2.1 Classification of circRNAs

CircRNAs exhibit three primary subtypes based on their contained sequences: exonic circRNAs (ecircRNAs), formed from exonic sequences in precursor mRNAs (pre-mRNAs); circular intronic RNAs (ciRNAs), formed from intronic sequences in pre-mRNAs; and exon-intron circRNAs (EIciRNAs), formed from both exonic and intronic sequences in pre-mRNAs.⁵ EcircRNAs predominantly reside in the cytoplasm, functioning as competing endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs), thereby protecting mRNAs from miRNA-mediated inhibition.^58-60 Conversely, ciRNAs and EIciRNAs are primarily located in the nucleus, where they regulate transcription.^{61, 62} All three circRNA subtypes consist of sequences derived from a single gene. Recently, two distinct types of circRNAs, fusion circRNAs (f-circRNAs) and read-through circRNAs (rt-circRNAs), have been identified, incorporating sequences from two different genes.^{63, 64} F-circRNAs originate from fusion genes formed by chromosomal translocations in cancer cells.^65-67 Rt-circRNAs result from read-through transcription, producing hybrid transcripts that include coding exons from two adjacent and similarly oriented genes.^{64, 68} While f-circRNAs are interchromosomal chimeras between distant genes, rt-circRNAs are intrachromosomal chimeras involving adjacent genes on the same strand.⁶³

2.2 Biogenesis of circRNAs

Maintaining cellular physiological balance involves the regulated generation of circRNAs through multiple cis-acting elements and trans-acting factors, mirroring the control of canonical splicing.^{69, 70} Cis-acting elements govern circRNA biogenesis through lariat-driven and intron pairing-driven circularization.⁶⁹ In lariat-driven circularization, pre-mRNA partially folds, enabling the downstream donor splicing site to attack the upstream receptor splicing site, forming circRNA with the spliced folded region.^{71, 72} In intron pairing-driven circularization,⁷³ reverse complementary sequences on the flanks of exons, acting as cis-acting elements, facilitate back-splicing to directly form circRNA.⁷⁴ CircRNA generation is also subject to regulation by diverse trans-acting factors under specific conditions. During RNA-binding protein (RBP)-driven circularization, RBPs promote circRNA generation by binding to specific sites in the flanking introns, bringing the donor and receptor sites together. Notably, certain RBPs facilitate circRNA generation, while others exert the opposite effect.⁶

2.3 Features of circRNAs

The foremost distinctive feature of circRNA is its stability, stemming from its closed-loop structure, rendering it resistant to exonucleases.^{75, 76} Second, circRNAs exhibit abundant expression across various species, with several circRNAs expressed at much higher levels than their cognate linear mRNAs.^{5, 6, 77} Third, some circRNAs demonstrate conservation among diverse species. Both the circularized exon sequences and the flanking intronic sequences of conserved circRNAs are conserved,⁷⁸ along with their splice sites and the effects of RBPs on circRNA biogenesis.^78-80 Fourth, circRNAs display species-, cell-, tissue-, developmental stage-, and disease-specific expression.^{49, 55, 81, 82}

2.4 Functions of circRNAs

2.4.1 miRNA sponging

Sponging miRNA stands out as a pivotal function of circRNA (Figure 1).^{5, 70} By binding and sequestering target miRNAs, circRNAs can modulate the expression and function of mRNAs targeted by these miRNAs. For instance, CDR1as harbors over 70 miRNA response elements (MREs) for miR-7.^83-87 Similarly, circHIPK3 targets multiple miRNAs, regulating various downstream mRNAs.^{75, 88} In specific situations, circRNAs act as reservoirs by sponging miRNAs for transportation.⁸⁹

2.5 Transport and turnover of circRNAs

After circRNA generation, EIciRNAs and ciRNAs often remain in the nucleus, whereas ecircRNAs are typically transported to the cytoplasm.⁵ Huang et al.¹¹¹ demonstrated an evolutionarily conserved length-dependent pathway controlling the export of circRNAs. Shorter circRNAs (<400 nucleotides) are preferentially exported by URH49, while the transport of circRNAs >1200 nucleotides is mediated by UAP56.¹¹¹ The transport regulation of circRNAs between 411 and 1099 nucleotides in length is complicated by the influence of RNA secondary structures.¹¹¹ Additionally, m⁶A modification is involved in the export of circRNAs from the nucleus.¹¹² Furthermore, the NXF1–NXT1 pathway plays a crucial role in the nuclear export of repeat-containing ciRNAs,^{110, 113} and the G-rich sequences and secondary structures of expanded repeats in the ciRNA are important for its stabilization and export mediation from the nucleus to the cytoplasm.¹¹⁰

CircRNAs have a much longer half-life than linear mRNAs, and their degradation mechanisms remain unelucidated.^{90, 114} Generally, degradation is thought to be initiated by endonucleases followed by a cascade of exonucleases or endonucleases.⁹⁰ Hansen et al.¹¹⁵ proposed that miR-671 directly cleaves CDR1as in an Ago2-slicer-dependent manner by binding to CDR1as at the near-perfect target site with high conservation for miR-671. Park et al.¹¹⁶ found that m⁶A modification of circRNAs is recognized by m⁶A reader protein YTHDF2, which interacts with the adaptor protein HRSP12 to recruit the RNase P/MRP complex that degrades YTHDF2-bound circRNAs. Liu et al.¹¹⁷ reported that circRNAs are degraded by RNase L upon viral infection or poly (I:C) treatment. In addition to RNase L-mediated circRNA degradation under immune conditions, Fischer et al.¹¹⁸ discovered a structure-mediated circRNA decay mode under normal conditions. Besides, as a key component of P-body and RNAi machinery, Drosophila GW182 and its human homologs, TNRC6A/TNRC6B/TNRC6C, have been shown to regulate circRNA degradation by ribonucleases, in a process thought to be independent of the P-body and RNAi machinery.^{119, 120}

Furthermore, circRNAs are enriched in exosomes and released into the extracellular space upon the fusion of multivesicular bodies with cell membranes.^{121, 122} The discharge of circRNAs from cells into the extracellular space via exosomes is another mechanism for circRNA clearance.¹²³

2.6 Secondary structures of circRNAs

Secondary structures in circRNAs can bind to special proteins¹¹⁷ and modulate circRNA stability, nuclear export, and decay,^{110, 119} influencing the bond between circRNA and its parental linear mRNA.⁹⁹

Through bioinformatic analysis, Sun et al.¹²⁴ found that multiple circRNAs contain internal complementary base-pairing sequences (ICBPS). Complementary paired ICBPSs might enable circRNA to form secondary double-stranded structures. The maximum length of the ICBPS in most circRNAs is under 15 or even 10 nucleotides. Researchers have discovered more than 2000 circRNAs containing over 20 pairs of ICBPS. As the overall length of circRNAs increases, the number and maximum length of ICBPS also tends to increase.¹²⁴ CircRNAs with a higher probability of internal base pairing are under 200 nucleotides in length.

In circRNAs, ICBPS overlaps with both MREs and open reading frames. Therefore, the double-stranded structure of circRNAs might influence their translation and miRNA sponging. Furthermore, the double-stranded structure of circRNA may also promote its bond with RBPs, facilitating its nuclear export and degradation.¹²⁴ However, these hypotheses require confirmation in further studies.

3.1 Sustaining proliferative signaling

The sustained proliferation of cells is a fundamental feature of malignant tumors.²² While normal cells rely on external growth-promoting signals to maintain an active proliferative state, tumor cells can autonomously generate proliferative signaling by disrupting the production of growth factor ligands, expression of receptor molecules, and activation of downstream signaling pathways.^{22, 125}

In HCC, hsa_circRNA_0104348 promotes the proliferation and inhibits apoptosis of HCC cells by regulating the miR-187-3p/RTKN2 axis and modulating Wnt/β-catenin pathways.¹²⁶ CircSMO promotes the proliferation and migration of glioblastoma (GBM) cells via binding to miR-326 to upregulate CEP85.¹²⁷ In gastric cancer (GC), circNFATC3 binds to IGF2BP3 to enhance the stability of IGF2BP3 by suppressing TRIM25-mediated ubiquitination, enhancing the IGF2BP3-CCND1 regulatory axis and elevating CCND1 mRNA stability to promote the proliferation of GC cells.¹²⁸ CircNFATC3 also functions as an oncogene in GC, which promotes cell proliferation via the miR-23b-3p/RAI14 axis.¹²⁹

Various circRNAs also contribute to the malignant proliferation of TC by activating downstream proliferative signaling cascades responsible for cell proliferation. Overexpression of hsa_circ_0007694 in TC cell lines suppresses cell proliferation, migration, and invasiveness. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicates that dysregulated genes in PTC cell lines overexpressing hsa_circ_0007694 are enriched in mTOR and Wnt signaling, as well as cancer-related pathways. These results suggest a role for hsa_circ_0007694 in TC cell proliferation, marked by the suppression of key proteins involved in these pathways, including p-ATK^Ser473, p-GSK3B^Ser9, and Vim.¹³⁰

The Wnt/β-catenin pathway is a pivotal oncogenic signaling pathway in PTC.¹³¹ The downregulation of circRNA_102171 enhances the interaction between CTNNBIP1 and β-catenin, which in turn, inhibits the interaction of β-catenin with TCF3/TCF4/LEF1, resulting in the suppression of target gene expression within the Wnt/β-catenin pathway. Consequently, this downregulation results in the inhibition of PTC cell proliferation both in vitro and in vivo. Notably, circRNA_102171 functions as a molecular sponge for CTNNBIP1 (Figure 1). However, it does not affect the expression of CTNNBIP1 mRNA, instead inhibiting the binding between CTNNBIP1 and β-catenin. Thus, circRNA_102171 facilitates the interaction between β-catenin and TCF/LEF, thereby activating the Wnt/β-catenin pathway and promoting the proliferation of PTC cells.¹³²

