common cause of cancer-related death globally, claiming the lives of at least 700,000 individuals worldwide each year (Ito and Nguyen, 2023). Patients with HCC have a dismal survival rate due to the disease's high capacity for metastasis and high rate of recurrence (Wang et al., 2018, Aishanjiang, 2021). HCC is treated typically with immunotherapy, liver transplantation, liver resection, and radiotherapy (Chakraborty and Sarkar, 2022, Shaalan, 2018, Abaza, 2023, Youness, 2023). Patients with HCC may live substantially longer if they receive an early diagnosis (El-Aziz, 2023). However, HCC is identified frequently at the late stages when there is no longer a chance for curative therapies (Gutiérrez-Cuevas, 2022). Research into the molecular mechanisms underlying HCC carcinogenesis and associated hallmarks is crucial to develop novel treatments and provide clinically useful diagnostics (Wang et al., 2018, Aishanjiang, 2021, Louis et al., 2022).

Non-coding RNAs (ncRNAs) are small RNA molecule that do not encode any protein, critical for cell development and growth regulation (ZeinElAbdeen et al., 2022, Dawoud, 2023, Youness and Gad, 2019, Abdel-Latif, 2022, Aboelenein, 2017, El Din, 2020, Soliman, 2023). There is a great variety of ncRNAs with varying structural and functional properties (Youness and Gad, 2019, ElKhouly et al., 2020, Selem, 2023, Abdallah, 2022, Awad, 2021). The two primary types of ncRNAs based on the length of the transcript are short ncRNAs (200 nucleotides) and long ncRNAs (lncRNAs, >200 nucleotides). Short ncRNAs, which include siRNA, miRNA, and piwiRNA, primarily regulate gene expression by interacting and binding to specific mRNAs, leading to their degradation or inhibiting their translation (Soliman, 2023, Selem, 2023, Youness, 2021, Nafea, 2021, Mekky, 2019, El Kilany, 2021). While lncRNAs, which include linear and circular RNAs, can bind and interact with DNAs, RNAs, or proteins and regulate gene expression at the transcriptional and post-transcriptional levels (Abdel-Latif, 2022, Selem et al., 2021, Mekky, 2023, Youness, 2018, Nafea, 2023, Youness, 2024). According to studies, ncRNAs not only regulate basic biological processes including growth, development, and organ function, but they also seem to be crucial for a complete spectrum of human disease, especially cancer, including HCC (Wang et al., 2018, Slack and Chinnaiyan, 2019, El-Daly, 2023).

Notably, the recently discovered subclass of ncRNAs known as circular RNAs (circRNAs) is found in a wide variety of species (Abaza, 2023, Dawoud, 2023, El-Daly, 2023). CircRNAs form a covalently closed continuous loop structure without 5’ caps or 3’ polyadenylated tails rendering them more stable than canonical linear RNAs (Villanueva). The biogenesis of circRNAs via non-sequential back-splicing of pre-mRNAs, in which a downstream splice donor is linked to an upstream splice acceptor, and their development is primarily mediated by three different mechanisms: intron pairing-driven circularization, RNA-binding proteins (RBPs)-mediated circularization and lariat-driven circularization. They can regulate gene expression through multiple mechanisms including; miRNA sponging, binding and sponging RBP, competing with linear splicing of pre-mRNAs, protein scaffolding, translation regulation through binding to PABP and eIF4G and others (Samant et al., 2021, Rahmoon, 2017). According to recent studies, circRNAs are important for the development and progression of HCC and other types of cancer. Some circRNAs have been found to have either oncogenic or tumor suppressive impacts on several HCC hallmarks, including deregulating cellular energy, self-sufficiency in growth signals, insensitivity to anti-growth signals, evading cell death, unlimited replicative potential, sustained angiogenesis, tissue invasion and metastasis (Elemam, 2023).

In this review, we briefly discuss the characteristics and functions of circRNAs with an emphasis on their biological significance in the process of HCC tumors development, initiation, and progression. The authors also shed the light onto the mechanistic role of circRNAs in tunning HCC hallmarks, including the new hallmarks of phenotypic plasticity and stemness of cancer cells, non-mutational epigenetic reprogramming, polymorphic microbiomes and senescent cells.

In the early 1970 s, circRNAs were discovered and it was thought that they were byproducts or spliced intermediates of pre-mRNA with no substantial biological functions (Hsu and Coca-Prados, 1979). However, with the development of high-throughput sequencing technology and computational analysis, several circRNAs with differential expression have been discovered in viruses, insects, plants, and mammals, shedding light on their diverse roles (Thölken, 2019, Zhang, 2020, Espindola, 2021, Jiang, 2021, Rao, 2023).

CircRNAs typically originate from fragments of linear precursor messenger RNAs or other RNA species (Rao, 2023, Liu and Chen, 2022). CircRNAs, unlike canonical linear RNAs, are more stable than linear RNAs due to their lack of 5’ caps and 3’ polyadenylated tails. They form covalently circular structures that protect them from degradation by RNA exonucleases or RNase R, enabling them to perform their paramount regulatory functions (Wang et al., 2018, Li, 2023).

CircRNAs can be classified into three types based on their structural composition into: i) Exonic circRNA (ecircRNA) originated from exons and account for the majority of known circRNAs; they are mostly located in the cytoplasm; ii) Exon-intron circRNA (EIciRNA) refers to circRNAs that have introns between their exons and are mostly found in the nucleus; and iii) Circular intronic RNA (ciRNA) refers to circRNAs that are made up entirely of introns and are mainly found in the nucleus as previously reviewed in (Wang et al., 2018, Li, 2023, Zhao et al., 2019).

Understanding circRNAs biogenesis is critical step for investigating various biological and pathological functions of circRNAs. In eukaryotes, most circRNAs share a lot of structural similarities with mRNA, for example, both originates from pre-mRNA. However, they differ in their splicing processes. If pre-mRNA undergoes canonical splicing, it gives rise to mRNA (Fig. 1a). Conversely, when pre-mRNA undergoes back-splicing, it results in the formation of a circRNA (Li, 2023).

CircRNAs are generated via back-splicing processes involving exons from protein-coding genes, introns, intergenic regions, antisense, or untranslated regions (Memczak, 2013, Granados-Riveron and Aquino-Jarquin, 2016, Zhang, 2013). The back-splicing process involves exon circularization between a 5′ splice site (donor) and an upstream 3′ splice site (acceptor), producing circRNAs (Wang et al., 2018). Nowadays, 3 circRNA biogenetic pathways have been widely accepted as described in Fig. 1: (1) intron pairing-driven cyclization, (2) RNA binding protein (RBP)-mediated cyclization, and (3) lariat-driven cyclization.

A circular structure is created by the base-pairing interaction between reverse complementary sequences, like the Arthrobacter luteus (ALU) repeats, found across exon-flanking introns. After intron pairing, pre-mRNAs undergo back-splicing, resulting in the formation of either eIciRNA, where introns remain without removal, or ecircRNA, where introns are removed from the structure (Fig. 1b) (Wang et al., 2018).

