Circular RNA in cancer development and immune regulation

Abstract Circular RNAs (circRNAs) are a class of single‐stranded RNAs with closed loop structures formed by covalent bonds of head and tail. Exploration of circRNAs is continually increasing; however, their functional relevance largely remains to be elucidated. In general, they are stable, abundant, conserved and expressed in tissue‐specific manner. These distinct properties and their diverse cellular actions indicate that circRNAs modulate transcription and translation, and may even function as translation templates. Growing evidence reveals that circRNAs contribute to various physiological and pathological processes, including the initiation and progression of cancer. In this review, we present the current knowledge about circRNAs in cancer development, as well as their potential for use as biomarkers and even therapeutic targets. CircRNA’s role in immune regulation and antitumour immunotherapy is also discussed. In addition, possible challenges in antitumour therapy are raised, and current progress and future perspectives are provided.

Both circular RNAs and linear RNAs are derived from precursor mRNAs and transcribed with the same efficiency. 5 Unlike linear RNAs' classical splicing, circRNAs are generated by different modes, primarily by back-splicing. According to the location of splice junction in genome, circRNAs are classified into four basic types, including exonic, intronic, exonic-intronic and tRNA intronic. Exonic circRNAs (ecircRNAs) are formed by either single or several exons and account for the main body of cirRNAs; circular intronic RNAs (ciRNAs) are made up of introns alone; exonic-intronic circRNAs (EIciRNAs) are composed of exons and introns; and tRNA intronic circRNAs (tricRNAs) are derived from splicing of pre-tRNA introns. 5,6 Different types of circRNAs are distributed in different sites of cells.
Generally, exonic circRNAs are located in cytoplasm, whereas some ciRNAs and EIciRNAs are in the nucleus, which is consistent with the location of their different biological functions, as shown below.
Furthermore, circRNAs are reported to be packaged and released in certain vesicles (exosomes and microvesicles), and compared with in cells, circRNAs in exosomes are more enriched and widely expressed. 7,8 Although most of the biological functions of circRNAs have yet to be elucidated, the current knowledge about circRNAs has shown that they can regulate gene expression (transcription and translation), interact with proteins, act as miRNA sponges, translate proteins or peptides, be involved in rRNA processing and generate pseudogenes (Figure 1). In the nucleus, circRNAs function in the following: (A) competition in splicing. When the circRNA contains the same exon as the parental gene, circRNAs can compete with linear splicing of pre-mRNA, thereby affecting the level of linear RNAs. 5

(B) Transcription regulation: ciRNAs and EIciRNAs
can bind to U1 snRNP through RNA-RNA interactions and further interact with the Pol II transcription complex to enhance parental gene expression. 9 (C) Regulation of parental gene by epigenetic mechanism: for example, circFECR1 from the FLI1 gene can bind to FLI1 promoter, recruit TET1 demethylase and bring extensive DNA demethylation in the CpG islands of promoter to activate FLI1.
FECR1 can also directly inhibit the gene transcription of DNMT1 methyltransferase, an essential enzyme in DNA methylation maintenance, by binding to its promoter region, which is enriched in H3K27ac. 10 In cytoplasm, circRNAs function in (D) translation regulation: for example, CircYap can bind with Yap mRNA and the proteins associated with translation initiation, eIF4G and PABP, thereby suppressing Yap translation initiation. 11 (E) Binding to proteins: some circRNAs that have binding sites for RNA-binding proteins may serve as protein sponges or scaffolds to affect their functions or translocations. For example, circ-Foxo3 can bind both Foxo3, p53 and sponge for MDM2. This allows it to prevent Foxo3 F I G U R E 1 Illustration of Circular RNA function. Circular RNAs fulfil multiple functions. In nucleus, circRNAs can regulate transcription A, compete with the mRNA for the available splicing machinery and interfere in the alternative splicing process; B, regulate the transcription of their gene of origin through direct (circRNAs) or U1 snRNP-mediated (EIcircRNA) interaction with the RNA polymerase II; C, regulate parental gene by epigenetic mechanism. In cytoplasm, circRNAs can D, regulate the translation of mRNAs by interacting with some translation initiation associated proteins; E, bind to some proteins as protein sponge or scaffold to affect their functions or translocations; F, act as miRNAs sponges, a majority of circRNAs function that interact with miRNA-AGO2 complexes to affect miRNA functions; G, encode proteins or peptides, possibly with an internal ribosome entry site (IRES) or with m 6 A modification; H, interfere with the processing of pre-rRNA subunits; I, generate pseudogenes. J, circRNAs may be released for cells communication or acting as vectors for miRNAs or proteins an essential 60S-preribosomal assembly factor, resulting in the impairment of exonuclease-mediated pre-rRNA processing and ribosome biogenesis. (J) CircRNAs can also generate pseudogenes like linear RNAs, such as circRFWD2-derived pseudogenes. 18 In addition, circRNAs in exosomes can also be released for cell communication or can act as vectors for miRNAs or proteins, functions which are described below. 19 Based on these biological functions, circRNAs contribute to diverse physiological and pathological processes, notably in the onset and progression of cancer. 20,21 In this review, we discuss the crucial roles of circRNAs pertaining to cancer, including their potential value as biomarkers and therapeutic targets. In view of the involvement of immune system in cancer, we also review the role of circRNAs in immune regulation and antitumour immunotherapy.