Recently, the tumor suppressor circITCH had been identified to be downregulated in PTC tissue.¹³¹ Overexpression of circITCH can impair the proliferation and invasiveness of PTC cell lines, promoting apoptosis. This effect is partially reversed by the transfection of miR-22-3p mimics. Additional experiments revealed that circITCH overexpression suppresses the Wnt/β-catenin pathway through the degradation of β-catenin, achieved by upregulating CBL. Therefore, circITCH regulates the Wnt/β-catenin pathway via the miR-22-3p/CBL axis, resulting in the suppression of PTC cell proliferation.¹³¹

Yao et al.¹³³ showed that hsa_circ_0058124 exhibits the highest fold-change in PTC tissue compared with normal tissue and is significantly upregulated in invasive tumors compared with that in noninvasive tumors. Knockdown of hsa_circ_0058124 results in significant inhibition of proliferation, migration, and invasiveness, accompanied by increased apoptosis. According to ceRNA theory, hsa_circ_0058124 upregulates the NOTCH pathway suppressor, NUMB, by sponging miR-218-5p. Besides, silencing hsa_circ_0058124 upregulates NOTCH3 and GATAD2A. Further in vitro and in vivo experiments suggested that hsa_circ_0058124 regulates NUMB expression and the downstream NOTCH3/GATAD2A signaling axis by sponging miR-218-5p in PTC.¹³³ Additionally, Liu et al.¹³⁴ found that hsa_circ_0058124 promotes the proliferation of PTC cells by modulating the miR-370-3p/LMO4 axis (Figure 1).

Consistent with the microarray profiling results of Peng et al.,⁵⁵ Jin et al.,¹³⁵ and Zhu et al.,¹³⁶ circPSD3 (hsa_circ_0004458) has been identified as upregulated in PTC tissue and cell lines.¹³⁷ Following circPSD3 knockdown in PTC cells, significant suppression of cell proliferation, migration, and invasiveness is observed. Silencing circPSD3 results in the downregulation of PI3K and Akt phosphorylation by increasing miR-637 and decreasing HEMGN levels. Therefore, circPSD3 acts as a sponge for miR-637, modulating HEMGN expression to regulate the PI3K/Akt signaling pathway and promoting PTC progression.¹³⁷ Moreover, circPSD3 might function as an oncogene, promoting the proliferation of PTC cells by modulating the miR-7-5p/METTL7B/MMP2/MMP9¹³⁶ and miR-885-5p/RAC1 axis.¹³⁵

Knockdown of hsa_circ_0067934 induces lower rates of proliferation, migration, and invasiveness while promoting higher apoptosis rates in TC cell lines. These effects were attributed, in part, to the modulation of the epithelial–mesenchymal transition (EMT) and PI3K/Akt signaling pathways.¹³⁸ Additionally, Zhang et al.¹³⁹ found that downregulating hsa_circ_0067934 suppresses TC proliferation by regulating the miR-1304/CXCR1 axis. Hsa_circ_0009294, with the highest expression levels among the ecircRNAs generated from SSU72 in thyroid cell lines, was designated as circSSU72.^{140, 141} Zhang et al.¹⁴¹ confirmed its participation in the proliferation, migration, and invasion of PTC by modulating the miR-451a/S1PR2 axis and downstream Akt pathway. Besides, circNRIP1 could promote the development of PTC via miR-195-5p to regulate the P38 MAPK and JAK/STAT pathways.¹⁴² Silencing the upregulated hsa_circ_0005273 in PTC cell lines suppresses proliferation, migration, and invasiveness. Further experiments revealed that hsa_circ_0005273 acts as an oncogene, accelerating PTC proliferation via the miR-1183/SOX2 axis.¹⁴³ Similarly, CDR1as overexpression promotes proliferation, migration, and invasion while inhibiting apoptosis of PTC cells in vitro by modulating the miR-7/EGFR axis.¹⁴⁴ Silencing circRNA_104565 inhibits PTC cell proliferation in vitro and in vivo. Rescue experiments demonstrated that circRNA_104565 promotes cell proliferation by sponging miR-134 to release ELF2.¹⁴⁵ Silencing circFAT1(e2) inhibits proliferation, migration, and invasiveness of PTC cells by sponging miR-873 to regulate downstream ZEB1.¹⁴⁶

With emphasis on TC, the functional mechanisms of dysregulated circRNAs in hallmarks of TC are summarized in Table 1. To better review the roles of circRNAs in the hallmarks of cancers, representative circRNAs are reviewed based on the roster of hallmark capabilities, providing insights into their involvement in the distinctive features of cancer progression (Figure 2).

3.2 Evading growth suppressors

In addition to achieving self-sufficiency in proliferative signaling, tumor cells possess the ability to circumvent suppressor programs that negatively regulate cell proliferation.^{22, 125} For instance, canonical suppressor genes TP53 and RB often exhibit functional subversion in various tumors.²² Hanahan and Weinberg¹²⁵ highlighted that during tumor development, cell cycle arrest induced by antigrowth signals to block proliferation is evaded and short circuited. Several circRNAs have been demonstrated to be involved in the evasion of growth suppressors and cell cycle arrest in different cancers.

Inactivation of p53 is essential for glioma tumorigenesis, in particular GBM. CDR1as can directly bind to the p53 DBD domain, disrupting the formation of the p53/MDM2 complex to protect p53 from ubiquitination and degradation. Thus, CDR1as contributes to the inhibition of gliomagenesis by directly interacting with proteins rather than serving as miRNA sponges.¹⁹⁰ In BC, hypoxia-inducible circWSB1 binds to the deubiquitinase USP10, suppressing USP10-mediated p53 stabilization and promoting the progression of BC.¹⁹¹ The upregulated circZFR in cervical cancer promotes p-Rb phosphorylation by binding to SSBP1 and activating CDK2/cyclin E1 complexes, which release activated E2F1 to promote the expression of DNA replication-associated genes and accelerate cell cycle progression.¹⁹²

In TC, several circRNAs also participate in the mediation of growth suppressor evasion. CircTP53, derived from TP53, exhibits upregulation in TC tissue compared with that in normal tissue.¹⁴⁷ CircTP53 levels show a negative correlation with p53 expression in TC tissue. Overexpression of circTP53 promotes the viability and proliferation of PTC cells, reducing p21 at both the mRNA and protein levels, and decreasing p53 expression at the protein level without affecting mRNA levels.¹⁴⁷ Cytological experiments confirm that circTP53 might function in TC by sponging miR-1233-3p to release MDM2, acting as an E3 ubiquitin ligase for p53 degradation in the proteasome,¹⁹³ thereby regulating the p53 signaling pathway.¹⁴⁷

CircWDR27 (hsa_circ_0078738) is significantly upregulated in PTC tissue and cell lines compared with that in normal controls,¹⁴⁸ consistent with the microarray profiling results of Ye et al.¹⁵¹ Suppression of circWDR27 arrests cells in the G0/G1 phase, promotes apoptosis, and inhibits proliferation, migration, and invasiveness of PTC cells. In vitro and in vivo experiments indicated that circWDR27 serves as a tumor promoter in PTC by modulating the miR-215-5p/TRIM44 axis to accelerate the cell cycle.¹⁴⁸

Silencing hsa_circ_0058129 inhibits PTC progression by regulating the miR-873-5p/FSTL1 axis to induce cell cycle arrest.¹⁴⁹ Similarly, circFNDC3B inhibition causes G1-phase cell cycle arrest, restrains PTC cell proliferation, migration, and invasion, and promotes apoptosis.¹⁵⁰ Rescue experiments confirm that circFNDC3B promotes PTC cell cycle progression via the miR-1178/TLR4 axis.¹⁵⁰ Overexpression of circFOXM1 (hsa_circ_0025033) in vitro promotes cell cycle progression and enhances the proliferation of PTC cells, while circFOXM1 knockdown has the contrary effects. Predominantly enriched in the cytoplasmic fractions of PTC cells, circFOXM1 participates in PTC tumorigenesis by regulating the miR-1179/HMGB1 network.¹⁵¹ Moreover, Pan et al.¹⁵² found that overexpressing circFOXM1 inhibits apoptosis and enhances the viability, proliferation, migration, and invasiveness of PTC cells by suppressing miR-1231 and miR-1304. The interaction of miR-1231/miR-1304 with circFOXM1 has a synergistic effect.¹⁵² This phenomenon confirms that circRNAs could function as ceRNAs by sponging different miRNAs to evade cell cycle arrest modulated by tumor suppressors.

