Advances in the research on circular RNA in pediatric pancreatic tumors

Abstract

Pancreatic cancer (PC), characterized by its insidious onset and extreme malignancy, is one of the most aggressive and lethal malignancies and poses significant challenges in clinical management, with a dismal 5-year survival rate due to late diagnosis and limited treatment options. Although advances have been made in understanding adult pancreatic tumors, pediatric pancreatic tumors (PPTs) remain understudied, with research on their molecular mechanisms and potential biomarkers still in the early stages. Circular RNAs (circRNAs), a newly discovered class of endogenous noncoding RNA molecules, have recently been identified as key regulators in tumorigenesis, progression, and therapeutic response. Unlike linear RNAs, circRNAs possess a unique covalently closed-loop structure, which confers exceptional stability and resistance to degradation. This feature makes them promising candidates as diagnostic biomarkers and therapeutic targets. A growing body of evidence suggests that circRNAs play crucial roles in various cancers, including pancreatic tumors, by modulating gene expression, interacting with microRNAs, and influencing signaling pathways. In PPT, the exploration of circRNAs is still nascent, yet preliminary studies indicate their involvement in tumor development and progression. Some circRNAs have been found to promote cell proliferation, invasion, and metastasis, while others may act as tumor suppressors. Identifying specific circRNA signatures in PPT could enhance early detection, improve prognostic assessment, and uncover novel therapeutic strategies. This review summarizes the current understanding of circRNAs in PPT, highlighting their potential as diagnostic markers and therapeutic targets. Elucidating their molecular mechanisms may inspire innovative approaches in managing this rare but devastating disease. Further investigations are needed to validate the findings gleaned thus far and translate them into clinical practice.

Introduction

Pediatric pancreatic tumors (PPTs) are a relatively rare and extremely malignant group of tumors. The incidence of pancreatic malignancy in children is approximately 0.018 per 100,000, while that in adults is 12.6 per 100,000 (1). PPT includes benign tumors, common cystadenoma, and intraductal papilloma but also malignant tumors, such as pancreatic cancer (PC), cystadenocarcinoma, solid pseudopapilloma, and neuroendocrine carcinoma, among other types. Pancreatoblastoma is both the most common pancreatic malignancy among younger children (aged 4–18 years, with a mean age of 10 years) and older children (aged 8–19 years, with a mean age of 14 years) (2). Owing to the difficulty in early diagnosis, poor prognosis, high mortality, and complex pathological types of PPT, clinical treatment is considerably challenging. In recent years, due to the rapid advancement of high-throughput sequencing technology and bioinformatics methods, circular RNA (circRNA), a newly identified type of RNA molecule, has been discovered to be aberrantly expressed in tumor tissues and cells in PPT and might be implicated in the occurrence and development of the tumors. Therefore, an in-depth exploration of the mechanism of circRNA’s effect in PPT may be highly valuable to clarifying the biological characteristics of tumors and identifying new therapeutic targets.

circRNA

Overview of circRNA

The human genome is complex and diverse, consisting of 3 billion base pairs. The sequencing of the human genome revealed that approximately 98% of non-protein-coding genes can be transcribed into noncoding RNA (3), while only 2–3% of the genome can be transcribed into protein-coding genes (4). circRNA, a type of noncoding RNA, was initially identified in plant eukaryotic cells under electron microscopy by Sanger in 1976 (5), and in 1993, circRNA was found to be present in the mouse Sry gene (6). circRNA is a single-stranded, covalently closed RNA molecule, which distinguishes it from ordinary linear RNA (7). Its formation mainly occurs when precursor messenger RNA (mRNA) undergoes RNA polymerase II (Pol II) back-splicing (8). Specifically, the 3' terminal poly(A) tail of an exon of a gene attaches to the 5' terminal cap of the same exon or adjacent exons, forming a closed ring structure through covalent bonds. This characteristic enables it to be unaffected by exoribonuclease, ensuring more stable expression, less degradation, and the ability to exist and function stably in cells for a long period of time. In most genes, the expression of circRNA is 10% higher than that of mRNA, and even in some cases, the expression of circRNA is 10 times higher than that of mRNA. Studies have demonstrated that the abundance of circRNAs in human platelets is 17–188 times that in nucleated tissues (9), and circRNAs also show significant tissue specificity and stage specificity (10), which makes them an important means for studying disease-related gene expression. With the advancement of high-throughput sequencing technology, a growing number of circRNAs have been identified. Wang et al. (11) identified 8321 circRNAs in the human placenta through RNA-sequencing technology (RNA-seq). Furthermore, two significant research papers published by Memczak et al. (12,13) in Nature in 2013 indicated circRNA to be a microRNA (miRNA) sponge, which led to a surge in circRNA research.

