Circular RNA in colorectal cancer

Abstract Circular RNA (circRNA) is a highly abundant type of single‐stranded non‐coding RNA. Novel research has discovered many roles of circRNA in colorectal cancer (CRC) including proliferation, metastasis and apoptosis. Furthermore, circRNAs also play a role in the development of drug resistance and have unique associations with tumour size, staging and overall survival in CRC that lend circRNAs the potential to serve as diagnostic and prognostic biomarkers. Among cancers worldwide, CRC ranks second in mortality and third in incidence. In order to have a better understanding of the influence of circRNA on CRC development and progression, this review summarizes the role of specific circRNAs in CRC and evaluates their potential value as therapeutic targets and biomarkers for CRC. We aim to provide insight in the development of therapy and clinical decision‐making.


| INTRODUC TI ON
The incidence and mortality of cancer are increasing every year, and projections for 2020 in the United States alone suggest 1.8 million new cancer cases and 600,000 cancer deaths. 1 Colorectal cancer (CRC) ranks second in mortality and third in incidence of cancers worldwide and has a higher burden of disease in regions with high socioeconomic development. 2 With 19 million disabilityadjusted life-years (DALYs) and over 18 million years of life lost (YLLs) in 2017 alone, CRC continues to have a significant impact on global public health. 3 Westernized diets, sedentary lifestyle behaviour and obesity are common modifiable risk factors. 4 As the world becomes more digitalized in addition to increasing obesity rates and globalization of Western diets, the incidence of CRC is expected to only rise. This emphasizes the necessity to have a better understanding of CRC development and progression, with recent research suggesting that circular RNA (circRNA) has a crucial role to play.
CircRNAs are a type of single-stranded non-coding RNA that are highly abundant in mammalian cells. 5 The majority of circRNAs are endogenously produced through a process known as back-splicing which can result in exon shuffling. 6 Given their covalently closed loop structure, lack of free terminals, and resistance to digestion by exonucleases and RNase R, circRNAs are more stable and evolutionarily conserved than linear RNAs. 7 Research in recent years has discovered functions of circRNAs ranging from being microRNA (miRNA) sponges and transcription regulators to having protein interactions and allowing for translation. [8][9][10][11] Although the function and properties of many specific circRNAs remain unknown, novel research has eagerly explored the roles of circRNAs in human cancer ( Figure 1). Some studies have discovered circRNAs with general oncogenic and tumour suppressor roles, with specific circRNAs changing gene expression in cancer cells. 12,13 Increasing research has shown that circRNAs are dysregulated in various stages of CRC and that circRNA levels could also be used as biomarkers for diagnosis and prognosis, even having the potential to serve as therapeutic targets. Certain circRNAs have been identified to be differentially expressed in drug-resistant CRC cells. 14 This suggests that circRNAs could serve as prognostic biomarkers and also aid in clinical decision-making by focusing the treatment scope. This review aims to evaluate the potential value of circRNA in the diagnosis and treatment of CRC in clinical practice.

| Biogenesis of circRNA
The co-transcriptional production of circRNAs could be mediated by three major mechanisms: intron-pairing-driven circularization, RNAbinding protein (RBP)/trans-factor-driven circularization and lariatdriven circularization. 15 CircRNAs are generally derived from the back-splicing of RNA transcripts at canonical splice sites (Figure 2), which occurs in intron-pairing-driven and RBP/trans-factor-driven circularization. 5,16 Back-splicing joins a downstream splice-donor site with an upstream splice-acceptor site. The region flanked by the splicedonor and acceptor sites is then spliced out as a covalently closed circular form. 17 The formation of a circRNA relies on the successful back-splicing that competes against the splicing of pre-mRNA. 17,18 In intron-pairing-driven circulation, the base pairing of long intronic sequences that contain inverted repeats, such as ALU elements, promote circularization. 19 These events could result in exonic cir-cRNAs (ecircRNA) or exon-intron circRNAs (EIciRNAs) depending on the removal or retainment of introns in the circularized region. 20 In RBP/trans-factor-driven circularization, RBPs (such as FUS and Quaking (HQK)) and trans-factors dimerize and bind to specific motifs present in flanking introns to mediate circularization. 21,22 Furthermore, research in Drosophila demonstrated that circRNA biogenesis could also be influenced by the combinatorial actions of multiple heterogenous nuclear ribonucleoprotein and serinearginine proteins. 23 In lariat-driven circularization, lariats are first generated from exon skipping, where alternative exons are spliced out of the mRNA during canonical RNA slicing as part of the lariat. The lariat undergoes subsequent internal back-splicing and intron removal to produce ecircRNAs. 5,24 Furthermore, intronic lariats containing a 7 nt GU-rich element at the 5' splice site and an 11 nt C-rich element near the branchpoint can escape debranching and generate intronic circRNAs (ciRNAs). 25 EcircRNA, ciRNA and EIciRNA constitute the three major classifications of circRNAs resulting from circRNA biogenesis ( Figure 2C).
The majority of circRNAs are ecircRNAs, which are located in the cytoplasm. In contrast, ciRNA and EIciRNA are mostly located in the nucleus. 20

| Detection of circRNA
The detection of circRNA can be challenging given that they have the same sequences as their linear RNA equivalents. Current techniques for the detection and quantification of circRNAs include cir-cRNA microarray profiling, RNA-sequencing (RNA-seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with divergent primers, northern blotting and in situ hybridization with probes specific to the back-splice junction. 26

| General function of circRNA in cell activities
CircRNAs have been found to serve many functions, from acting as miRNA sponges and interacting with proteins to regulating transcription and allowing for translation. New functions and properties of circRNA continue to be discovered.