3.3 Resisting cell death

The maintenance and expansion of tumors are influenced by both cell proliferation and death.¹²⁵ Generally, three major pathways are involved in cell death: apoptosis, autophagy, and necrosis.^{1, 22}

In retinoblastoma, knocking down circFAM158A promotes apoptosis in vitro and in vivo by modulating the miR-138-5p/SLC7A5 axis.¹⁹⁴ Downregulation of circCCS in lung cancer cells promotes apoptosis in vitro via regulating the miR-383/E2F7 axis.¹⁹⁵ In gastrointestinal stromal tumors (GISTs), circSMA4 inhibits apoptosis via the miR-494-3p/KIT axis and by modulating the downstream JAK/STAT signaling pathway.¹⁹⁶ CircPTPN22 stimulates the phosphorylation of Akt and Erk via the miR-6788-5p/PAK1 axis, thus mediating autophagy in GC cells.¹⁹⁷ Additionally, circDHX8 can competitively bind to RNF5, inhibiting the interaction between ATG2B and RNF5 to maintain the stability of ATG2B protein, thus promoting autophagy and tumor development in GC.¹⁹⁸

In TC, most research has focused on the roles of circRNAs in apoptosis. Silencing or overexpression of circNCOR2 (hsa_circ_0000461) increases or inhibits PTC cell apoptosis, respectively. Primarily distributed in the cytoplasm of PTC cells, circNCOR2 functions via the post-transcriptional regulation of the miR-615a-5p/MTA2 axis.¹⁵³ Knockdown of hsa_circ_0000644 or circPRMT5 promotes apoptosis and inhibits the proliferation, migration, and invasiveness of PTC cell lines, which could be reversed by overexpressing E2F3 or inhibiting miR-1205 or miR-30c. Considering that E2F3 is a master regulator of DNA damage-induced apoptosis,¹⁹⁹ circPRMT5 and hsa_circ_0000644 might serve as carcinogenic circRNAs to suppress PTC apoptosis through modulating the miR-30c/E2F3 and miR-1205/E2F3 axes, respectively.^{154, 155}

Silencing of hsa_circ_0001666 promotes apoptosis and inhibits cell proliferation both in vitro and in vivo, which could be reversed by overexpression of EVT4 or inhibition of miR-330-5p, miR-193a-5p, or miR-326 in vitro. Upregulation of hsa_circ_0001666 in PTC prevents cell death and plays an oncogenic role by regulating EVT4 via acting as a miRNA sponge.¹⁵⁶ Silencing circRPS28 (hsa_circ_0049055) induces apoptosis in PTC cells and blocks their proliferation, migration, and invasiveness. CircRPS28, mainly distributed in the cytoplasm, might function as an oncogene in PTC by sponging miR-345-5p to modulate FZD8, thereby preventing cell death.¹⁵⁷

Knockdown of upregulated circTIAM1 (hsa_circ_0061406) promotes apoptosis of PTC cells and decreases their migration and proliferation abilities, which can be reversed by inhibiting miR-646. HNRNPA1 is a target of miR-646, and overexpression of HNRNPA1 reverses the antitumor effects of miR-646 overexpression in PTC cells. Therefore, circTIAM1 inhibits PTC apoptosis via the miR-646/HNRNPA1 axis.¹⁵⁸ Silencing of the upregulated hsa_circ_0011385 promotes apoptosis and induces cell cycle arrest in PTC cells by modulating miR-361-3p.¹⁵⁹ Similarly, silencing circBACH2 enhances apoptosis of PTC cells, and a mechanistic study suggested that circBACH2 might promote PTC by regulating apoptosis via the miR-139-5p/LMO4 axis.¹⁶⁰ Tao et al.¹⁶¹ found that circPVT1 plays an essential role in PTC apoptosis and progression by modulating miR-126, a tumor suppressor in PTC.^{200, 201} Meanwhile, hsa_circ_0102272 might serve as an oncogene in TC by regulating apoptosis; however, the underlying regulatory mechanism requires further investigation.¹⁶² These results suggest that circRNAs may be a novel therapeutic target for promoting cell death in TC.

Few studies have been conducted on the role of downregulated circRNAs in PTC apoptosis. CircNEURL4 (hsa_circ_0041821) is downregulated in PTC tissue and cell lines.¹⁶³ Overexpression of circNEURL4 stimulates PTC cell apoptosis and inhibits PTC cell proliferation in vitro and tumor formation in vivo, which could be reversed by overexpression of miR-1278. CircNEURL4, mainly located in the cytoplasm, might function as a “sponge” to target miR-1278, liberating LATS1 to modulate the apoptosis progress of PTC.

Recent research has focused on the roles of circRNAs in autophagy. The upregulated expression of circEIF6 (hsa_circ_0060060) in TC tissue⁵⁵ was confirmed in five pairs of ATC tissue and paired normal tissue and in ATC and PTC cell lines compared with that in normal cell lines.¹⁶⁴ Cisplatin treatment results in the upregulation of circEIF6 and downregulation of miR-144-3p. TGF-α levels and LC3 II/LC3 I ratios are increased, and cleaved poly (ADP-ribose) polymerase (PARP), cleaved Caspase3, and p62 levels are decreased by overexpression of circEIF6. Furthermore, the effect of circEIF6 overexpression could be reversed by miR-144-3p mimics. Together with additional GFP-LC3 puncta detection used for testing autophagy, these results suggest that circEIF6 induces autophagy and promotes proliferation by upregulating TGF-α during cisplatin treatment, which could be reversed by miR-144-3p.¹⁶⁴

Ferroptosis, an iron-dependent form of nonapoptotic regulated cell death, has garnered considerable attention.²⁰² As a reactive oxygen species (ROS)-dependent form of cell death, ferroptosis is characterized by two main biochemical features: lipid peroxidation and iron accumulation. Recently, Li et al.²⁰³ found that circSTIL suppresses ferroptosis in colorectal cancer via the miR-431/SLC7A11 signaling axis. CircLRFN5 could modulate ferroptosis in GBM by binding to the PRRX2 protein and promoting its degradation, which downregulates the ferroptosis suppressor GCH1.²⁰⁴ In PTC cells, Wang et al.¹⁶⁵ observed that silencing hsa_circ_0067934 increases the levels of ferroptosis-related markers, including Fe²⁺, iron, and ROS, producing an effect similar to that of erastin stimulation. Further experiments indicated that hsa_circ_0067934 regulates ferroptosis, apoptosis, and proliferation of PTC by modulating the key ferroptosis-negative regulator SLC7A11 through sponging miR-545-3p.¹⁶⁵

3.4 Enabling replicative immortality

Generally, cultured cells undergo senescence and subsequently enter a crisis phase after repeated cycles of cell division. This process is regulated by telomeres, which protect the ends of chromosomes and shorten progressively in nonimmortalized cells during each round of DNA replication.⁵ In tumor cells capable of immortalized division, the specialized DNA polymerase known as telomerase is expressed to circumvent this replicative barrier. By adding telomere repeat segments to telomeric DNA, telomerase extends telomeres.²² As the core catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) plays essential roles in the tumorigenesis and development of various cancers.²⁰⁵

In HCC, Zhang et al.²⁰⁶ investigated the effects of TERT promoter mutations on the expressions of ncRNAs. In the mutant promoter group, 21 circRNAs were significantly upregulated, and 23 circRNAs were significantly downregulated. Among them, bioinformatic analysis indicated that hsa_circ_0003154, hsa_circ_0008952, and hsa_circ_0031584 could play essential roles in the tumorigenesis of HCC.²⁰⁶ In colorectal cancer, hsa_circ_0020397 enhances cell viability and invasion of cancer cells and suppresses their apoptosis by regulating the expression of miR-138 target genes, including PD-L1 and TERT.²⁰⁷ Wang et al.²⁰⁸ found that silencing hsa_circ_0000263 in Hela cells inhibits telomerase activity and promotes apoptosis and radiosensitivity by modulating the miR-338-3p/TERT axis. Additionally, circWHSC1 was shown to promote ovarian cancer progression by upregulating TERT through the sequestration of miR-1182.²⁰⁹

3.5 Inducing or accessing vasculature

Similar to normal tissue, tumor tissue requires vascularization to provide nutrients and oxygen, meeting the increasing metabolic demands of malignant cells while facilitating the removal of metabolic waste and carbon dioxide.^{22, 210} Tumor vasculature can develop through angiogenesis or by co-opting normal tissue vessels, principally via invasion and metastasis.²³ Several circRNAs are known to participate in angiogenesis.

The upregulated circFNDC3B in oral squamous cell carcinoma (OSCC) stimulates angiogenesis by accelerating the ubiquitin degradation of FUS and promoting VEGFA expression and angiogenesis.²¹¹ In GC, after being transported from the nucleus to the cytoplasm, m⁶A-modified circPAK2 interacts with IGF2BPs, forming a circPAK2/IGF2BPs complex to stabilize VEGFA mRNA, thereby promoting angiogenesis and lymph node metastasis (LNM).²¹² Hsa_circ_0000520 acts as a scaffold, promoting the binding of UBE2V1/UBC13 to Lin28a, which facilitates the ubiquitous degradation of Lin28a in bladder cancer. By increasing PTEN mRNA stability and suppressing the PI3K/AKT pathway, the vasculogenic mimicry (VM) formations of bladder cancer cells are significantly inhibited.²¹³ Similarly, hsa_circ_0000758 accelerates the angiogenesis of bladder cancer by regulating the miR-1236-3p/ZEB2 axis.²¹⁴ Additionally, hsa_circ_0084043, derived from colorectal cancer-associated fibroblasts (CAFs), had been shown to induce angiogenesis by sponging miR-140-3p, thereby regulating the functions of the VEGF signaling pathway.²¹⁵

Members of the fibroblast growth factor (FGF) family are endowed with potent proangiogenic activities. Activation of the FGF/FGF receptor (FGFR) system may lead to neovascularization in various human tumors, supporting tumor progression and metastatic dissemination.²¹⁶ Among them, the role of FGFR1 has been demonstrated previously.²¹⁷ In PTC, circRAPGEF5 (hsa_circ_0001681) modulates FGFR1 expression by regulating miR-198,⁵³ implying its potential influence on angiogenesis in PTC cells. The tumor-promoting roles of circRAPGEF5 that enhance the proliferation, migration, and invasiveness of PTC cells have been described⁵³; however, further experimental evidence of circRAPGEF5 stimulating angiogenesis is required. Another circRNA derived from RAPGEF5, hsa_circ_0079558, could also regulate FGFR1 expression by modulating the expression of miR-198, which in turn could modulate the angiogenesis of PTC cells.¹⁶⁶ Similarly, members of the VEGF family have been identified as inducers of tumor angiogenesis.²¹⁶ In vivo and in vitro experiments confirmed that circPVT1 regulates the expression of miR-195 to modulate the activities of VEGFA and the Wnt/β-catenin signaling pathway.¹⁶⁷ Hence, circPVT1 might play a carcinogenic role in PTC by inducing angiogenesis.