RNA-binding proteins (RBPs) play an indisputable role as trans-factors involved in the orchestration of circRNA biogenesis (Fig. 1c). Based on the presence of double-stranded RNA-binding domains (dsRBDs), we can classify RBPs into two categories: (1) RBPs with dsRBDs mediating circRNA biogenesis which are further sub-classified based on their effects on circRNA biogenesis as illustrated below and (2) RBPs lacking dsRBDs mediating circRNA biogenesis.

These RBPs could suppress circRNAs formation by influencing the RNA pairing stability. Adenosine deaminase 1 acting on RNA (ADAR), is a strong suppressor of circRNAs formation. ADAR binds to complementary RNA pairs, catalyzing adenosine-to-inosine (A-to-I) RNA editing, therefore reducing RNA stability, and ultimately hindering circRNAs formation (Nishikura, 2010, Shi, 2017). DExH-box helicase 9 (DHX9), a nuclear RNA helicase contains both a dsRBD and an RNA helicase domain. DHX9's role is to target Alu sequences and uncouple complementary RNA pairs, thereby preventing circRNA production (Aktaş, 2017, He and Zhu, 2023).

In contrast, some RBPs such as the immune factors nuclear factor 90/110 (NF90/NF110), also contain two dsRBDs that function as pivotal regulators in the circRNA biogenesis. In the nucleus, NF90/NF110 exhibits the capability to bind to double-stranded RNA (dsRNA) to promote the formation of circRNA (He and Zhu, 2023).

RBPs that do not have dsRBDs can play a prominent role in orchestrating circRNAs production through binding to specific RNA sequences. Quaking (QKI), is an alternative-splicing regulator (Teplova, 2013). Despite its absence of dsRBDs, when QKI binds to the recognition element it forms a dimeric structure that drives exons closer together through dimerization, encouraging reverse splicing (He and Zhu, 2023, Wu, 2002).

As previously stated, introns are spliced from pre-mRNA to form mRNA during the splicing process. However, in some cases, both introns and exons are removed from pre-mRNA, resulting in a structure known as “skipped exons”. Within this structure, the non-adjacent exons are brought close to each other to form a lariat intermediate. The introns are subsequently removed, followed by back-splicing occurring to produce ecircRNAs (Gong et al., 2015). Sometimes, the lariat's introns are not entirely spliced out but remain within the encircled exons, resulting in the formation of eIciRNAs (Fig. 1d) (Wang et al., 2018, Conn, 2015).

During the splicing process, introns are excised from pre-mRNA to form mRNA (Fig. 1a). Typically, these introns undergo a debranching reaction and are subsequently degraded by exonucleases. However, in some cases where the 5′ branch site of pre-mRNA contains a 7 nt GU-rich motif and the 3′ branch site contains an 11 nt C-rich motif, these motifs can interact to produce a lariat structure with the help of small nucleus RNA (U1 snRNA). This interaction can avoid debranching and exonucleolytic degradation. Following this, the intronic lariat is spliced to generate ciRNA (Fig. 1e) (Li, 2023, Yu, 2023).

It is also worth noting that recent studies have revealed a new type of circRNA, referred to as tricRNA. During pre-tRNA maturation, the tRNA splicing endonuclease (TSEN) complex can cleave the pre-tRNA at the bulge–helix–bulge (BHB) motif. Subsequently, RtcB ligase connects the exon halves and the intron ends to produce a mature tRNA and a circular intron RNA called tRNA intronic circular RNA (tricRNA), respectively (Fig. 1f) (Shen, 2021).

CircRNAs have been demonstrated to provide a variety of functions in eukaryotes. Despite most circRNAs having a low copy number within living organisms, their stability gives them unique and indispensable functions in tissues and cells (Liu, 2020). In the cytosol, some circRNAs can serve as microRNA sponges (Wang et al., 2018). In addition, some circRNAs have been shown to interact with proteins (Altesha, 2019). In the nucleus, circRNAs primarily regulate gene transcription, alternative splicing, and chromatin loops (Zhang, 2013, Li, 2015, Conn, 2017, Liu, 2020, Liu, 2023).

The biological or physiological functions of circRNAs can be classified into five main types (Fig. 2): circRNAs acting as miRNA sponges, circRNAs acting as transcriptional regulators, circRNAs compromise a translational ability, circRNAs acting as scaffolds and circRNAs acting as RBP sponges.

miRNAs possess the capability to modulate gene expression by binding to the 3’ untranslated region (3′UTR) of mRNA, leading to inhibition of the translation of mRNA (Li, 2023). CircRNAs have been discovered to include miRNA response elements (MREs), indicating their ability to competitively bind miRNAs (Bezzi et al., 2017). This binding is known as the “sponging effect” (Fig. 2a) (Valencia-Sanchez, 2006). For instance, the human cerebellar degeneration-related protein 1 transcript (CDR1as) also, called circRNA sponge for miR-7 (CiRS-7) harbored more than 70 conserved binding sites for miR-7 (Memczak, 2013). cDR1as interact with miR-7, impairing its ability to bind to its target mRNAs (Wang et al., 2018).

It is also worth shedding light on the fact that the interaction between circRNAs and miRNAs does not always result in the inhibition of miRNAs. Nonetheless, circRNAs might potentially store miRNAs or act as shuttles for transporting miRNAs. For instance, miRNA-671 has the capability to degrade CDR1as. CDR1as acts as a shuttle by binding to miR-7 and transporting it to a specific site. Thereafter, CDR1as is degraded by miRNA-671, resulting in the release of miR-7 (Wang et al., 2018).

CircRNAs, primarily located in the nucleus like eIciRNAs and ciRNAs, could influence the transcription of their parent genes through the presence of these intronic sequences (Fig. 2e). For instance, circEIF3J and circPAIP2 could interact with the U1 small nuclear ribonucleoprotein (U1 snRNP) and RNA Polymerase II in the promoter region of their host genes. As a result of this interaction, the expression of the parent genes, namely eukaryotic translation initiation factor 3 J (EIF3J) and poly(A)-binding protein-interacting protein 2 (PAIP2), is improved (Li, 2015). Furthermore, circRNAs have been found to encourage the early termination of gene transcription by forming R-loops, which are RNA-DNA hybrids (Xu, 2020, Pisignano, 2023).

The majority of circRNAs are primarily composed of exons, raising the possibility of their translation into proteins. Nevertheless, bioinformatic predictions estimated that only a small percentage of circRNAs possess both open reading frames (ORFs) and internal ribosome entry site (IRES) elements or incorporate the m6A RNA modification in their 5′ UTR, enabling them to be translated via a cap-independent mechanism (Fig. 2b) (Pisignano, 2023). For instance, circARHGAP35 is one of the most abundantly expressed forms of circRNA in HCC and CRC cell lines. Recent studies elucidated that circARHGAP35 encodes a long (1289aa) oncogenic protein through an alternative m6A-dependent translation. Furthermore, the studies have demonstrated that circARHGAP35 has a pivotal role in promoting tumor cell growth, migration, invasion, and metastasis (Pisignano, 2023, Li, 2021). Another study has identified CircMAP3K4 as a highly expressed circRNA in HCC. Driven by m6A modification, circMAP3K4 produces circMAP3K4–455aa, which provides protection to HCC cells from cisplatin exposure and is associated with a poorer prognosis in HCC patients. Targeting circMAP3K4–455aa may provide a promising therapeutic strategy for HCC patients, particularly those with chemoresistance (Duan, 2022). Despite recent insights, unraveling the functions of circRNA translation products remains an enigma, demanding further future investigations.