| CIRCUL AR RNA IN C AN CER DE VELOPMENT
As a complex pathological process, cancer development contains many hallmarks, such as genome instability and mutation, reprogramming energy metabolism, sustaining proliferative signalling, enabling replicative immortality, evading growth suppressors, resisting cell death, activating invasion and metastasis, inducing angiogenesis, promoting tumour inflammation and evading immune destruction.

| Tissue specificity
Research into the relationship between circRNAs and cancer found that circRNAs are often dysregulated in cancers which allows tumour tissue to be distinguished from adjacent normal tissue. 20,27 Studies have identified multiple functional cancer-associated cir-cRNAs, with differential expression, in different cancers. These circRNAs act as tumour suppressors or promotors and impact can-

| Angiogenesis
Tumour progression usually requires formation of new blood vessels

| Genome mutation
In general, accumulation of mutations accompanies the vast major

| CIRCUL AR RNA IN C AN CER IMMUNE REG UL ATION
The immune system is responsible for defending the host from exogenous invasion and maintaining internal homeostasis. The emerging research on circRNAs has suggested that they are involved in the modulation of different immunocytes and diverse immune responses. For example, circular RNA100783 functions in CD8(+) T cell ageing and immunosenescence, and circRNA-003780 and circRNA-010056 function in macrophage differentiation and polarization. 44,45 A gene ontology analysis for 422 circRNAs identified from healthy human saliva categorized them into several groups, including establishment of T cell polarity, chemotaxis, inflammatory response and the integrin-mediated signalling pathway, which indicated that these circRNAs may participate in intercellular signalling and immune responses. 22 However, inflammation can play vital roles during all stages of tumour development, especially in early tumour initiation as well as tumour promotion, tumour progression and metastasis. Immunosuppressive cells, inflammatory cytokines and signalling, chemokines and inhibitory receptors all contribute to tumour-promoting inflammation. 46

| Checkpoint
Immune checkpoints, such as PD-1 and CTLA-4 (inhibitory receptors), account for tumour immune escape. The rising hotspot of checkpoint blockade therapy and the emerging role of circRNAs has attracted studies seeking to elucidate the relationship between them. For instance, hsa_circ_0020397 was found to be up-regulated in CRC and could mediate colorectal cancer cell viability and invasion by inhibiting miR-138 and subsequently enhancing telomerase reverse transcriptase (TERT) and PD-L1. 26 In another case, circ-

| Chemokine
Chemokines are a type of cytokine that can recruit leucocytes and other types of cells. Evidence shows that some chemokines and chemokine receptors are frequently involved in cancer, even as tumour promoters. In one experiment, two human CRC cell lines

| Inflammatory cytokines
Cytokines in tumour development are typically derived from cancer cells or immunosuppressive cells. In the process of tumour inflammatory microenvironment formation, caspase-1 contributes to the activation of proinflammatory cytokines IL-1β and IL-18, which induce the key downstream inflammatory response. 51

| Anti-viral
A number of viruses have been proven to generate circRNAs after infecting the host, and some oncogenic viruses can encode circRNAs, which may contribute to oncogenesis, such as circEBNA_W1_C1 de-

| CIRCUL AR RNA A S A C AN CER B IOMARKER
Considering the harmful effects of cancer, finding new and effective biomarkers to utilize in diagnostics, prognosis and/or to predict treatment response is vitally important. As described above, circR-NAs are closely linked with cancer onset and progression, and there are differential expressions between tumour tissues and adjacent normal tissues. Furthermore, circRNAs have distinct properties of abundance, stability and tissue-specific expression. More importantly, circRNAs can be secreted in human body fluids, such as saliva, urine, blood and cerebrospinal fluids, and are enriched in exosomes.
Taken together, these characteristics indicate the advantages of cir-cRNAs as biomarkers for detection and surveillance in cancer, which may be more accurate, convenient and noninvasive. 3,4,60