Knockdown of hsa_circ_0011058 inhibits angiogenesis in PTC cells, which manifests as reduced tube formation and downregulation of the angiogenesis activators, VEGFA and FGF-2. The proliferation of PTC cells is also inhibited, whereas apoptosis and radiosensitivity of PTC cells are enhanced. Mainly distributed in the cytoplasm, hsa_circ_0011058 has been shown to regulate YAP1 by sponging miR-335-5p. In vivo experiments suggested that silencing hsa_circ_0011058 inhibits the formation of xenograft tumors and decreases microvessel density in xenograft tumors. In summary, hsa_circ_0011058 is involved in angiogenesis, proliferation, apoptosis, and radioresistance in PTC by modulating the miR-335-5p/YAP1 axis.¹⁶⁸

Knockdown of circRASSF2 (hsa_circ_0059354) in PTC cells suppresses angiogenesis, which was assessed using a tube formation assay of human umbilical vein endothelial cells cultured in a PTC cell suspension. Besides, silencing circRASSF2 suppresses cell proliferation, migration, and invasion and promotes apoptosis in PTC cells.¹⁶⁹ Rescue experiments demonstrated that circRASSF2 serves as an oncogene in PTC by modulating the miR-766-3p/ARFGEF1 axis.¹⁶⁹ Additionally, Wu et al.¹⁷⁰ found that circRASSF2 regulates proliferation, migration, invasiveness, cell cycle progression, and apoptosis of PTC cells through the miR-1178/TLR4 axis.

In brief, the above studies suggest that circRNAs play crucial roles in vascular dysregulation, promoting the progression of this hallmark in TC.

3.6 Activating invasion and metastasis

Invasion and metastasis are representative hallmarks of malignant tumors and are usually associated with poor prognosis.^{22, 24, 125} During the multistep process of invasion and metastasis, cancer cells undergo morphological alterations and changes in cell-cell or cell-matrix interactions, accompanied by dysregulation of E-cadherin, N-cadherin, and extracellular proteases.^{1, 22, 125} EMT is arguably essential for modulating invasion and metastasis.²²

The upregulated circFNDC3B in OSCC promotes EMT and lymphangiogenesis by sequestering miR-181c-5p, leading to the upregulation of SERPINE1 and PROX1.²¹¹ Additionally, another circRNA derived from FNDC3B, hsa_circ_0003692, could be translated to a novel protein-FNDC3B-267aa in GC, which inhibits GC migration and metastasis by directly binding to c-Myc and promoting its degradation, thereby suppressing the downstream c-Myc-Snail/Slug axis.²¹⁸ Similarly, circYAP encodes a novel truncated protein, YAP-220aa, which binds to LATS1 and leads to YAP dephosphorylation and nuclear translocation, thereby activating a host of metastasis-promoting genes in colorectal cancer.²¹⁹ Furthermore, circYAP transcription is activated by YAP, thus forming a positive feedback loop promoting the liver metastasis of colorectal cancer.²¹⁹ In addition, hsa_circ_0088036 promotes the invasion and metastasis abilities of bladder cancer cells through the miR-140-3p/FOXQ1 signaling axis.²²⁰

LNM is a well-known risk factor for TC recurrence and poor outcomes.^{46, 221, 222} Mounting evidences suggest that circRNAs can promote EMT in TC to facilitate the invasion-metastasis cascade. Overexpression of circRNA_102002 in PTC causes a noticeable shift in cellular morphology to a spindle shape with increased intercellular mass. This is accompanied by the downregulation of E-cadherin and the upregulation of N-cadherin, Vimentin, Slug, Twist, MMP2, and MMP9, promoting the EMT process and enhancing the migration and invasion of PTC cells. In addition, silencing of circRNA_102002 inhibits lung metastasis of PTC cells in vivo. Further mechanistic investigation suggested that circRNA-102002 promotes EMT, as well as the migration and invasiveness of PTC cells, to facilitate PTC metastasis by modulating the miR-488-3p/HAS2 axis.¹⁷¹

Consistent with the microarray profiling results of Peng et al.,⁵⁵ circLDLR (hsa_circ_0003892) is upregulated in PTC tissue and cell lines.^{172, 173} Silencing circLDLR causes decreased migration, invasion, and proliferation and promotes PTC cell apoptosis, resulting in xenograft tumors of smaller size and lighter weight.^{172, 173} Focusing on the underlying mechanism, Gui et al.¹⁷² found that overexpression of circLDLR increases Twist1 levels and decreases E-cadherin expression by modulating LIPH through sponging miR-195-5p to promote the migration and invasion of PTC cells. Jiang et al.¹⁷³ also observed that circLDLR knockdown decreases the expression of MMP2 and MMP9 via the miR-637/LMO4 pathway, inhibiting migration and invasiveness of PTC cells. Knockdown of hsa_circ_0008274 significantly decreases the expression of ICAM-1, fibronectin, and vitronectin, thereby suppressing cell migration and adhesion, which is abrogated by SLC7A11 overexpression. Mechanistic studies showed that hsa_circ_0008274 modulates SLC7A11 expression by acting as a sponge for miR-154-3p to promote PTC migration and invasion.¹⁷⁴

Chu et al.¹⁷⁵ discovered that circRUNX1 (hsa_circ_0002360) is upregulated in PTC tissue and cell lines compared with those of normal controls. Higher expression levels of circRUNX1 in PTC tissue are associated with larger tumor size, advanced TNM stage, extrathyroidal extension, and LNM, implying that circRUNX1 is related to stronger migration and invasiveness of PTC. This was confirmed in an in vitro study, as overexpressing circRUNX1 promoted migration, invasion, and proliferation of PTC cells through the miR-296-3p/DDHD2 axis, whereas silencing it exerted opposite functions on PTC cells.

Similarly, hsa_circ_0001018 expression is remarkably increased in PTC tissue and cell lines and is associated with TNM staging, LNM, and distant metastasis in PTC tissue. Overexpression of hsa_circ_0001018 reduces the expression of E-cadherin, enhances the expression of vimentin and fibronectin, promotes the migration and invasion of PTC cells, reduces cell cycle arrest at the G1 phase, and inhibits cell apoptosis by modulating the miR-338-3p/SOX4 axis.⁵⁴

High circVANGL1 expression is associated with LNM and advanced TNM stages.¹⁷⁶ Overexpression of circVANGL1 enhances the migration, proliferation, and invasiveness of PTC cell lines and increases the expression levels of N-cadherin and vimentin, whereas decreasing the expression levels of E-cadherin by modulating the miR-194/ZEB1 axis and the downstream EMT pathway.¹⁷⁶ In addition, the knockdown of circCCDC66 suppresses migration, invasiveness, and mouse xenograft tumor generation in PTC cells through the miR-129-5p/LARP1 axis and the downstream EMT pathway in the development of PTC.¹⁷⁷ Wei et al.⁵⁷ found that the migration, invasion, and proliferation of PTC cells are suppressed by circZFR (hsa_circ_0072088) knockdown, which is attenuated by the ectopic expression of TC1. Further results suggest that the circZFR/miR-1261/TC1 cascade might act as a potential target for inactivating invasion and metastasis in PTC therapy.

In vitro and in vivo experiments revealed that overexpressing circEIF3I promotes the migration, invasion, and proliferation of PTC cell lines via the miR-149/KIF2A axis.¹⁷⁸ Hsa_circ_0062389 stimulates PTC migration and development partly via the miR-1179/HMGB1 axis,¹⁷⁹ whereas hsa_circ_0039411 promotes the migration and invasion of PTC by regulating the expression of ABCA9/MTA1 via miR-1179/miR-1205.¹⁸⁰ Therefore, these interactome ceRNA networks are implicated in the invasion and metastasis of PTC and have potential as therapeutic targets in clinical practice.

3.7 Deregulating cellular metabolism

During dysregulated cancer cell proliferation, the energy metabolism of cancer cells is reprogrammed to meet the demands of rapid cell growth and division.²² By consuming more glucose and producing more lactate, cancer cells prefer glycolysis even in the presence of oxygen and functioning mitochondria.²²³ This phenomenon, termed the Warburg effect, is an inefficient means of generating ATP compared with oxidative phosphorylation.²²³ Dysregulation of energy metabolism is closely associated with other cancer hallmarks, such as sustained proliferative signaling and evasion of growth suppressors.²²⁴

In non-small cell lung cancer (NSCLC), circSLC25A16 stimulates glycolysis and proliferation of NSCLC cells via the miR-488-3p/HIF-1α axis, facilitating the transcription of LDHA.²²⁵ Ma et al.²²⁶ found that circLIPH promotes glycolysis in pancreatic cancer by sponging miR-769-3p and modulating the downstream GOLM1/PI3K/AKT/mTOR pathways. In pancreatic ductal adenocarcinoma (PDAC), circRREB1 increases PGK1 phosphorylation, enhancing glycolytic flux by disrupting the interaction between PTEN and PGK1. Additionally, circRREB1 directly binds to YBX1, promoting its nuclear translocation and stimulating WNT7B transcription, thereby activating the Wnt/β-catenin pathway to maintain stemness in PDAC.²²⁷ Hsa_circ_0004674 promotes the glycolysis and progression of osteosarcoma by regulating the expression of glycolysis-related genes through the miR-140-3p/TCF4 axis.²²⁸ Suppression of this axis reduces glucose consumption and lactate accumulation in cancer cells.