Classification of circRNA

Depending on the source, circRNA can be classified into three main categories: exonic circRNA (ecircRNA), intronic circRNA (ciRNA), and exon-intron circRNA (eiciRNA) (14). Additionally, there are other smaller subpopulations, such as fusion circRNA (f-circRNA), readable circRNA (rt-circRNA) (15), intergenic circRNA, and transfer RNA (tRNA) intron circRNA (16) (Figure 1). Among them, ecircRNA constitutes 80% of circRNA and plays a prominent role in the occurrence and development of tumors (17).

Figure 1.

Formation and classification of circRNA (original, created using Biorender.com). ciRNA, intronic circRNA; circRNA, circular RNA; ecircRNA, exonic circRNA; eiciRNA, exon-intron circRNA; mRNA, messenger RNA.

Biological function of circRNA

Molecular sponging

circRNA molecules are abundant in complementary binding sites of miRNAs and can function as miRNA sponges to absorb and modulate the activity of specific miRNAs. This prevents them from binding with downstream target genes and thereby indirectly regulates the expression these genes (18), realizing the regulatory mechanism of competing endogenous RNA (ceRNA). This mechanism, known as the circRNA-miRNA-mRNA axis, occurs in various tumors. A considerable number of studies have demonstrated that multiple genes participate in the adsorption function of miRNA sponges. For instance, circNRIP1 serves as the gene miRNA-149-5P in gastric cancer, and circHSDL2 promotes the occurrence of breast cancer through miR-7978 ZNF704 (19). The expression of miRNA and its downstream genes is regulated by circRNA through the ceRNA mechanism, thereby influencing biological processes. Compared to normal tissues, circRNA-0007334 is significantly upregulated in pancreatic tumors and acts as a ceRNA to relieve MMP7 by sponging miR-144-3p/577, facilitating tumorigenesis (20). This further substantiates the notion that circRNA functions as a molecular sponge and contributes significantly to regulating the differentiation, proliferation, apoptosis, and disease progression of tumor cells.

Interacting with RNA-binding proteins

circRNA can interact with transcription factors or RNA-binding proteins (RBPs), competitively bind to RBPs, regulate the intracellular transport of corresponding RBPs, influence the structure of chromatin and the activity of transcription factors, and thereby regulate the selective cleavage, transport, transcription, and translation levels of genes. The interaction between circRNA and RBPs can regulate the concentration of free RBPs, and subsequently regulate pathophysiological processes such as cell proliferation, apoptosis, metastasis, and differentiation (21). Guo et al. (22) have demonstrated that the degradation of circRNA is regulated by two RBPs: UPF-1 and G3BP1, which is known as structure-mediated RNA decay (SRD). The degradation of circRNA by SRD reduces its ability to competitively bind to RBPs, thereby lowering the level of gene transcription and translation, and inhibiting the proliferation and differentiation of cancer cells.