| Prognosis
Many groups have also investigated the prognostic value of circR-NAs in cancer. Okholm et al 64 identified 113 differentially expressed circRNAs between low-and high-risk groups of patients with nonmuscle-invasive bladder cancer (NMIBC). They also discovered 13 circRNAs that independently correlated with BC progression, especially circCDYL and circHIPK3, which exhibited high prognostic value for early-stage BC. Exosomes can be perfect carriers for cir-cRNAs and exo-circRNAs derived from tumours and can be secreted into human body fluids, ultimately impacting cancer development. 8 Li et al 65 found that exo-circ-PDE8A derived from the blood of patients with pancreatic cancer was significantly up-regulated, and these blood exosomes extracted from pancreatic ductal adenocarcinoma (PDAC) patients exhibited high correlation with duodenal invasion, vascular invasion, TNM stage, and finally, survival expectancy.
In this way, exo-circPDE8A may be a superior early diagnostic and prognostic biomarker for PDAC.
In addition, circRNAs also exhibit certain potential in pre- with OS during treatment with cisplatin, indicating its potential in dynamic monitoring. 67 The sensitivity and specificity of circRNAs for monitoring drug resistance should be taken into account.

| CIRCUL AR RNA IN C AN CER THER APY
Exploring novel antitumour methods is urgent due to the harm and severity of cancer and the limitations of traditional treatment methods. CircRNAs are widely involved in tumorigenesis, making them an attractive target in the field of cancer therapy.

| Gene therapy
There have been multiple circRNAs identified as tumour promotors or suppressors, which influence cancer phenotypes in different ways, especially angiogenesis, proliferation or growth, invasion and metastasis. Exogenous up-regulation or down-regulation of relevant circRNAs, by changing their gene expression, is common in studying their potential in ameliorating harmful phenotypes. In contrast, plasmid or lentiviral vectors are often used to increase cir-cRNAs levels. For example, circ-Foxo3 was reported to be downregulated in bladder cancer, whereas overexpression of circ-Foxo3 by the above vectors could induce bladder cancer apoptosis through directly inhibiting miR-191. 68 Similarly, as a translation template of FBXW7-185aa, the circ-FBXW7 level was found to be reduced in glioblastoma, whereas overexpression of FBXW7-185aa could help inhibit cancer cell proliferation. 16  Therefore, such tools need to be evaluated carefully, and further research may help us better apply gene therapy with this strategy. 83

| Therapeutics vectors
In addition to being targets in gene therapy, circRNAs can also be used as promising therapeutic vectors due to their unique stability and capacity for binding miRNA or proteins. They can be designed with miRNAs and/or proteins binding sites, which might be a sim- considering is designs with cell-specific promoters or disease-activated control elements, which may make the circRNAs optionally expressed in certain cells. Moreover, as some circRNAs can act as coding templates, it is expected that applying their expression cassettes in tumour suppressor proteins could be another approach to assist antitumour efforts.

| Immuno-oncology
Although both the innate and  41,60,75 As coding template, proteins or peptides translated from abnormal circRNAs may also act as tumour antigens. 19,58 As is known, for cancer vaccine therapy or CAR T cell adoptive therapy, it is more attractive to explore novel and specific antigens to improve the treatment outcome. In this way, this new potential tumour antigen may be applied in cancer vaccine therapy or CAR T cell adoptive therapy.
Because research on miRNAs in antitumour immunity is clearer than that on circRNAs, the close relationship between circRNAs and miRNAs can help research into the role of circRNAs in antitumour immunity. Using circRNA databases (starBase v2.0 and circBasecan), we can predict a potential circRNA regulating tumour immunity-associated miRNAs and identify potential new tumour antigens. 76,77 In Taken together, this evidence exhibits the potential of circRNAs in regulation of antitumour immune responses and even as therapeutic targets. However, the underlying mechanisms of circRNAs in this field remain largely unknown and need to be further clarified.
Additionally, with increased recognition of circRNAs in antitumour-drug resistance, targeting specific circRNA may ameliorate the drug resistance during antitumour treatment. As mentioned above, up-regulated circPVT1 in gastric cancer is associated with multidrug resistance, such as resistance to cisplatin, paclitaxel, and oxaliplatin; thus, targeting circPVT1 may have the potential to treat drug resistance in gastric cancer.