Cellular metabolism dysregulation also plays an important role in TC. Glucose metabolic profiling demonstrated that hsa_circ_0011290-depletion suppresses glucose uptake, decreases lactate production, and increases ATP levels. Hsa_circ_0011290 depletion also inhibits proliferation and induces apoptosis in PTC cell lines. FSTL1 transcripts are markedly downregulated in response to hsa_circ_0011290 knockdown, which could be reversed by concurrent miR-1252 inhibition. Similarly, the compromised malignant phenotypes induced by hsa_circ_0011290 silencing could subsequently be stimulated by miR-1252 inhibition. In summary, hsa_circ_0011290 modulates cellular metabolism of PTC via the miR-1252/FSTL1 axis.¹⁸¹

HK2, a critical participant in the Warburg effect, is upregulated in PTC tissue and cell lines and is negatively correlated with miR-15a-5p expression in PTC tissue. HK2 overexpression could reverse the inhibitory effect of miR-15a-5p on glycolysis and the malignant phenotypes of PTC cells.¹⁸² Besides, silencing circNUP214 inhibits cell glycolysis, proliferation, migration, and invasion while it induces apoptosis in PTC cell lines, which can be reversed by inhibiting the expression of miR-15a-5p. Therefore, circNUP214 promotes anaerobic glycolysis and PTC progression via the miR-15a-5p/HK2 axis.¹⁸² At the same time, Li et al.¹⁸³ found that circNUP214 promotes PTC development by modulating the miR-145/ZEB2 axis.

Silencing circPRKCI (hsa_circ_0122683) inhibits glucose uptake and lactate production, suppresses the proliferation of PTC cells, and arrests them in the G0/G1 phase, which can be reversed by inhibiting miR-335.¹⁸⁴ Further experimental results suggested that circPRKCI acts as an oncogenic participant in PTC carcinogenesis and development by regulating cellular metabolism with precise spatiotemporal control of the miR-335/E2F3 axis.¹⁸⁴ Similar to the results of Yao et al.¹³³ and Liu et al.,¹³⁴ Sun et al.¹⁸⁵ found that hsa_circ_0058124 is upregulated in PTC tissue and cell lines compared with those of normal controls. Silencing of hsa_circ_0058124 significantly suppresses the oxygen consumption rate of basal and maximum respiration of TC cells, thereby inhibiting their metabolic activity and suppressing their proliferation, migration, and invasiveness. These effects can be replicated by miR-940 overexpression, and miR-940 inhibition can reverse the suppressive effect of silencing MAPK1 in TC cells.¹⁸⁵ Thus, hsa_circ_0058124 may function through the miR-940/MAPK1 axis during PTC metabolism and progression.

Silencing the upregulated circRAD18 inhibits cell glucose uptake, lactate production, and proliferation, as well as metastasis of PTC cells. The underlying downstream molecule was confirmed to be PDK1, a metabolic protein involved in glucose intake, regulated by the circRAD18/miR-516b axis.¹⁸⁶ Similarly, circPUM1 accelerates PTC tumorigenesis by dysregulating cellular metabolism via the miR-21-5p/MAPK1 signal axis.¹⁸⁷ Thus, circRNAs are essential players in the dysregulated cellar metabolism of various cancers, including TC.

3.8 Avoiding immune destruction

According to the theory of immune surveillance, human cells are dynamically monitored by the immune system, which is capable of discerning and eliminating the newly transformed malignant cells.²² Disruption at any step of the cancer-immunity cycle can impair the immune system's ability to generate effective anticancer immune responses to control tumor progression.²²⁹ Moreover, the tumor microenvironment (TME) has been shown to play a critical role in modulating the anticancer immune response.²²⁹

Liu et al.²³⁰ found that circIGF2BP3 upregulation in NSCLC inhibits CD8⁺ T-cell responses and causes tumor immune evasion by regulating the miR-328-3p/miR-3173-5p/PKP3 axis and stabilizing the PD-L1 protein in an OTUB1-dependent manner. Serving as a scaffold to enhance the interaction between TRIM25 and IGF2BP, circNDUFB2 inhibits the progression of NSCLC by promoting ubiquitination and degradation of IGF2BPs. In addition, circNDUFB2 overexpression triggers immune responses in NSCLC cells by mediating RIG-I–MAVS signaling cascades, increasing the recruiting of CD8⁺ T cells and DCs into the TME.²³¹ Furthermore, circFAM53B can be translated into a specific peptide that can be presented by DCs to prime naive T cells, driving antigen-specific adaptive anticancer immunity in BC cells.²³² Similarly, circFAT1 can regulate the recruitment of CD8⁺ cells into the TME and promote immune evasion by activating STAT3.²³³ In colorectal cancer, circREEP3 promotes tumor progression by recruiting the chromatin remodeling protein CHD7 to the FKBP10 promoter, activating its transcription. Additionally, circREEP3 inhibits anti-tumor immunity by enhancing RNF125-dependent degradation of RIG-1, thereby regulating IFN-β production and CD8⁺ T cell infiltration into the TME.²³⁴

3.9 Genome instability

Genome instability is inherent in most types of cancer cells.²² The irreversible activation of oncogenes and the silencing of tumor suppressor genes are necessary for the initiation of various cancers.²³⁵ More than 500 oncogenic driver mutations have been identified in over 28,000 cancer exomes.²³⁶ It is important to note that these mutations could also be found in nontumor tissues, which suggests that part of these mutations can drive tumor generation only when they cooperate with other irritants or hallmarks of cancer.²³⁵

In a Cadmium (Cd)-induced lung tumor model, DNA damage was identified as a key factor promoting tumorigenesis. The downregulation of circCIMT in this model functioned as a tumor suppressor that inhibited DNA damage by directly binding to APEX1, thereby regulating the DNA base excision repair (BER) pathway, which can remove small and nonhelix-distorting base damages.²³⁷ CircSMARCA5 can form an R-loop with its parent gene locus in BC, inhibiting the transcription of its parent gene, SMARCA5. As SMARCA5 is a key player in chromatin remodeling by providing a structural basis for recruiting different DNA damage repair factors in DNA damage regions, circSMARCA5 inhibits DNA damage repair in BC.²³⁸ Hsa_circ_0007919 has been shown to increase LIG1 transcription by recruiting FOXA1 and TET1, thereby promoting multiple DNA repair pathways in PDAC.²³⁹

3.10 Tumor-promoting inflammation

As an enabling characteristic, tumor-promoting inflammation can provide bioactive molecules to the TME that contribute to multiple hallmark capabilities, including growth, survival, proangiogenic factors, extracellular matrix-modifying enzymes, and inductive signals.²² CircRNAs have been shown to participate in the regulation of immune cells and inflammatory cytokines, modulating the inflammatory TME in various tumors that support tumor progression.^{224, 240}

Chronic inflammation is an essential promoter of all steps in tumor progression and is associated with about 20% of cancer deaths worldwide.²⁴¹ Sun et al.²⁴¹ showed that TNFα accelerates the expression of circDMD, which enhances tumorigenesis by activating the canonical NF-κB pathway and promoting VEGFR3 expression through R-loop formation at its promoter, while also regulating the miR-4711-5p/KDM5A axis. In NSCLC, circNOX4 promotes the secretion of interleukin 6 (IL-6) to establish an inflammatory niche via the miR-329-5p/FAP axis, enhancing tumor progression.²⁴² Similarly, IL-6 is also regulated by circFOXK2 in NSCLC, which can sponge miR-149-3p.²⁴³ In BC, tumor cell-derived exosomal circSERPINE2 can be absorbed by tumor-associated macrophages (TAMs), enhancing their IL-6 secretion. The increased IL-6, in turn, elevates EIF4A3 and CCL2 levels within tumor cells, which upregulate circSERPINE2 biogenesis in tumor cells and promote the recruitment of TAMs in a positive feedback mechanism.²⁴⁴

In TC, circRNAs also regulate the secretion of inflammatory cytokines and chemokines. IL-6 expression is noticeably upregulated in PTC tissue, positively correlated with circRNA_103598 expression, and negatively correlated with miR-23a-3p expression. Knockdown of circRNA_103598 markedly suppresses the proliferation of PTC cells, which is reversed by the introduction of miR-23a-3p. MiR-23a-3p expression is markedly decreased and negatively correlated with upregulated circRNA_103598 or IL-6 expression in PTC tissue. Suppression of PTC cell proliferation and replication of the oncolytic vaccinia virus (OVV) mediated by miR-23a-3p inhibition is abolished by IL-6 overexpression. Therefore, circRNA_103598 is involved in the progression of PTC and OVV-mediated antitumor effects via modulation of the miR-23a-3p/IL-6 axis.¹⁸⁸

However, the role of circRNAs in the regulation of various immune cells and the TME inflammation of TC requires further exploration, considering that inflammation is an essential player in oncogenesis and recurrence.^{245, 246}

3.11 Unlocking phenotypic plasticity

Normal cells are destined to follow a pathway that results in terminal differentiation to maintain the homeostatic functions of the organs. Evasion from end-stage differentiation by unlocking the normally restricted phenotypic plasticity has been recorded as a critical process in tumorigenesis.²⁴⁷ Phenotypic plasticity manifests through three main mechanisms: dedifferentiation, blocked differentiation, and transdifferentiation.²³

In GBM, exosomal circCMTM3 derived from GBM stem cells (GSCs) has been shown to promote the phenotypic transition from differentiated glioma cells (DGCs) to VM. Once internalized by DGCs, circCMTM3 binds to CNOT4, suppressing the ubiquitination and degradation of STAT5A and STAT5B. This binding enhances the phosphorylation of STAT5A via the protein scaffold function of circCMTM3, which further activates the transcription of provasculogenic factors.²⁴⁸ In NSCLC, circNOX4 plays an essential role in the phenotypic conversion of normal fibroblasts (NFs) to CAFs.²⁴² Similarly, in PDAC, upregulation of circCUL2 in NFs induces the transition to inflammatory CAF phenotype, which promotes tumor development through IL-6 secretion by regulating the miR-203a-3p/MyD88/NF-κB/IL-6 axis.²⁴⁹ Additionally, the levels of circZEB1 in melanoma cells remain high during phenotypic switching from cancer cells lacking cancer stem cells (CSCs) markers to those expressing CSCs markers, underscoring its regulatory role in the phenotypic plasticity of melanoma.²⁵⁰