CircRNAs also serve as molecular scaffolds, enabling the interaction and assembly of proteins (Fig. 2c). CircFoxo3 is a protein scaffolding circRNA. The interaction between circ-Foxo3, p21, and CDK2 results in the creation of a ternary complex, which in turn halts the activity of CDK2 and prevents the progression of the cell cycle (Du, 2016, Zhang and Wang, 2021).

Recent studies have unveiled that circVAMP3 could act as a scaffold that negatively regulates the proliferation and metastasis of HCC cells in-vitro and in-vivo. It accomplishes this by driving phase separation of CAPRIN1 and facilitating stress granule formation in cells, which can reduce the protein level of Myc proto-oncogene protein by inhibiting c-Myc translation. Furthermore, there is a noteworthy correlation between reduced circVAMP3 expression and poor prognosis of HCC patients, suggesting that circVAMP3 might be used as a prognostic biomarker for HCC (Liu, 2023, Chen, 2022).

CircRNAs can bind to RBPs and function as RBP sponges (Fig. 2d). HuR is an example of RBP that can bind to PABPN1 mRNA and enhance its translation. circPABPN1, derived from the pre-mRNA nuclear poly(A)-binding protein 1 (PABPN1), can extensively interact with HuR. This interaction affects the translation rate of the parent gene by preventing HuR from binding to the corresponding mRNA (Altesha, 2019, Pisignano, 2023). As demonstrated, circRNAs play substantial roles in various biological processes; nonetheless, their functions remain largely enigmatic, requiring further efforts to unravel their full functions and their potential impacts.

Indeed, early diagnosis is essential for effective curative care for all malignant cases (Fahmy, 2022, Youness, 2021, Youness, 2023, Youness, 2023, Sedky, 2023). It is still difficult to diagnose HCC in its early stages because there are few symptoms to be detected until the cancer progresses to a late stage (Rao, 2023). Crucially, the discovery of circRNAs in biological tissues and fluids raises the prospect of their application as molecular biomarkers for diagnosis and prognosis (Dawoud, 2023). CircRNAs that could be utilized as biomarkers can be isolated from serum, HCC tissue biopsies, exosomal circRNA and from other body fluids (Rao, 2023).

Zhang et al. also showed that circRNA_104075 was highly expressed in HCC tissues and serum samples and also demonstrated better predictive performance for HCC than alpha fetoprotein (AFP) which is one of the conventional non-specific biomarkers for HCC and other liver diseases (Zhang, 2018), suggesting that circRNA_104075 could be a more useful serum and tissue biomarker for HCC diagnosis than AFP (Aishanjiang, 2021). Moreover, the three-circRNA signature: hsa_circ_0004001, hsa_circ_0004123, and hsa_circ_0075792 that is highly expressed in HCC patients compared to healthy controls was recently discovered as well as a promising circRNA signature with a strong diagnostic efficacy for HCC patients (Sun, 2020). It was also reported that hsa_circ_0016788 (also known as hsa_circTRIM11_001), which is isolated form HCC tissues, has high diagnostic value for HCC and can be used as diagnostic biomarker (Rao, 2023, Guan, 2019).

Another form of isolated circRNA biomarkers is the exosomal form, where exosomes are a kind of nanoscale secreted vesicles that are found in all body fluids in both healthy and diseased states and they are known to carry a variety of biomolecules, including circRNAs (Aishanjiang, 2021). One example of these exosomal circRNAs is hsa_circ_0070396, which has been shown to be a more accurate diagnostic biomarker than AFP for identifying early-stage HCC patients (Lyu, 2021).

Therapeutic, prognostic, and diagnostic implications of circRNAs in HCC or as recently denoted as theranostic agents include the notable upregulation of circCAMSAP1 expression in HCC tissues and as summarized in Table 1 (Luo, 2021). Mechanistically, circCAMSAP1 facilitates HCC progression through the miR-1294/GRAMD1A pathway, suggesting its potential as a prognostic tool and therapeutic target for HCC (Luo, 2021). Similarly, circRHOT1 expression is markedly upregulated in HCC tissues, and elevated levels are associated with the clinical stage and an unfavorable prognosis, suggesting its potential as a prognostic biomarker for HCC (Wang, 2019). Additionally, increased expression of SCD-circRNA 2 and hsa_circ_104348 can predict an adverse prognosis in HCC patients, highlighting their potential as prognostic biomarkers for HCC (Dong, 2019, Huang, 2020). In addition to upregulated circRNAs expression, downregulated expression of circRNAs also holds imperical potential to be used as biomarkers for HCC. For instance, decreased expression of circTRIM33–12 and circDLC1 can predict a poor clinical outcome, underscoring their prognostic value in HCC (Liu, 2021). Furthermore, downregulated expression of hsa_circ_0091570 could serve as a potential diagnostic and prognostic marker for HCC (Wang, 2019). Another study reveals that circ_0014717 expression is significantly decreased in HCC, and its reduced expression is associated with overall survival and time to tumor recurrence. Overexpression of circ_0014717 inhibits the growth, migration, and invasion of HCC cells in-vitro and in-vivo, suggesting its potential as a prognostic biomarker for HCC (Liu, 2021). Significantly decreased expression of circPABPC1 in HCC tissues was evidenced and it was reported to be strongly correlating with shortened overall survival and disease-free survival. As a tumor suppressor in HCC, circPABPC1 physically links ITGB1 (β1 integrin) to proteasomes for ubiquitin-independent degradation, inhibiting cell adhesion and migration. Therefore, circPABPC1 may be a potential prognostic and therapeutic tool for HCC (Shi, 2021). Another example of a prognostic circRNAs is circZKSCAN1, whose down-regulation in HCC tissues was linked to a poor prognosis and a poor overall survival rate, making it a potential prognostic marker for HCC patients.

In another study it was reported that reduced circSMARCA5 expression in HCC tumor tissues was strongly associated with a worse prognosis because it was correlated with poor tumor differentiation, a more advanced stage of the tumor, a larger tumor, and the presence of microvascular invasion (Yu, 2018). Moreover, according to a recent study, tumor size and TNM stage were positively correlated with hsa_cic_0005397, an up-regulated plasma circRNA in HCC patients (Liu, 2021). The level of plasma hsa_cic_0005397 was significantly reduced in HCC patients following surgery, but it was noticeably elevated in patients with recurrent or metastatic HCC (Liu, 2021). This suggests that plasma hsa_cic_0005397 could act as a prognostic biomarker for post-operational recurrence and metastasis in HCC patients, according to a dynamic monitoring study conducted on HCC patients who had undergone surgical resection. Furthermore, in patients with HCC, the plasma hsa_cic_0005397 level was negatively correlated with overall Survival (Liu, 2021).