| CHALLENG E S IN C AN CER THER APY
Using circRNAs as novel therapeutic targets will undoubtedly expand the field of potential "druggable" targets. However, despite the rapid growth of related research in recent years, knowledge of circRNAs is not sufficient compared to that of miRNAs and mRNA. Interaction with protein Inhibiting cell proliferation through PI3K/AKT/mTOR signalling pathway. Over-Overexpression of hsa_circ_0079299 suppressed tumour growth in vitro and in vivo, retarded cell cycle progression while had no effect on cell migration and apoptosis  Therefore, prior to clinical application, we should investigate the issues and challenges that currently exist in antitumour therapy.

| Potential targets
Simply, discovery of a novel circRNA in one type of cancer cell or tissue and experimentally finding that it acts as an oncogene or tumour suppressor does not rule out the possibility that the circRNA may be involved in other vital physiological processes or has other targets that have yet to be identified. In addition, one circRNA may target multiple miRNAs in one cancer; for example, circ-ITCH can sponge both miR-7 and miR-214 to inhibit the Wnt/β-catenin pathway and repress proliferation of lung cancer cells. 28 Additionally, a specific circRNA may be down-regulated in different tumours, which implies that its dysregulation cannot be uniquely related to a specific disease. 12,68 Furthermore, the same circRNA may exert opposite functions by targeting different miRNAs in different cancers. CircHIPK3 can target miR-558 to downregulate HPSE and subsequently inhibit angiogenesis, migration and invasion of BC cells, but also can target miR-7 to promote CRC growth and metastasis. 78,79 Thus, it is important to study the circRNA spectra as much as possible before focusing on single molecules related to a certain disease and to carry out enough trials to minimize the harm induced by other potential targets.

| Specificity
Specificity is one of the most important matters to consider when drugs or therapeutic targets are applied clinically so as to avoid possible off-target effects and assure safety and efficacy. First, targeting tumour-associated circRNAs should preferably be exerted without disturbing expression of other RNAs, especially the corresponding linear mRNA. RNAi and ASOs are currently the two main approaches used to target RNAs, and minimizing the toxicity of offtarget is a major concern. 80 Considering the structure and biogenesis of circRNAs, RNAi or ASOs should be designed to be perfectly com-

| Gene therapy
In gene therapy, overexpression of circRNA is common, and plasmid or lentiviral vectors are frequently used. 82

| Delivery
How to safely deliver engineered circRNAs in vivo is an important question. As mentioned above, the abundance and stability of circR-NAs in exosomes has been identified. Exosomes have a lipid bilayer structure, which can protect RNAs against degradation and ensure TA B L E 1 (Continued) their effective concentration. 8 Furthermore, the small size and membrane structure of exosomes facilitate their absorption and fusion by cancer cells. Transporting circRNAs via exosomes can support intercellular communication in cancer. 19 For therapy purposes, modified exosomes may be an effective approach, because they contain engineered circRNAs or siRNAs targeting circRNA. 83 However, even if cancer-derived exosomes have higher affinity with malignant cells, the possibility of targeting healthy ones still cannot be excluded. In recent times, nanoparticles, as a promising tool to deliver nucleic acids into cells, have emerged for application in therapy. Du et al 12 reported the application of gold nanoparticles to deliver circ-Foxo3 in mice to inhibit tumour progression. However, this approach can only be applied to exonic circular RNAs, because nanoparticles have no ability to enter the nucleus. A recent study of combination of nanoparticles and microfluidics may provide an exciting strategy to increase nuclear entry for resolving this limitation. 84 Hence, it is expected that circular RNAs will become a new therapy target or tool to treat diverse cancers as the field continues to grow and delivery problems are resolved.

| Current progress
Although various studies have indicated the promising potential of circRNAs in cancer therapy (Table 1), such as in gene therapy, immunotherapy and a combination of the two, there are still many drawbacks to overcome. Thus far, there have been no preclinical reports in which circRNAs are applied alone as therapeutic targets or vectors in cancer, but we believe the ongoing research will change this in the future. With the development of new technologies, more circRNAs will be discovered as diagnosis-and prognosis-related markers, and these can be used to guide clinical medication selection. Therapeutic strategies that depend on circRNAs will be formulated, thereby providing new perspectives and directions for cancer therapy. Each experimental report will bring us closer to a patient-tailored approach to achieve maximum anticancer effects with minimal side effects.
Additional work with larger sample sets and long-term follow-up clinical information is needed for further validation.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analysed during the current study are available in the figshare repository.