Dedifferentiation of PTC cells is related to decreased expression or loss of the sodium iodide symporter (NIS) and deficiencies of NIS in the plasma membrane, which result in the failure of iodine uptake in thyroid cells.²⁵¹ Recently, Sa et al.¹⁸⁹ found that higher expression levels of the aryl hydrocarbon receptor (AhR) are associated with the dedifferentiation of PTC. AhR antagonists inhibit proliferation and increase ¹²⁵I uptake and the expression of NIS in PTC cells, localized to the membrane of PTC cells, suggesting that AhR antagonists promote the differentiation of PTC cells. CircSH2B3 (hsa_circ_0006741), upregulated in PTC cell lines compared with that in normal cell lines, is downregulated after treatment with AhR antagonists. Furthermore, silencing circSH2B3 upregulates the expression of NIS in PTC cells, increases ¹²⁵I uptake, and inhibits proliferation. However, overexpression of circSH2B3 leads to contrary effects and reverses the differentiation effects induced by AhR antagonists. MiR-4640-5p suppression might partially reverse the differentiation effect of silencing circSH2B3, whereas IGF2BP2 inhibits the differentiation effect induced by miR-4640-5p overexpression. In addition, as an m⁶A reader, IGF2BP2 enhances the translocation of AhR from the cytoplasm to the nucleus to promote its function. Therefore, circSH2B3 induces PTC dedifferentiation by modulating the miR-4640-5p/IGF2BP2 axis.¹⁸⁹

3.12 Senescence

Senescence is an irreversible process that occurs during aging, wherein dysfunctional or otherwise unnecessary cells are inactivated or deleted, serving as a protective mechanism for maintaining tissue homeostasis. During this process, cell morphology and metabolism undergo changes, and cell division is inhibited. Most importantly, senescence-associated secretory phenotype (SASP) is activated during cellular senescence. While cellular senescence is generally accepted as a tumor-antagonizing player, increasing evidence suggests that senescence can act as a tumor-promoting factor in certain contexts.²³ SASP is the principal mechanism through which senescent cells promote tumor development, which could transmit hallmark capabilities to adjacent cells in the TME via paracrine signaling with various molecules.²³

Many malignant tumors are associated with aging and senescence, including lung cancer, HCC, colorectal cancer, GC, and BC, among others.²⁵² Various studies have provided evidence that circRNAs play important roles in those tumors.²⁵² One recent review summarized the pathways through which circRNAs regulate cellular senescence.²⁵³ On the other hand, Li et al.²⁵⁴ found that patients with nasopharyngeal carcinoma (NPC) who suffered from distant metastasis display senescence-related phenotypes. Silencing circWDR37 enhances cisplatin- or gemcitabine-induced cellular senescence in NPC but suppresses the migration and invasion capabilities of senescent NPC cells in vitro.²⁵⁴ Mechanistically, circWDR37 initiates PKR homodimerization and autophosphorylation. Phosphorylated PKR then induces IKKβ phosphorylation, which binds to and releases p65 from IκBα, triggering NF-κB activation. This activation stimulates the transcription of CCND1 and SASP component genes. Taken together, circWDR37 regulates the senescence-driven metastasis in NPC by modulating PKR activity.²⁵⁴

However, overexpression of circLARP4 induces senescence and inhibits tumor progression in HCC by regulating the miR-761/RUNX3 axis and the downstream p53/p21 pathway.²⁵⁵ CircDnmt1 inhibits cellular senescence and promotes tumor growth by stimulating cellular autophagy through the nuclear translocation of p53 and Auf1.²⁵⁶ In colorectal cancer, circDNA2v directly binds to IGF2BP3, maintaining its stability and sustaining the mRNA levels of c-Myc. Silencing circDNA2v results in the downregulation of c-Myc, which induces tumor cell senescence, release of proinflammatory mediators, and recruitment of cytotoxic T cells.²⁵⁷

Thus, the authentic roles of circRNAs and senescence in various cancers require further exploration. Furthermore, Ding et al.²⁵⁸ demonstrated that human umbilical cord mesenchymal stem cell-derived exosomes (UMSC-Exos) prevent cardiac senescence by delivering circHIPK3, which serves as a scaffold to recruit ubiquitin ligase to degrade HuR. However, whether UMSC-Exos could exhibit similar antisenescence functions in various tumors necessitates further research.

3.13 Nonmutational epigenetic reprogramming

The notion of nonmutational epigenetic reprogramming of gene expression is well acknowledged as the critical mechanism regulating embryonic development and organogenesis.²³ Complementary to the theory that tumors result from genomic instability and mutation, named permanent genetic alterations, nonmutational epigenetic reprogramming refers to the gene expression changes modulated by epigenetic manipulations independent of genome reprogramming.²³ Surging studies demonstrated that circRNAs promote tumor development and progression by participating in epigenetic reprogramming.

In TNBC, multiple oncogene transcription processes are regulated by YBX1, whose O-GlcNAcylation is modulated by the circZEB1/miR-337-3p/OGT axis.²⁵⁹ Moreover, Lan et al.²⁶⁰ showed that circBRAF can recruit KDM4B to enhance MMP9 and ADAMTS14 expression via H3K9me3 modification in TNBC. Furthermore, circBRAF interacts with IGF2BP3 to regulate mRNA stability through m⁶A modification, enhancing the expression of VCAN, FN1, CDCA3, and B4GALT3 in TNBC.²⁶⁰ In GC, circRHBDD1 binds to IGF2BP2 to inhibit IGF2BP2 ubiquitination and degradation, thereby IGF2BP2 can enhance PD-L1 mRNA stability through m⁶A modification.²⁶¹ Besides, hsa_circ_0000119 promotes ovarian cancer progression by increasing the methylation of CDH13 by regulating the miR-142-5p/DNMT1 axis.²⁶² Furthermore, circGNAO1 can sequester DNMT1 to reduce the methylation of GNAO1 promoter, upregulating the expression of GNAO1 to suppress HCC.²⁶³

3.14 Polymorphic microbiomes

Gut microbiota, the bacteria settled in the human gastrointestinal system, is well acknowledged to be an essential player in the onset and progression of major depressive disorders, Alzheimer's disease,²⁶⁴ and various tumors, including gastrointestinal cancers.^265-269 The interactions between gut microbiota and circRNAs are gaining momentum in research, which would shed light on the oncogenic mechanisms underlying cancers and provide clues to the development of novel therapeutic interventions.²⁷⁰

Gut microbiota modulated by NLRP3 inflammasome deficiency can ameliorate depressive-like behaviors by affecting astrocyte dysfunction via the regulation of circHIPK2.²⁶⁶ Zhu et al.²⁷¹ demonstrated that gut microbiota inhibits tumor metastasis in mice models by regulating the IL-11/circRNA/miRNA axis to modulate the expression of genes involved in the stemness of CSCs and EMT. In GC, helicobacter pylori (H. pylori) upregulates the expression of circMAN1A2 in GC cell lines, which accelerates the progression of GC by sequestering miR-1236-3p to modulate MTA2 expression.²⁷² Interestingly, the induced overexpression of circMAN1A2 by H. pylori is not dependent on CagA, one of the most crucial virulence factors of H. pylori.²⁷² Similarly, circPGD can be upregulated by H. pylori infection, which serves as a tumor promoter in GC.²⁷³ However, circRNA_15430 is downregulated by H. pylori infection, which functions as a tumor suppressor in GC by regulating the miR-382-5p/ZCCHC14 axis.²⁷⁴

In recent years, the intratumoral microbiome has been discovered in various cancer tissues that were previously considered sterile. Recent studies have revealed the roles of the intratumoral microbiome in tumorigenesis and progression, delineating the underlying carcinogenic mechanisms, including epigenetic modifications, metastasis induction, and immune dysfunctions, among others.^{275, 276} However, the interplay and crosstalk between circRNAs and the intratumoral microbiome warrant further research.

4.1 The landscapes of circRNA in TC tissue and predicted ceRNA networks

Although Xu et al.⁴⁹ identified 3777 circRNAs in normal thyroid tissue, the roles of circRNAs in malignant thyroid tissue were not investigated in this study. In a groundbreaking study, Peng et al.⁵⁵ distinguished the expression profiles of circRNAs between TC and benign thyroid tissue, marking the beginning of a novel chapter in understanding TC development and progression (Figure 3). They identified 88 significantly upregulated and 10 downregulated circRNAs in PTC tissue compared with matched normal thyroid tissue, and 129 upregulated and 226 downregulated circRNAs in PTC tissue compared with tissue of benign thyroid lesions.⁵⁵ Among them, 12 upregulated and four downregulated circRNAs were identified in PTC tissue in both comparisons. Notably, the downregulation of hsa_circRNA_100395 appeared to play a critical role in PTC by scavenging miR-141-3p and miR-200a-3p to regulate their downstream cancer-related genes. However, the authentic roles of these circRNAs in the pathogenesis of PTC require further confirmation.⁵⁵

Microarray data obtained by Peng et al.⁵⁵ were subsequently analyzed in other studies to profile the ceRNA network in PTC.²⁷⁷ Differential expression analysis of miRNAs and mRNAs between PTC and normal tissue from The Cancer Genome Atlas database led to the construction of a network involving 12 circRNAs, 33 miRNAs, and 356 mRNAs based on ceRNA theory.²⁷⁷ Five hub genes were selected to refine the ceRNA network, establishing a circRNA-miRNA-hub gene subnetwork, which included axes such as hsa_circ_0011385/hsa-miR-204/CDH2, hsa_circ_0011385/hsa-miR-6777/VCAN, and hsa_circ_0067934/hsa-miR-375/IGFBP3/FSTL3.²⁷⁷ KEGG pathway analysis revealed the mRNAs targeted by key circRNAs were enriched in pathways such as the p53 signaling pathway, cell adhesion molecules, cellular senescence, and transcriptional dysregulation in cancer.