Protein interaction

Certain circRNAs (20) can act as sponges for proteins, interact with then to form circRNA-protein complexes, facilitate a rapid response to extracellular stimuli, regulate protein stability and function, and achieve protein translation. Although the majority of circRNAs are noncoding, a small number, for example, circ-ZNF609 (23) and circ-CEP70 (24), possess the ability to encode proteins and peptides, thus exerting regulatory functions. Other circRNAs can encode proteins through cap-independent protein translation mechanisms such as internal ribosome entry sites and N6-methyladenine (25-27). Meanwhile, circRNA can be modified and translated via internal ribosome entry sites. The role of circRNA as a protein sponge was initially discovered from circRNA circMBL. When circRNA binds to the MBL protein, it can prevent its binding to other target genes and participate in the adjustment of the feedback loop between MBL and circMbl. The biogenesis of circMbl is promoted through the binding of flanking introns of circMbl containing conserved MBL binding sites and interaction with circMBL (28). In 2017, Pamudurti et al. (29) found that circMb13 in circRNA could translate head proteins, further verifying the translation function of circRNA for proteins.

Parental gene expression regulated by cis-elements

eiciRNA is mainly situated in the nucleus and is capable of interacting with Pol II to enhance the transcription of its parental genes. For instance, the intron-derived ankyloprotein repeat domain can exert an influence on the transcription of parental genes, while the cyclic transcriptor of eukaryotic initiation factors can regulate gene transcription through cis-interaction, thereby affecting gene expression (30). Studies have discovered that coding genes form linear RNA via typical splicing and transcription and form circRNA through atypical splicing and transcription. Regarding exon requirements, these two expressions are in a competitive dynamic equilibrium, and once the equilibrium is disrupted, abnormal expression of circRNA will result in abnormal expression of tumors (31,32). He et al. (33) found that circATG7 in circRNA can upregulate the expression of the parental gene ATG7 by targeting mir-766-5p, thereby promoting the proliferation, invasion, and metastasis of PC. In conclusion, targeting circATG7 may be an effective therapeutic strategy for patients with PC, with its reduced expression potentially inhibiting the proliferation, differentiation, and invasion of tumor cells, as shown in Figure 2.

Figure 2.

Biological function of ecircRNA (original, created using Biorender.com). circRNA, circular RNA; ecircRNA, exonic circRNA; miRNA, microRNA; mRNA, messenger RNA; RBP, RNA-binding protein; TF, tissue factor.

The role of circRNA in PPTs

Mechanism of action

circRNA, when functioning as a ceRNA, can absorb and sequester specific miRNAs, thereby alleviating the inhibition of miRNAs on their target genes. For instance, some circRNAs can upregulate the expression of genes associated with tumor growth, invasion, and metastasis through the ceRNA effect, thereby enhancing the incidence of tumors (34). circRNA can also directly engage in intracellular signaling pathways and influence physiological processes such as cell proliferation, apoptosis, and metabolism. One study demonstrated that circRNA can regulate the biological activity affecting the autophagy pathway of cancer cells, thus either inhibiting or promoting carcinogenesis (35). Li et al. (36) identified 14 upregulated and 35 downregulated circRNAs that play a key role in tumorigenesis and metastasis in pancreatic neuroendocrine tumors. In PPTs, certain circRNA decrease the apoptosis rate of tumor cells by inhibiting the expression of apoptosis-related genes. Meanwhile, some circRNAs may facilitate the malignant transformation and progression of tumor cells by regulating key molecules in key signaling pathways. circRNAs can influence multiple signaling pathways in PPT, such as the Wnt/β-catenin signaling pathway (37), the PI3K/Akt signaling pathway (38,39), and the KRAS signaling pathway (40), and thus participate in the occurrence and development of tumors.