Moreover, exosomal circRNAs serve as readily detectable potential biomarkers for HCC as previously mentioned. For instance, upregulated expression of exosomal circAKT3 in HCC may act as a prognostic marker following surgical treatment (Luo et al., 2020). Additionally, exosomal circ-0004277 expression is significantly increased in the plasma of HCC patients, demonstrating good diagnostic value with an area under the curve (AUC) of 0.816, sensitivity of 58.3%, and specificity of 96.7%. These results suggest that exosomal circ-0004277 might be a useful diagnostic biomarker for HCC patients (Zhu, 2020) (Table 1).

CircRNAs have been recently casted as tempting therapeutic target options because of their dominating role in the development and progression of HCC (Rao, 2023). Many therapeutic approaches for HCC have been put forth, based on the elementary role of circRNAs in oncology and theses approaches divided into two main categories: i) increasing the intracellular expression of tumor suppressor circRNAs by tornado approach (where the RNA's two ends are modified to create a loop), or by using synthetic circRNA (Schreiner et al., 2020, Kameda et al., 2023); and/or 2) targeting and inhibiting the oncogenic circRNAs expression using siRNA, shRNA or other antisense oligonucleotides either by transfection, drug delivery systems, or by using CRISPR/Cas9 technology (Rao, 2023, Piwecka, 2017).

As aforementioned, many circRNAs served as tumor suppressors and can be used as therapeutic agents. For instance, overexpression of circSMARCA5 resulted in decreased HCC progression and metastasis through increasing the expression of TIMP3, a well-known tumor suppressor, by inhibiting and sponging miR-181b-5p and miR-17–3p (Aishanjiang, 2021, Yu, 2018). In another study, it was reported that Lentivirus-mediated overexpression of CircZKSCAN1, tumor suppressor circRNA in HCC, resulted in decreased and suppressed HCC progression and this is by modulating cell stemness (Aishanjiang, 2021).

Also, an array of oncogenic circRNAs can be employed as therapeutic targets as previously mentioned. For instance, circIPO11, which is oncogenic circRNA that play role in self-renewal of HCC cells and also play a fundamental role in HCC progression and metastasis, such circRNA can be silenced by CRISPR/Cas9 and by antisense oligonucleotides (ASOs) resulted in inhibition of HCC progression (Gu, 2021). Furthermore, in order to knockdown of circMDK, which is highly expressed in HCC, Du et al., created poly β-amino esters (PAE)-siRNA nanomolecular particles which is constituted of PAEs loaded by siRNAs. On the functional level, in-vivo studies showed that the siRNAs-leaded nanoparticles dramatically slowed down the growth of the HCC tumor without having a major side effects (Du, 2022). Table 1 includes a summary of several circRNAs that can be used as therapeutic targets or tools.

CircRNAs serve a crucial function in tumor immunotherapy by influencing different facets of tumor immunity, particularly in the regulation of immune checkpoint molecules as previously reviewed by our research group (Abaza, 2023) and summarized in Table 2.

Sustained proliferation is one of the oldest and well-established hallmarks of cancer. With such characteristic, tumors grow beyond normal capacities, expanding their malignancy. HCC proliferation is mediated through several pathways that ultimately result in either positively regulated growth signals, or negatively regulated "off switches" to growth. It has been found in the literature that circRNAs ubiquitously influence this hallmark in HCC. For instance, circRHOT1 sustains HCC proliferation through increasing the transcriptional rate of NR2F6 by recruiting chromatin remodeling protein, TIP60 (Wang, 2019). Circβ-catenin is translated into an isoform of β-catenin that can suppress GSK3β-induced β-catenin phosphorylation and degradation of its target gene. Consequently, the target Wnt pathway is activated enabling uncontrolled growth of the tumor (Liang, 2019). Similarly, circRNA_0067934 increases the replicative capacity of HCC cells through manipulating the FZD5/Wnt/β-catenin signaling pathway. Additionally, it allows the tumor cells to evade apoptosis by sponging miR-1324 (Zhu, 2018). CircRNA ZFR promotes such phenomenon through upregulating MAP2 kinase 1 (Cedric, 2020), while hsa_circ_0091581 induces the chief regulator of cellular proliferation, c-Myc, through sponging miR-526b (Youness, 2016, Wei, 2020, Ahmed Youness, 2020).

CircRNA_0001955, through silencing miR-516a-5p, increases TRAF6 and MAP2 kinase 11 expression, resulting in increased stimulatory autocrine signals to HCC cells (Yao, 2019). Moreover, circ_ZEB1.33 downregulates miR-200a-3p, which sets Cyclin-dependent kinase 6 (CDK6) into action, activating uncontrolled cell proliferation (Gong, 2018). Likewise, hsa_circ_016788 mediates it through activating another cyclin-dependent kinase member, namely CDK4 (Guan, 2019).

Adding to the growing list of circRNAs regulating sustained proliferative signals hallmarks in HCC, some oncogenic circRNAs exhibit mechanistic duality, where not only do they increase the replicative power of HCC cells, but they also allow them to escape apoptotic cell death. One of the examples that fall under this class was the aforementioned circRNA_0067934 (Zhu, 2018), ssa_circ_0103809 (Cai, 2018) and circ_0000517 (Zang, 2020). Likewise, circRNA_0000502 induces HCC cellular proliferation and inhibits apoptosis in HCC cells proving the oncogenic effect of circRNA_0000502 which are mainly mediated through sponging the tumor suppressor miR-124 (Zhang, 2019). Overexpression of circIGF1R is correlated with increased solid tumor size in HCC patients and on the molecular level, it was reported to modulate HCC proliferative capacity through inducing PI3K/AKT signaling pathway (Fu, 2019).

As there are oncogenic circRNAs illustrated above, several sources have discovered tumor-suppressive circRNAs that mechanistically work on decreasing the clonal expansion of HCC cells. circSETD3 (hsa_circ_0000567) for instance, enhances the expression of cell division suppresser gene MAP kinase 14 through sponging miR-421. This pathway leads to suppressed proliferation, arresting the cell cycle at the G1/S stage (Xu, 2019). Exosomal hsa_circ_0051443, normally found downregulated in HCC cells, was discovered to play a role as an anti-proliferative and pro-apoptotic agent in HCC cells (Chen, 2020)

Tumor suppressor genes (TSG) control is the negative regulatory body that restrains cell growth and division to maintain division homeostasis. In malignancy, this control is disrupted; canceling the control imposed on a cell to remain normal, induces cancer. Most members of this class are functionally tumor suppressive, however, with evidence, they are found downregulated in HCC cells, dysregulating cell division for oncogenic consequences (Rahmoon, 2017, Youness, 2016, Shaalan, 2018). Recently, scientists had shed the light onto the modulatory role that circRNAs might impose on regulating HCC power to evade growth suppressors. CircHIAT1 levels inversely correlate with miR-3171, where the decrease of the latter controls cell division through PTEN gene expression (Wang, 2019). Moreover, major TSGs like RUNX3 and TP53 are silenced by miR-761 due to its decreased degradation that is normally mediated by circLARP4 in HCC (Chen, 2019). CircBACH1 (hsa_circ_0061395) also is an oncogenic circRNA that is found to be upregulated in HCC tissues. Molecularly, circBACH1/HuR/p27 axis is the pathway responsible for this evasion potency on tumor suppressors such as p27 (Liu, 2020).