Using a different approach, Lou et al.²⁷⁸ identified the key regulation axis in PTC. Analyzing the microarray profiling results of Peng et al.,⁵⁵ Lou et al.²⁷⁸ recognized circRNAs were significantly differentially expressed when comparing PTC with normal tissues, but not in comparisons between benign lesions and normal tissues. This selection included 13 upregulated and one downregulated circRNA. Among them, the upregulated hsa_circ_0088494 was predicted to target miR-876-3p, which was significantly downregulated in TC tissue and associated with a favorable prognosis. The hsa_circ_0088494/miR-876-3p/CTNNB1/CCND1 axis, deemed important in PTC emergence and progression, was confirmed.

Subsequent studies investigated differentially expressed circRNAs in PTC using microarray analysis.^{56, 151, 170, 279} A comprehensive analysis identified 678 significantly upregulated and 459 downregulated circRNAs in five PTC tissue samples compared with paired normal tissue samples.¹⁵¹ Similarly, Wu et al.¹⁷⁰ identified 478 upregulated and 446 downregulated circRNAs in five PTC tissue samples compared with paired normal tissue samples, while Guo et al.²⁷⁹ found 74 upregulated and 84 downregulated circRNAs in three PTC tissue samples compared with paired normal tissue samples. Ren et al.⁵⁶ observed that 206 circRNAs were significantly upregulated, and 177 circRNAs were downregulated in PTC tissue compared with that in normal tissue. Bioinformatic analysis indicated that downregulated hsa_circRNA_047771 targets elevated miR-522-3p/miR-153-5p in PTC tissue.⁵⁶

RNA sequencing (RNA-seq) also had been employed to investigate the differentially expressed circRNAs in PTC compared with that in normal thyroid tissue (Figure 3). Lv et al.²⁸⁰ identified 16,569 circRNAs from four paired PTC tissue and neighboring nontumor tissue samples using RNA-seq. Among them, 301 circRNAs were upregulated and 419 were downregulated in PTC tissue. In another study, Lan et al.²⁸¹ identified 41 upregulated and 46 downregulated circRNAs in PTC tissue samples from three women with PTC compared with matched normal tissue. KEGG analysis of the parental genes of dysregulated circRNAs showed that the most enriched pathway was autoimmune thyroid disease, a known risk factor for TC.³³

Apart from comparing circRNA expression patterns in TC and benign thyroid tissue, studies have delved into comparing circRNA expression patterns in invasive and noninvasive TC tissue. Using a ceRNA microarray, the expression profiles of circRNAs were compared in four invasive PTC tissue samples (with extrathyroidal extension and metastasis), four noninvasive PTC tissue samples (with no extrathyroidal extension or metastasis), and four matched adjacent normal tissue samples.¹³³ A total of 377 circRNAs were upregulated and 230 were downregulated in invasive tumors compared with that in adjacent normal tissue. Compared with that in noninvasive tumors, 42 circRNAs were upregulated and seven were downregulated in invasive tumors. Eleven circRNAs were consistently upregulated, and two were downregulated in both sets of comparisons.¹³³

However, the sample sizes of these studies were relatively small, and the methods and parameters used for identifying dysregulated circRNAs differed, limiting their generalizability. Consensus on the overall landscape of circRNAs in TC is lacking, highlighting the need for further studies to unravel the mystery of these closed rings in TC.

4.2 The landscapes of circRNA serum exosomes

Exosomes, ranging in size from 30 to 150 nm, are actively released by parent cells and taken up by recipient cells. They carry diverse contents, including RNAs, influencing the functions of recipient cells and participating in intricate cell-to-cell communication.^{282, 283} In PTC, exosomal long noncoding RNAs play pivotal roles in driving the EMT and inducing stemness in PTC cells.^{284, 285} Concurrently, exosomal miRNAs can orchestrate oncogenic behaviors in PTC cells.²⁸⁶ However, the potential roles of circRNAs in PTC exosomes remain largely unexplored.

Yang et al.²⁸⁷ collected serum samples from three patients with PTC and three patients with benign thyroid goiter. Exosomes were extracted from these samples using exosome isolation kits. The analysis revealed three upregulated circRNAs, including hsa_circ_007293, and 19 downregulated circRNAs in the serum exosomes of patients with PTC. KEGG analysis indicated that differentially expressed circRNAs were enriched in 16 signaling pathways, such as the thyroid hormone, PI3K-Akt, AMPK, ABC transport, pyruvate metabolism, calcium, and phosphatidylinositol signaling pathways. These findings suggest significant roles of exosomal circRNAs in the development and progression of PTC by influencing various pathways, warranting further research.

Lin et al.²⁸⁸ showed that overexpression of hsa_circ_007293 in PTC cell lines led to increased levels of hsa_circ_007293 in the exosomes generated by these cells. These exosomes were absorbed by recipient PTC cells, promoting their proliferation, migration, invasion, and EMT pathways. Hsa_circ_007293 was predominantly enriched in the cytoplasm of PTC cells and targeted miR-653-5p to regulate the expression of PAX6. Additionally, the enhanced proliferation, migration, invasion, and EMT pathways in PTC cells receiving exosomes were effectively suppressed by miR-653-5p overexpression or PAX6 inhibition. Therefore, exosomal hsa_circ_007293 may play a crucial role in promoting the malignant behavior of PTC through the miR-653-5p/PAX6 axis.²⁸⁸

5.1 CircRNAs as diagnostic biomarkers of circRNAs for cancers

For tumors originating from the digestive system, mounting evidences suggest the potential of circRNAs as diagnostic biomarkers. CircYAP is significantly upregulated in colorectal cancer tissues with liver metastasis. Receiver operating characteristic (ROC) analyses indicated that circYAP could predict liver metastasis in colorectal cancer with an area under the curve (AUC) value of 0.8433.²¹⁹ Higher expression of circMAN1A2 in GC tissues is associated with advanced stages of GC. Additionally, circMAN1A2 levels are significantly higher in the plasma of patients with GC than that in healthy plasma, indicating its potential as a novel diagnostic biomarker for GC.²⁷² CircMORC3 can serve as a diagnostic biomarker for hypopharyngeal squamous cell carcinoma (SCC) with an AUC of 0.834.²⁹⁰ Higher expression of hsa_circ_0006091 in HCC tissues can distinguish HCC from healthy controls with an AUC of 0.916, which could be improved if combined with levels of AFP or RGS12 as a combined biomarker.²⁹¹

For tumors located in the respiratory tract, the diagnostic values of circRNA are also investigated. In laryngeal SCC, hsa_circ_0036722 can distinguish laryngeal SCC from adjacent normal tissues with an AUC of 0.838.²⁹² Higher expression of hsa_circRNA_102231 is associated with advanced TNM stages and LNM, which can diagnose lung cancer with an AUC of 0.897.²⁹³ Furthermore, Wang et al.²⁹⁴ identified hsa_circ_0001821 and hsa_circ_0077837 as potential diagnostic biomarkers for NSCLC, demonstrating AUC values of approximately 0.90.

The circWSB1 expression level is significantly correlated with the T stage of patients with BC, and the ROC curve demonstrated that the circWSB1 level can identify BC effectively with an ROC of 0.705.¹⁹¹ In cervical cancer, increased expression of circZFR is positively associated with LNM, SCC antigen value, and Ki-67 value. Additionally, the ROC curve proved that the circZFR level can distinguish cervical cancer effectively with an AUC of 0.88.¹⁹² The positive relationships between circSMA4 expression and mitotic figures, as well as the malignant degrees of GISTs, were established. Additionally, the ROC curve analysis underlined the nearly perfect diagnostic efficiency of circSMA4 for GISTs with an AUC of 0.9824.¹⁹⁶

The diagnostic value of circRNAs in the serum were also investigated. Payervand et al.²¹⁵ validated the diagnostic potential of a batch of six circulating circRNAs in serum from patients with colorectal cancer, including hsa_circ_0060745, hsa_circ_001569, hsa_circ_007142, hsa_circ_0084043, circBANP, and CDR1as. The results indicated that all AUC values exceeded 0.75, with three values surpassing 0.90, demonstrating the effectiveness of these circRNAs in differentiating patients with colorectal cancer from healthy individuals.²¹⁵ Sun et al.²⁹⁵ validated the upregulation of hsa_circ_0004001, hsa_circ_0004123, and hsa_circ_0075792 in blood samples from patients with HCC. The levels of these three circRNAs were associated with TNM stages and tumor sizes, which could serve as a noninvasive diagnostic biomarker for HCC. Furthermore, the combined three-circRNA exhibited a higher AUC value for diagnosis, along with enhanced sensitivity and specificity.²⁹⁵ Additionally, circSLC39A5 expression in plasma was significantly associated with satellite nodules, LNM/vascular invasion, total bilirubin levels, and HBsAg levels in patients with HCC, enabling diagnosis of HCC with an AUC of 0.915.²⁹⁶ Additionally, plasma circELMOD3 could identify HCC patients with an AUC of 0.908.²⁹⁷ In addition, the ROC curve suggested that hsa_circ_0023179 could act as a serum marker of NSCLC with an AUC of 0.831.²⁹⁸ CircHERC1 expression in plasma could distinguish patients with lung cancer and cancer-free patients with an AUC of 0.746.²⁹⁹

The circRNAs in saliva and urine also demonstrated their potential as biomarkers. Song et al.³⁰⁰ validated that urinary hsa_circ_0137439 could differentiate patients with bladder cancer from healthy controls with an AUC of 0.890. Moreover, it could distinguish muscle-invasive bladder cancer from nonmuscle-invasive bladder cancer with an AUC of 0.798.³⁰⁰ Yang et al.³⁰¹ found that urine hsa_circ_0071196 could serve as a potential noninvasive biomarker for detecting bladder urothelial carcinoma with an AUC of 0.935. For the diagnosis of OSCC, the combination of salivary hsa_circ_0001874 and hsa_circ_0001971 showed an AUC of 0.922.³⁰²