circRNA can regulate the expression of target genes through competitive binding to miRNA, thereby influencing cell proliferation, apoptosis, migration, invasion, and growth inhibition of tumor cells. For instance, circPDK1 activates c-myc in PPT by regulating the miR-628-3p-BPTF axis and degrading BIN1, facilitating the proliferation and migration of PC cells (41). A comparative study with normal pancreatic tissues verified that the ciRS-7 gene enhances the metastasis and proliferation potential of PC (42). Similarly, circEIF3I, acting as a molecular scaffold, directly binds to SMAD3 and AP2A1 to form a ternary complex that promotes the metastasis of PC mediated by the transforming growth factor (TGF)-β signaling pathway (43). Furthermore, circRNA binds to proteins and affects their functions. For example, circCGNL1 regulates the localization and function of histone deacetylase 4 (HDAC4) in PC by binding to phosphatase NUDT4, thereby influencing the apoptosis process of PC cells (44). Moreover, it has been shown that the PI3K/AKT/mTOR signaling pathway affects tumor progression through the encoding of new proteins by circRNA (38). One study found that circTMEM59 inhibits the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of tumor cells by regulating the miR-147b-SOCS1 axis, with circTMEM59 exerting antitumor effects in pancreatic tumors (45) (Figure 3). Other research indicates that circRNA MYLK gene silencing may inhibit the invasion and migration of PANC-1 cells by suppressing the expression of multiple proteins (46). Finally, Wang et al. (47) found that circ-ANAPC7 regulates the CREB-miR-373-PHLPP2 precursor ring through the PHLPP2-AKT-TGF-β signaling axis, which can prevent the progression of PC and delay disease progression.

Figure 3.

The biological function of circRNA in pancreatic tumors (original, created using Biorender.com). circRNA, circular RNA.

Detection methods

High-throughput sequencing technology, also known as next-generation sequencing (NGS), has revolutionized the field by introducing reversible termination terminals to achieve synthesis during sequencing and determining the DNA sequence through capturing special markers carried by newly added bases during DNA replication (48,49). With the advancement of this sequencing technology, the genome-wide research on circRNAs has been expedited (50). An increasing number of studies have indicated that the expression level of circRNA in tissues is significantly higher than that in serum (51,52), and there are notable differences in circRNA between PPT tissues and normal pancreatic tissues. For instance, certain circRNAs, such as circPDK1 and circEIF3I (53) (Table 1), are highly expressed in PPT tissues and are closely associated with tumor staging, metastasis, and prognosis. As another example, circRREB1 stimulates PGK1 to induce glycolysis and activate the Wnt/β-catenin signaling pathway to maintain the stemness of PC, indicating the potential of circRREB1 as a biomarker and therapeutic target (54). These findings suggest that circRNA may serve as a potential biomarker for the early diagnosis of PPT. Other detection methods for circRNA include circRNA microarray analysis technology for detecting the expression profile of circRNA in cells (55), with the target sequence capture and sequencing technology being derived from the combination of NGS and microarray technology.

Table 1. circRNA signaling pathways and pathways related to pediatric pancreatic tumors.

circRNA	Expression	Function	Mechanism	Reference
circRNA-0007334	Up	Promotion of the proliferation of pancreatic tumor cells	miR-144-3p/577	(20)
circMb13	Up	Translation of proteins, promotion of the development of tumors	Regulation of the loop between MBL and circMbl	(29)
circATG7	Up	Promotion of PC proliferation, invasion, and metastasis	miR-766-5p	(31)
circFARP1	Up	Promotion of fibroblast enhancement and increase in gemcitabine resistance in pancreatic cancer	miR-6603p	(37)
Circ_GLG1	Up	Promotion of cancer cell proliferation, differentiation, invasion, and metastasis	KRAS signaling pathway	(40)
circPDK1	Up	Promotion of the proliferation and migration of PC cells	miR-628-3p/BPTF axis	(41)
ciRS-7	Up	Increase in the transfer and proliferation potential of PC	miR-432-5p	(42)
circEIF3I	Up	Promotion of TGF-β signaling pathway-mediated PC metastasis	Direct binding to SMAD3 and AP2A1 to form a ternary complex	(43)
circCGNL1	Down	Promotion of PC cell apoptosis	NUDT4-HDAC4-RUNX2-GAMT	(44)
circPVRL3	Up	Encoding of new proteins and promotion of the proliferation and metastasis of pancreatic ductal adenocarcinoma	PI3K/AKT signaling pathway	(38)
circTMEM59	Down	Inhibition of tumor cell proliferation, invasion, and EMT	miR-147b/SOCS1 axis	(45)
circ-ANAPC7	Down	Halting the progression of PC	PHLPP2-AKT-TGF-β axis	(47)

circRNA, circular RNA; EMT, epithelial-mesenchymal transition; MBL, mannose-binding lectin; PC, pancreatic cancer; TGF, transforming growth factor.