Unlike the aforementioned circRNAs, circMTO1 is a tumor suppressor circRNA that normally sponges miR-9, that when left unregulated, exhibits its oncogenic role in inhibiting cell cycle inhibitor p21, enabling unrestricted replication of the tumor (Han, 2017). Also, circRNA cSMARCA5 sponges miR-17–3p and miR-181b-5, promoting the expression of their respective tumor suppressor target TIMP3 resulting in tumor suppressor activity in HCC cells examined (Yu, 2018).

Although monitoring tissue homeostasis and protecting it against cell damage is the main function of immune system, cancer still occurs with high frequency in humans (de Visser et al., 2006). Such phenomenon can be attributed to the fact that cancer cells acquire specific feature allow it to evade immune destruction. Consequently, evading immune destruction by tumor cells was counted a next-generation hallmark that has emerged in the last decade (Hanahan and Weinberg, 2011). However, the cross interaction between cancer cells and immune system is still elusive. For instance, abundance of infiltrating lymphocytes has been correlated to favorable prognosis whereas abundance of infiltrating innate immune cells, such as neutrophils, macrophages, and mast cells been linked to poor prognosis and increased angiogenesis (Abdel-Latif and Youness, 2020, Elemam, 2023, de Visser et al., 2006). Attempts to understand the role of the immune system in HCC have shown that CD8+ T cells and lymphotoxin β are pivotal mediators of HCC tumorigenesis whose inhibition whether by antibodies or pharmacologically resulted in marked delay of tumor development in mice (Endig, 2016).

The association of circRNAs with evading immune destruction in HCC is yet to be determined. Nonetheless, hsa_circ_0082002 (circMET) is an overexpressed onco-circRNA in HCC tissues that was found to be involved in immunosuppression via the miR-30–5p/snail/DPP4/CXCL10 trajectory (Huang, 2020). Mechanistically, Huang et al.,. have found that circMEt sponges miR-30–5p family (miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-30d-5p and miR-30e-5p) leading to an increase in the transcription factor Snail which upregulates the expression of dipeptidyl peptidase 4 (DPP4). Eventually, these molecular events lead to the degradation of C-X-C motif chemokine ligand 10 (CXCL10) which is believed to be a key player in CD8+ lymphocyte trafficking (Liu, 2015). Collectively, circMET upregulation led to HCC immune tolerance. Along this line of reasoning, trimming this pathway by administration of the DPP4 inhibitor, sitagliptin, in parallel with administering anti-PD1 antibody improved the antitumor immunity in immunocompetent mice. This was clinically supported by finding that higher CD8+ T cell infiltration in HCC lesions from diabetic patients receiving sitagliptin compare to lesions collected from HCC patients with diabetes who are not receiving sitagliptin treatment (Huang, 2020). Accordingly, using sitagliptin in HCC patients receiving anti-PD1 therapy in a subgroup of patients with HCC may reduce anti-PD1 therapy resistance and enhance its anti-tumor efficacy.

As a consequence of evading cell death and senescent bypass, cancer cells enter a state of cellular immortality by undergoing continuous proliferation. This phenomenon is driven by genetic and epigenetic alteration that fuel autonomous growth (Yaswen, 2015). Telomere maintenance is believed to be the workhorse in replicative immortality (Hahn and Meyerson, 2001). Telomerase is the enzyme that is responsible for the maintenance of telomeres without which chromosomes shorten with progressive cell division, inducing either cellular senescence or apoptosis. Given the scarcity of identified regulatory circRNAs for replicative immortality in HCC, genetic targets such as Telomerase Reverse Transcriptase (TERT) represent a promising approach for HCC immortality control (Nault, 2019). Mechanistically, telomerase reactivation in HCC has been identifies in previous studies to be through TERT promoter mutations, TERT translocation, TERT amplification, and viral insertion into the TERT gene. Hence, up- and downstream molecular regulator, including circRNAs, of these genetic changes represent a hot research area that needs intensive investigation (ZeinElAbdeen et al., 2022, Dawoud, 2023, ElKhouly et al., 2020, El-Daly, 2023, El-Aziz, 2023).

Being increasingly recognized as an integral component of tumorigenesis, inflammation has gained great interest to further understand its underling origin (Samadi, 2015). Notwithstanding the fact that inflammatory response is an anti-tumor immune response, prolonged release of the inflammatory mediators was found as a key promotor for cancer hallmarks such as proliferation, invasion, and angiogenesis. Furthermore, inflammation was found to has a substantial role in genetic and epigenetics changes of cancerous cells (Fahmy, 2022, Abdel-Latif, 2022, Selem, 2023, Mekky, 2023, Sowers, 2014). Anti-inflammatory targets such as cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNFα), nuclear factor-κB (NF-κB), macrophage migration inhibitory factor (MIF), inducible nitric oxide synthase (iNOS), protein kinase B (PKB), and CXC chemokines are being intensively investigated to unravel the relationship between cancer and tumor-promoting inflammation (Elemam, 2023, Youness, 2021, Nafea, 2021, Youness, 2023, Samadi, 2015, Youness, 2022, Youssef, 2022).

As previously mentioned, HCC remained to be one of the leading causes of cancer-related deaths. This was found to be mainly due to chronic liver inflammation after the substantial efforts against hepatitis B and hepatitis C viruses (Youness, 2016, Youness, 2016, Mekky, 2019). Therefore, while working to improve therapeutic modalities for HCC, it is critical to better understand the mechanisms causing chronic inflammation and how these contribute to the emergence of HCC (Youssef, 2022).

CircRNAs governing tumor-promoting inflammation process are still not well-investigated. However, circRNA RSF1 (circRSF1) was found to promote inflammation in the hepatic stellate cells via modulating miR-146a-5p activity (Chen, 2020, Galun, 2016). High expression of circRSF1 has inhibited miR-146a-5p by acting as a miRNA sponge, increasing Ras-related C3 botulinum toxin substrate 1 (RAC1) expression.

Primary tumors usually release angiogenic factors initiating vascularization events. Cancerous cells at this time start to obtain motile nature and secrete matrix metalloproteinases allowing them to invade the tissue stroma. Then, the invasive tumor cells undergo intravasation and enter the circulation, migrating or walking to distant areas; however, the migration could be also through the lymphatic vessels (Chambers et al., 2002). After survival of the migrating cells in the circulation and landing in a new organ, they extravasate into the surrounding tissue, stabilize, and initiate a maintained growth supported by vascularization (Zhou, 2014). In HCC, a significant barrier to further progress in improving long-term survival following curative HCC resection is the high risk of recurrence and due to intrahepatic metastatic spread (Tang, 2001).