5.2 CircRNAs as diagnostic biomarkers for TC

6.1 CircRNAs as prognostic biomarkers for cancers

CircIGF2BP3 expression levels were positively correlated with LNM, advanced tumor stages, and shorter overall survival (OS) in NSCLC patients. Multivariate regression analysis results suggest that a higher expression level of circIGF2BP3 is an independent prognostic biomarker for NSCLC.²³⁰ Besides, higher expression of hsa_circRNA_102231 is associated with poorer OS of patients with lung cancer.²⁹³

Higher expression of circFAM53B and circFAM53B-219 peptides are associated with smaller tumor size and better disease-free survival (DFS) in patients with BC. In multivariate Cox regression analyses, circFAM53B serves as an independent prognostic factor for BC.²³² Higher levels of circWSB1 are related to poorer OS of patients with BC and could be a standalone risk biomarker.¹⁹¹ Furthermore, circROBO1 is upregulated in BC-derived liver metastases compared with the BC primary sample and is related to shorter OS.³¹⁴ In TNBC, lower circFBXW7 expression is negatively associated with tumor size, LNM, poorer OS, and DFS. Additionally, multivariate Cox regression analysis indicated that lower circFBXW7 is an independent prognostic factor for patients with TNBC.¹⁰⁹ In patients with HER2⁺ BC, higher levels of circCDYL2 predicted rapid recurrence and shorter DFS and OS following anti-HER2 therapy.³¹⁵

The prognostic values of circRNAs were also evaluated in tumors located in the organs of the digestive system. Lower expression of circLARP4 independently predicted poor survival outcomes in patients with HCC.²⁵⁵ Higher circNFATC3 expression levels are associated with poorer OS in GC patients.¹²⁹ CircROBO1 is significantly upregulated in HCC and higher circROBO1 in HCC is correlated with worse OS and relapse-free survival (RFS).³¹⁶ Higher expression of hsa_circ_0007919 is significantly associated with vascular invasion, nerve invasion, T stage, LNM, and TNM stage in patients with PDAC.²³⁹ Moreover, higher expression of hsa_circ_0007919 can predict poorer OS and DFS of patients with PDAC.²³⁹

Higher expression of circCCT3 in patients with multiple myeloma is associated with longer progression-free survival and OS intervals, which could serve as a reliable, independent predictor for prognosis.³¹⁷ However, Papatsirou et al.³¹⁸ found that higher levels of circulating CDR1as are correlated with a poorer prognosis in multiple myeloma. Additionally, CDR1as expression levels are negatively associated with glioma grade and serve as an independent molecular biomarker of OS in glioma, particularly in GBM.¹⁹⁰

The prognostic value of circRNAs in circulation and other body fluids is also noteworthy. Higher levels of exosomal circAKT3 are associated with poorer OS and RFS in patients with HCC.³¹⁹ Higher expression of hsa_circ_0004831, circ_PVT1, and hsa_circRNA_001569, in circulation is associated with poorer OS in patients with colorectal cancer.^{320, 321} Yan et al.³²² confirmed that higher levels of hsa_circRNA_100199 in serum are an independent predictor of RFS and OS in patients with acute myeloid leukemia. Moreover, the levels of hsa_circRNA_100199 in serum are significantly lower in patients who achieved complete remission than in those who did not, indicating its value for predicting the therapeutic response.³²² Additionally, circVMP1 expression levels in the serum samples of patients with cisplatin-resistant NSCLC are significantly higher than those in cisplatin-sensitive patients, implying its potential as a novel biomarker for cisplatin sensitivity in patients with NSCLC.³²³

Hsa_circ_0137439 in urine can act as an independent prognostic predictor of RFS and OS for patients with bladder cancer.³⁰⁰ Furthermore, highly expressed circPRMT5 in the serum and urinary exosomes is positively associated with LNM and advanced tumor progression in patients with urothelial carcinoma of the bladder, suggesting its potential as a promising prognostic biomarker.³²⁴ Chen et al.³²⁵ found that hsa_circ_0000231 expression level was an independent predictor for the poor prognosis of patients with tongue squamous cell carcinoma (TSCC), which was also upregulated in saliva samples of patients with TSCC.

6.2 CircRNAs as prognostic biomarkers for TC

The clinical potential of circRNAs as prognostic biomarkers for TC has been established. Liu et al.¹⁶² identified significantly elevated levels of hsa_circ_0102272 in TC tissue and cell lines compared with that in normal controls. High expression of hsa_circ_0102272 is strongly correlated with adverse factors such as LNM, higher TNM stage, and histological grade, leading to poorer OS and progression-free survival. Multivariable regression analysis confirmed hsa_circ_0102272 as an independent predictor of prognosis in patients with TC.¹⁶² Similarly, Wang et al.¹³⁸ observed higher levels of hsa_circ_0067934 in TC tissue associated with larger tumor size, LNM, and higher American Joint Committee on Cancer (AJCC) stage. Kaplan–Meier analysis indicated that elevated hsa_circ_0067934 expression predicted lower survival rates, and Cox proportional hazards regression analysis identified it as an independent risk factor for poor OS.¹³⁸ Zhang et al.¹³⁹ further validated the prognostic significance of hsa_circ_0067934.

In patients with TC, higher expression levels of circBACH2, circFNDC3B, circCCDC66, circLDLR, hsa_circ_0124055, and hsa_circ_0101622 are significantly associated with larger tumor size, higher TNM stage, LNM, and poor OS.^{150, 160, 173, 177, 309} Similarly, increased expressions of circRNA_102002, circPVT1, hsa_circ_0008274, hsa_circ_0011058, circZFR, and circPUM1 are linked to positive LNM, higher TNM stages, and poorer OS.^{57, 161, 168, 171, 174, 187} Among them, the expressions of hsa_circ_0008274 and hsa_circ_0011058 are closely associated with tumor infiltration and nodular goiter, respectively.^{168, 174} Additionally, higher expression of circRNA_103598 is associated with a more advanced TNM stage, larger tumor size, metastasis, and poorer OS in patients with PTC.¹⁸⁸ Besides, higher expression levels of hsa_circ_0005273 and hsa_circ_0011290 are linked to poor OS in patients with PTC.^{143, 181}

Limited research has explored the prognostic role of downregulated circRNAs in TC. Ding et al.¹⁶³ found that downregulated circNEURL4 expression in patients with PTC is associated with advanced TNM stages, LNM, and poorer survival.¹⁶³ The expression level of circITCH is associated with the clinical stage and LNM, with proportional hazards analysis suggesting that circITCH is an independent prognostic marker for PTC.¹³¹ Higher expression of hsa_circ_0015278 is significantly correlated with younger age, absence of extrathyroidal invasion, pathological LNM, and lower pT and pTNM stages in patients with PTC.³⁰⁶ Furthermore, higher hsa_circ_0015278 expression is significantly associated with a lower relapse rate and better DFS.

While various studies have reported the differential expression of circRNAs in TC tumor tissue and matched normal tissue and demonstrated their value as prognostic biomarkers in patients with TC (Table 3),^{57, 163, 168, 174, 187, 306} little attention has been paid to the prognostic role of dysregulated circRNAs in peripheral circulation for TC. This potential, which could serve as candidates for liquid biopsy,³²⁶ requires further exploration.

7.1 Functions of circRNAs in chemoradiotherapy resistance

7.2 CircRNAs and their role in TC resistance: potential therapeutic targets

¹³¹I therapy is a common treatment for recurrent and metastatic DTC based on cancer cell iodide uptake.³⁶² However, resistance to ¹³¹I treatment poses a significant challenge, with patients exhibiting refractory DTC facing a mean survival of less than five years and a 10-year survival rate of less than 10%.^{189, 363}

Addressing ¹³¹I resistance in DTC, Chen et al.³⁶⁴ identified circNEK6 (hsa_circ_0088483) as one of the most upregulated circRNAs in TC, particularly in ¹³¹I-resistant DTC tissue and cells. CircNEK6 suppression enhances the ¹³¹I radiosensitivity of DTC cells by upregulating the inhibitory effect of miR-370-3p on MYH9 expression, resulting in the inhibition of cell proliferation, migration, and invasiveness, induction of cell apoptosis, and DNA damage in ¹³¹I-resistant DTC cells.³⁶⁴ Additionally, circNEK6 regulates the miR-370-3p/FZD8 axis and downstream c-myc and cyclin D1, activating the Wnt signaling pathway and influencing TC progression.³⁶⁵

The involvement of circRNAs in the chemosensitivity and radiosensitivity of TC has also been explored. For instance, circEIF6 upregulation in ATC and PTC cells after cisplatin treatment led to suppressed cisplatin sensitivity. CircEIF6 knockdown regulates miR-144-3p/TGF-α, enhancing the chemosensitivity and suppressing proliferation and autophagy in TC cells.¹⁶⁴ Besides, hsa_circ_0011058 knockdown enhances the radiosensitivity of PTC cells through the miR-335-5p/YAP1 axis, modulating angiogenesis, proliferation, and apoptosis.¹⁶⁸

Several other circRNAs have been implicated in TC treatment resistance. For example, circSH2B3 induces PTC dedifferentiation by modulating the miR-4640-5p/IGF2BP2 axis, suggesting its potential as a target for redifferentiation treatment in radioiodine-refractory PTC.¹⁸⁹ Hsa_circ_0079558, promoting the proliferation and migration of PTC cells, was identified as a potential therapeutic target by regulating the miR-26b-5p/MET axis and the downstream MET/Akt signaling pathway, as well as the miR-198/FGFR1 axis. These effects were reversible with PHA665752, a MET-specific small-molecule inhibitor, highlighting its therapeutic potential.¹⁶⁶

Concludingly, circRNAs and their associated pathways play pivotal roles in the progression and resistance of TC, presenting promising opportunities as therapeutic targets.