The application prospects of circRNAs in the treatment of PPTs

According to the International Society of Paediatric Oncology (SIOP), the diagnostic screening of pancreatic tumors includes tumor markers such as human chorionic gonadotropin (HCG), carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 125 (CA125), among others, with surgery being the predominant treatment (56,57). For children with inoperable or recurrent pancreatic tumors, oral gemcitabine, vinorelbine, and oral cyclophosphamide are administered (58). circRNA plays a significant role in PPT, and researchers have begun to evaluate its utility in tumor therapy. For one, certain inhibitors or activators designed for differentially expressed circRNA can regulate the expression of related genes, thereby inhibiting the proliferation of tumor cells and promoting apoptosis or inducing differentiation. For another, the high stability and ease of detection of circRNAs facilitate their use as tumor biomarkers for the early diagnosis and prognosis assessment of PPT (59). Since the expression level of circRNA in tissues is significantly higher than that in serum, the combined detection of preliminary screening blood and biopsy of tumor tissue can be employed to enhance the detection rate. Additionally, as novel strategy for the treatment of PPT, circRNAs can also serve as carriers to directly deliver therapeutic genes or drugs to tumor cells, improving the therapeutic effect and reducing side effects (60). This strategy may address the current limitations in diagnosing the organs of children, which include the immaturity of the organs involved, the insufficient means for examination, and the need for patient compliance.

Conclusions

circRNA is a distinctive type of RNA that has garnered significant attention due to its exceptional structural specificity, immunogenicity, stability, and biological attributes. circRNA offers a stable alternative to mRNA-based therapies and can serve as a complement to related carrier technologies. The exploration of the role of circRNA in PPT has provided crucial clues for uncovering the mechanism of tumor occurrence and developing novel therapeutic strategies. Through high-throughput sequencing techniques and series of molecular biology experiments, researchers have identified a set of differentially expressed circRNAs in PPT and preliminarily clarified their mechanisms of action in tumorigenesis and development (18). These discoveries not only provide a novel biomarker for the early diagnosis of PPT but also lay the groundwork for the development of targeted therapeutic strategies against circRNA. In current clinical practice, many DNA, RNA, and protein-based biomarkers are employed as biological tools for disease diagnosis. Since circRNA is not readily degraded by exonuclease and has a considerably longer half-life than does linear RNA, the selection of biomarkers for early tumor detection through circRNA is targeted and reliable. Detection of circRNA expression, when combined with an analysis of the developmental characteristics of children, can assist clinicians in diagnosing tumors more accurately, assessing the prognosis of children, reducing side effects, and formulating personalized treatment plans. circRNA may also emerge as a new target for PPT therapy, and drugs designed to target specific circRNAs may inhibit tumor growth and spread and enhance therapeutic efficacy. Although circRNAs hold considerable potential in PPT research, there remain certain challenges, including the lack of clarity in the mechanisms related to circRNA; the need to improve its biological distribution, pharmacokinetics, and long-term safety; the limitations of the related therapeutic materials; and uncertainty regarding the means to effectively applying circRNA in clinical diagnosis and therapy. Moreover, research into circRNAs should not only focus on their sponge adsorption function but also on protein translation and gene transcription. Developing methods to enhance circRNA synthesis and transport, designing and optimizing circRNA overexpression vectors, and alleviating circRNA-induced cellular immune responses remain challenges. In the future, with the in-depth exploration of circRNA and the advancement of gene chip and RNA-seq technology, the optimal signaling pathway of circRNA will be identified, the occurrence and development of tumor cells will be inhibited, and hydrogels and other drug-carrying carriers will be employed to precisely treat pancreatic tumors in children. We believe that circRNA’s significance in the early diagnosis and molecular targeted therapy of PPT will continue to grow.

Supplementary

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