The role of circRNAs in inducing invasion and metastasis in HCC is quite well-studied. Given the tumor-suppressive role of androgen receptor (AR) in HCC, Ouyang and colleagues has revealed that AR/circ-LNPEP/miR-532–3p/RAB9A signaling pathway is involved in hypoxia-induced HCC cells invasion (Ouyang, 2021). Intriguingly, they found AR acts as a transcription factor resulting in circ-LNPEP downregulation. This reduction of circ-LNPEP attenuates its sponging potential for miR-532–3p. Thus, miR-532–3p downregulates Ras-related protein Rab-9A (RAB9A) inhibiting its role in HCC cells invasion. Similarly, circ_0091579 was found to promote proliferation, invasion, migration, and glycolysis in HCC cells via sponging miR-490–5p which in turn results in upregulation of cancer susceptibility candidate 3 (CASC3) (Liu et al., 2021).

Hsa_circ_0085616 (circASAP1) was also found to be associated with pulmonary metastasis in HCC patients (Hu, 2020). In-vitro, circASAP1 promoted not only migration and invasion, but also cell proliferation and colony forming ability. Mechanistically, the studies revealed that circASAP1 acts as an endogenous RNA competitor for the tumor-suppressor miRNAs: miR-326 and miR-532–5p which are endogenous suppressor for mitogen-activated protein kinase (MAPK) and colony stimulating factor (CSF). MAPK and CSF are associated with cell proliferation and invasion, as well as mediating tumor-associated macrophage infiltration, respectively. Another miRNA-sponging circRNA which is involved in inducing HCC invasion and metastasis is hsa_circ_0003258 (circ-ZNF652) (Guo, 2019). Patients with high circ-ZNF652 expression profiles were found to be more susceptible for vascular invasion, intrahepatic and distant metastasis with general poor outcome. Mechanism studies reported circ-ZNF652 to downregulate miR-203 and miR-502–5p, thereby increasing the expression of gene Snail, a common target for both miRNAs. Snail is a key transcription factor. When upregulated, it promotes the process of epithelial-mesenchymal transition (EMT), which in turn promotes HCC metastasis.

On the other hand, hsa_circ_0001445 (cSMARCA5) was characterized as an inhibitor for proliferation and migration of HCC cells as previously mentioned (Yu, 2018). cSMARCA5 exerted its tumor-suppressive activity and upregulates the tissue inhibitor of metalloproteinase 3 (TIMP3) which is well-known tumor suppressor via sponging miR-17–3p and miR-181b-5p (Yu, 2018). Hsa_circ_0020007 (circ-ADD3), is another downregulated circRNA was found to be involved in HCC intrahepatic and distant metastasis, as well as vascular invasion (Sun, 2019). Mechanistically, circ-ADD3 increase EZH2 ubiquitination and subsequently its degradation. This action is achieved by circ-ADD3 reinforcing the interaction between EZH2 and CDK1. Downregulation of EZH2 increases the expression a group of anti-metastatic genes including dampening circ-ADD3 itself. This is accomplish by reducing the levels of H3K27me3 (a histone tri-methylation marker) on the promoter regions of the anti-metastatic genes (Sun, 2019).

Another way cancer cells are able to support their high processes demand is by driving the formation of new blood vessels in the tumor’s direction, in order to exploit physiological functions for itself. Starting with hsa_circ_0000092: this stimulatory ncRNA promotes the formation of vasculature by binding to regulatory molecule miR-338–3p. The decoying strategy allows for the overexpression of HN1 (hematopoietic- and neurologic-expressed sequence 1), MMP9, and VEGF molecules involved in the angiogenic process (Pu, 2020). Hsa_circ_0046600 creates a similar effect but through modulation of another angiogenic factor, namely HIF1a (Zhai, 2019, Farhadi, 2021). Circ-EPHB4 (hsa_circ_0001730) has the same mechanism target as hsa_circ_0046600 but in a tumor suppressive fashion, suppressing HIF1 to resist angiogenesis (Tan, 2019). Yet, the oncogenic circGFRA1 promotes blood vessel formation by sponging to miR-149 and thus facilitating vasculature induction hallmark for HCC tumors (Yu et al., 2020). A 2020 study has also studied and demonstrated the correlation of circRNA-100338 on positively influencing angiogenesis (Huang, 2020). CircCRIM1 promotes this hallmark by sponging miR-378a-3p and as a consequence, elevating SKP2 levels (Ji, 2021). Another proposed mechanism is through the circCMTM3/miR-3619–5p/SOX9 axis which was demonstrated to promote angiogenesis and carcinogenesis of HCC cells (Hu, 2021). Oppositely, the presence of circ_4911 and circ_4302 in the liver cancer tumor microenvironment (TME) controlled the angiogenic process by inhibiting human umbilical vein endothelial cells (HUVECs) proliferation and migration steps (Yan, 2020).

Genomic instability and mutations are associated progression and development of many cancers including HCC. Using next-generation sequencing methods, we were able to create a detailed picture of the most frequently altered and mutated genes and their associated genomic instability and chromosomal instability in HCC. The genes most commonly mutated in HCC are: TERT, TP53, CTNNB1, AXIN1, LAMA2, ARID1A, ARID2, WWP1, RPS6KA3, ATM, CDKN2A, KMT2D, NFE2L2, ERRFI1 and others (Valiante and Grammatico, 2022). One of the most frequent mutations that occur in HCC is TERT activating or gain function mutation (with mutation rate of 60% in HCC) which increase the telomerase enzyme that play role in replicative immortality of cancer (Valiante and Grammatico, 2022, Rao et al., 2017). In addition, TP53 produces p53, one of the most well studied tumor suppressors with a variety of roles. When paired with mutations that cause mitotic errors, such as spindle checkpoint deficiencies and/or Rb faults, loss of p53 activity can allow aneuploid cells to survive or proliferate. According to Laurent-Puig et al., p53 mutation is related with increased CIN (chromosomal instability) in HCC (Rao et al., 2017, Laurent–Puig, 2001). Also it has been consistently noted that HCC cells exhibit dysregulation of the mitotic checkpoint, which ultimately causes aneuploidy and cellular rearrangements. The master regulators that have been discussed in HCC include CENP-A/E, BUBR1, MAD1/2, and BUB3 (Schvartzman et al., 2010). Another mutated gene (loss function mutation) is the KMT2D gene that produces lysine-specifc methyltransferase that methylates histone H3 lysin-4 residue and it is a tumor suppressor protein. Its mutations cause genomic instability in areas of the genome that contain replicating fragile spots (Valiante and Grammatico, 2022, Kantidakis, 2016, Ng, 2010) and there are many other mutations and chromosomal instabilities that contribute to the HCC tumorgenesis and development. Here, the involvement of circRNA in genomic instability and mutation in HCC is still not well understood, and more research is required to understand how circRNA can be involved in this characteristic either as an upstream or downstream modulator.

One of the purposes of apoptosis is to kill and avoid the propagation of damaged or genetically abnormal cells. This tightly controlled cell death is dysfunctional in cancer cells whereby a magnitude of mechanisms, they can escape it. Mechanisms can be categorized as a dysfunctional damage detection system, defects in apoptotic signaling, or dysregulated anti- and pro-apoptotic protein balance (Hanahan, 2022). Carrying out these mechanisms are several circRNAs, some of which were already mentioned including circRHOT1, circRNA_0067934, circ_0000517, circRNA_0000502, circIGF1R, and circZFR (Wang, 2019, Zhu, 2018, Zang, 2020, Zhang, 2019, Fu, 2019, Yang, 2019) Added to that list is circ-BIRC6. It has been found that through silencing its direct target miR-3918, the cascade of Bcl-2 gene expression is increased, promoting the anti-apoptotic survival of HCC cells (Yang, 2019). Another proof of the large influence of circRNAs on programmed cell death is circ-PRKCI functioning through diminishing AKT3 (Qi, 2019). Hsa_circ_0051443 promotes programmed cell death in HCC tissue by blocking miR-331–3p, regulating Bak1 levels (Chen, 2020). circHIAT1 inhibits apoptosis through targeting the miR-3171/PTEN axis (Wang, 2019). CircRNAs like circITCH and circ-0001649 reduce the level of their respective oncogenic miRNAs. Hence, the miRNAs mediated degradation of the subsequent axis targets is lowered, allowing anti-apoptotic proteins such as MAFF and SHPRH to produce their effect (Guo, 2017, Su, 2019). Yet, it is worth noting that HCC patients showed a repressed expression of the aforementioned protective, pro-apoptotic circRNAs, further promoting death escape (Chen, 2020, Wang, 2019, Guo, 2017, Su, 2019)

Circ_0000105 was found by Sun et. al. to act as oncogenic mediator through its dual activity in inducing proliferation and repressing apoptosis. This is brought about by decoying miR-498 to stimulate the expression of PIK3R1 (Sun et al., 2020). However, circPVT1 dual activity is validated where it influences proliferation and apoptosis, decreasing the former and increasing the latter via the miR-3666/SIRT7 signaling axis (Li, 2020).

The abnormal and overdriven cellular functions of cancerous cells, including progression, replication, invasion, and metastasis, are supported by increased and dysregulated energy metabolism. Previous research showed that circRNAs primarily affect glucose utilization and glycolysis. circMAT2B (hsa_circ_0074854) dysregulates glycolysis, leading to increased glucose utilization under hypoxic conditions (Li, 2019). Li et. al demonstrated that this dysregulation is mediated via the activation of the miR-338–3p/PKM2 axis (Li, 2019). The same effect is achieved by circ-PRMT5 but through activating a key enzyme found early in the glycolysis pathway, namely hexokinase 2 (HK2) (Ding, 2020). Additionally, the infamous circ_0000517 was discovered to play a metabolic role in HCC as well, where by sponging miR-326, it results in upregulated IGF1R gene expression (He, 2020). CircZFR enhances glycolysis through circZFR/miR-511/AKT1 axis as elucidated by Yang et. al.(Yang, 2019) While another group has found a different pathway through which increased glycolysis in HCC is mediated which is circZFR/miR-375/HMGA2 axis (Xu, 2021). Additionally, circSPECC1 promoted carcinogenesis by acting on target TGFβ2, while circC3P1, when expressed in cells, inhibits it by acting on PCK1, a regulator enzyme of gluconeogenesis- both being players in HCC cell metabolism (Zhang, 2020, Zhong, 2018, Starling, 2019).

A newly discovered circRNA was found to play a role in modulating aerobic respiration in metastatic HCC as demonstrated by Li. et. al. CircRPN2 is an anti-tumor sequence that was found to be reduced in metastatic stages of HCC. It suppresses aerobic glycolysis and metastasis if expressed in sufficient levels through 2 different pathways. The first pathway is by binding to and degrading enolase 1 (ENO1), which reprograms the glucose metabolic pathway through Akt/mTOR. Secondly, CircRPN2 upregulates forkhead box protein O1 (FOXO1) by functioning as a ceRNA to miR-183–5p. Moreover, inhibited metastasis and glycolysis occur respectively as a consequence of the mentioned mechanisms (Li, 2022).

A recent publication released in 2022 explored and concluded the anti-HCC effect of Fuzheng Xiaozheng prescription (FZXZP) through re-calibrating the hepatic glucose and lipid metabolic profile is mechanistically mediated by circRNA-miRNA-mRNA networks (Liu, 2022). As of yet, new studies need to be established on the possible effects of circRNAs on other metabolic pathways such as the Krebs cycle, protein, and adipogenesis pathways.

One of the newly proposed hallmarks of cancer is polymorphic microbiomes (Hanahan, 2022). It is suggested that the unique flora profile each individual possesses may influence the status of their cancer, both positively and negatively (Dzutsev, 2017, Helmink, 2019). This phenomenon is extended to various liver diseases such as NAFLD, cirrhosis, and HBV which lead to the progression to HCC (Meroni et al., 2019, Liu, 2019, Nolan, 2010). Dysbiosis of the intestinal microflora has shown evidence to promote liver diseases and cancer, where it has also been found that 20–75% of chronic liver disease patients develop intestinal disorders proving the crucial role and link of the gut-liver axis (Yu, 2010, Dapito, 2012, Compare, 2012, Schwabe and Greten, 2020). The available body of evidence proposes that this link is due to the constant contact of the liver with intestinal microbes and their products through the hepatic portal vein, where changes in the compositional mix of the biome and its byproducts reflect back on the liver’s immunity and inflammatory signals (Chassaing et al., 2014, Ponziani, 2019). The persistent inflammatory status is considered the ignite to initiate and promote liver tumorigenesis through the acquisition of one or more of other cancer hallmarks (Hanahan, 2022, Yu et al., 2018).

The crosstalk between circRNAs and this hallmark is still very limited. There are only few articles demonstrating the effects of certain circRNAs on microbial plethora affecting CNS disorders via the gut-brain axis. These diseases, like depression and AD, were studied in mice models (Diling, 2020, Zhang, 2019). In regards to the hepatic system, circRNAs were found to have a role in HBV-mediated HCC and liver metastasis from colorectal cancer (Wu, 2020, Xu, 2019). Up to our knowledge, there have been no reports of circRNAs involved in HCC, either negatively or positively, through manipulating the individual’s flora polymorphism yet.

It is commonly known that cancer is a mutational genetic disease, hence the hallmark "Genetic Instability". Yet, one of the newly proposed hallmarks by Hanahan et. al. is “non-mutational epigenetic reprogramming”. Briefly, it demonstrates that for a cluster of cells to be transformed to malignant is not due to permanent genetic variations, but due to reversible epigenetic changes that impart cancer hallmarks to the tissue of interest (Hanahan, 2022). This proposed pillar has considerable precedence out of the new hallmarks, going back to around a decade, with increasing interest as it explains striking cases where cancer is non-mutational or is genotypically identical with phenotypic variance (Huang, 2012). HCC, in particular, exhibits high phenotypic variance, which is due to a combination of large pools of genetic mutations and epigenetic manipulations (Hlady and Robertson, 2018, Wu et al., 2020). Epigenetic reprogramming is the process by which an organism’s genotype interacts with the environment to produce its unique phenotype and provides a tailored framework for explaining individual variations and the uniqueness of cells, tissues, or organs, diseases despite identical genetic information which is the core of personalized/precision medicine (Kiriacos, 2022, Tang and Ho, 2007). Inter- and intra-heterogeneity between HCC patients definitely highlights the importance of studying epigenetic reprogramming in such notorious disease (Fahmy, 2022, ZeinElAbdeen et al., 2022, Youness, 2023, Mekky, 2022). The main epigenetic mediators are histone modification, DNA methylation, and ncRNAs (Abdel-Latif, 2022, Abdallah, 2022, Awad, 2021, Selem et al., 2021, Youssef, 2022).

Focusing on DNA methylation as a type of epigenetic reprogramming. YAP1 initiates the transcription of several proliferative and anti-apoptotic genes. Its mRNA is silenced by PCBP2 regulating those functions. The presence of circCPSF6 (hsa_circ_0000417) competitively binds to PCBP2 instead to stabilize the expression of YAP1. This imparts oncogenic characteristics to the HCC cells such as growth, motility, and metastasis (Chen, 2022). Another DNA-methylation modifier is circMEMO1. It adsorbs miR-106b-5p resulting in the demethylation of TCF21. This negatively affects three cancer hallmarks- proliferation, invasion, and migration. However, it is found that its basal level in HCC tissue samples is deficient (Dong, 2021) Likewise in mechanism and expression is circTRIM33–12, affecting an additional hallmark which is immune evasion (Zhang, 2019). Furthermore, Xu. et. al. published a study that screens an array of circRNAs to identify additional and potential methyl modifiers in HCC (Xu, 2021). CircSOD2 and circ-LRIG3 both progress HCC malignancy via the epigenetic modifications of STAT3 (Sun, 2020, Zhao, 2020).

Another epigenetic effect lies not on HCC cells' genetic material but on the circRNA itself; circSORE half-life increases by METTL3 and METTL14 methylating it, resulting in the creation of sorafenib-resistant cells (Xu, 2020). On the ncRNAs level, dozens of circRNAs affects HCC through its sponging capacity on miRNAs. For instance, Cdr1as/ciRS-7 sponges miRNAs-7, releasing CCNE1 and PIK3CD to be freely expressed, allowing expansion and invasion of the solid tumor mass (Yu, 2016). Moreover, circ-0001649 binds to three different miRNAs to activate SHPRH, non-traditionally affecting nucleosome remodeling through responsible proteins (Su, 2019).

Cellular senescence is an irreversible process believed to be physiologically involved in the regulation of embryogenesis, tissue remodeling, and injury, through which the cell enters a state of cell cycle arrest. Usually, the cells that are subjected different endogenous or exogenous stresses such as in cases of rapid proliferation undergo the senescence process as a defense mechanism to avoid unnecessary damage (Collado et al., 2007). Indeed, cellular senescence could be triggered by multiple factors such as telomere shortening, DNA damage, or any change that would limit uncontrolled or aberrant cellular proliferation (Collado et al., 2007, Calcinotto, 2019). Some of the molecular key players that are observed to be highly expressed in senescent cells and used as miscellaneous senescence biomarkers are p53, p21, p19ARF, and plasminogen activator inhibitor-1 (PAI-1) (Campisi, 2013). Senescence also works as a natural potent barrier to prevent tumorigenesis at a premalignant level (Collado et al., 2007). Moreover, it could be induced by upregulation of oncogenes in what so-called oncogene-induced senescence (OIS) (Mittermeier et al., 2020, Chandeck and Mooi, 2010). Thus, when senescence mediators are downregulated or mutated in cancer cells, it gives a mark of poor prognosis.

In HCC and other cancer types, induction of senescence and its mediators have been targeted for drug development (Mittermeier et al., 2020). Therefore, pharmacological and molecular regulators for senescence have been investigated. The role of circRNAs in regulating cellular senescence in HCC is still in its infancy; however, circLARP4 was identifies as a mediator of cell cycle arrest in HCC by inducing senescence in vitro through circLARP4/miR-761/RUNX3/p53/p21 signaling axis(Chen, 2019). CircLARP4 is a downregulated circRNA in HCC tissues and cell lines and considered as a marker for poor survival outcome. When ectopically expressed in HCC cell lines, it suppressed their proliferation and induced cellular senescence resulting in cell cycle arrest. Further investigation in the downstream axis through which circLARP4 exerts its tumor-suppressive activity was found to be via sponging miR-761. Downregulation of miR-761 results in an increase of the expression of Runt-related transcription factor 3 (RUNX3) which in turn induces the transcription of p53and p2, the mainstay of cellular senescence (Chen, 2019). These findings provide a circRNA-dependent potential survival marker and therapeutic target in HCC, encouraging further investigations in this field.

The most important characteristic of cancer cells is their phenotypic plasticity; it gives the cancer cells the ability to temporarily change both its morphological and functional characteristics (Hanahan, 2022). Physiologically, during developmental stages such as organogenesis, cells gain sort of genetic restriction that limit the organization and function within of the cells within their respective tissues (Yuan et al., 2019). Throughout phenotypic plasticity, the cell does not only loss its normal identity and function, but also undergoes epigenetic alterations and genetic changes to a lesser extent. In HCC, unlocking phenotypic plasticity has become a promising therapeutic target in which researchers aim to induce cell differentiation (Zheng, 2022). For instance, targeting liver enriched transcription factors (LETFs), including FOXA3, HNF6, HNF1, HNF4, and CEBPA which are key regulators of the hepatocyte differentiation process whether pharmacologically and molecularly represent a promising approach for Differentiation therapy (Zheng, 2022). The role of circRNAs in unlocking phenotypic plasticity in HCC remains largely unknown and need intensive focus, particularly, the upstream circRNAs regulator for such molecular targets.

In order to determine the patterns of circRNA expression, Zhu et al. used sequencing techniques. They discovered that the expression of circZKSCAN1 is lowered in HCC tissues. They discover a negative connection between circZKSCAN1 expression and epithelial cell adhesion molecule (EpCAM which is stimulator for tissue plasticity and stemness) mRNA utilizing 112 pairs of HCC (EpCAM high and low expression) samples. So circZKSCAN1 is tumor suppressor circRNA that suppresses stemness and plasticity of HCC by sponging RBP which is RBP fragile X mental retardation protein (FMRP), and consequently turning off the Wnt/β-catenin signaling pathway (Zhu, 2019, Fagotto, 2020).