| CircRNA as miRNA sponges
One of the identified roles of circRNA is to function as miRNA sponges, with some circRNAs having multiple direct binding sites for miRNAs. 8 Hence, the significance of those circRNAs in pathogenic pathways can be studied in the context of their target miRNAs.
MiRNAs function to silence their target gene expression by partially base pairing with their home gene and negatively regulating gene expression. 27 By sponging miRNAs, circRNAs are able to reverse the silencing effect of their target miRNA, thereby increasing the expression of the gene targeted by the miRNA.

| Regulation of transcription
Recent functional studies have found that circRNAs play a role in regulating the expression of RNA transcripts. Some circRNA can bind to RNA polymerase II, an enzyme for transcription, promoting and further regulating transcription. 15 CircRNA can also affect their home gene expression through epigenetic regulation by binding to the promoter region. 28 Given the co-transcriptional biogenesis of circRNA, the expression of linear RNA often decreases when the majority of spliced transcripts circularises. 18 CircRNAs can serve an important role in modulating gene expression given that they have been found to have differential expression relative to linear RNAs depending on their source tissue type. 29,30

| Translation of circRNA
The ability for circRNA to be translated has been heavily debated in the past given that circRNAs lack 5' and 3' terminal ends. 11 However, increasing studies indicate the translation of circRNAs without a need for the 7-Methylguanosine cap. 31 The presence of a single N6methyladenosine site is sufficient for the initiation of translation, and through mass spectrometry, computational predictions and F I G U R E 1 Roles of circRNA in colorectal cancer. CircRNAs have been identified to be involved in colorectal cancer growth, proliferation, apoptosis, migration, invasion, metastasis and drug resistance. Many circRNAs have also been proposed as potential diagnostic and prognostic biomarkers F I G U R E 2 CircRNA biogenesis and interactions. A, The splicing of pre-mRNA and removal of introns to form linear RNA. B, Back-splicing of pre-mRNA to form circRNA competing against linear RNA splicing. C, The three types of circRNA: Exon circRNA, exon-intron circRNA and intron circRNA. D, Interactions of circRNA in transcription regulation through RNA polymerase II and promoter region binding, miRNA sponging, translation, protein scaffolding and RNA-binding proteins (RBP) polysome profiling, a few endogenous circRNAs have been found to have the potential for translation. 32

| CircRNA-protein interactions
CircRNAs facilitate protein contact and assembly by acting as dynamic scaffolding molecules. 10 Through these interactions, circRNA exert various functions. CircRNAs have been found to regulate transcription by mediating the recruitment of transcription factor complexes to promotor regions for transcription initiation. 33 CircRNAs can also interact with transcription factors, serving as a protein scaffold for the formation of ternary complexes, thus increasing both overall expression and nuclear translocation. 34 Moreover, circRNAprotein interactions can suppress translation of proteins by competitively binding to mRNA transcripts, lowering protein translation rates. 35 RBPs play a key role in post-transcriptional gene expression by regulating the splicing, processing, localization and stabilization of circRNA. 36 Some RBPs bind to RNA and promote back-splicing, facilitating the production of circRNAs. Furthermore, there are circRNAs that are produced directly from RBP genes themselves, containing binding sites for their respective RBPs. Interestingly enough, since circRNAs are not translated in the same manner that linear mRNAs are, ribosomes do not displace any RBPs bound to circRNAs. As such, some RBPs can be bound to certain circRNAs with high specificity, significantly affecting biosynthesis, miRNA binding and expression levels. 18

| Abundance
CircRNAs are abundantly expressed across a diversity of eukaryotes, from humans and mice to zebrafish, Drosophila, and plants. 37 Over one million circRNAs across six vertebrate species (human, macaque, mouse, rat, pig and chicken) are currently recorded in the circAtlas database. 38 CircRNAs are widely expressed across numerous human cell types. There are 148,811 circRNAs originating from 12,251 genes in lung cells recorded in the circ2GO database as of 2020. 39 In human fibroblast cells, 14.4% of expressed genes were found to produce circRNA and the expression of some circRNAs was 10fold higher compared to that of their associated linear transcripts. 5 It was estimated that the total circRNA abundance was roughly 1% of the poly-adenylated RNA abundance in A549, AG04450 and HeLa cells. CircRNA isoforms were more abundantly expressed than linear isoforms for around 50 genes in each cell line, while most genes expressed circRNA at 5-10% of their linear isoforms. 40 In leukocytes, circular isoforms were shown to constitute a substantial fraction of spliced transcripts belonging to hundreds of genes. 16 Overall, the abundance of circRNAs provide a novel and valuable resource to investigate physiology and pathology of the body.

| Stability
One of the outstanding properties of circRNAs is their exceptional stability. CircRNAs are much more resistant to degradation by exonucleases as opposed to their associated linear mRNAs. RNase R is an exonuclease that requires a 3' overhang of a minimum of seven nucleotides for tight binding and nuclease activity. Due to its circular nature, the absence of a 3' overhang allows circRNAs to be preserved under RNase R treatment. 41 Similarly, ribonuclease II and polynucleotide phosphorylase also preserve circRNA integrity while degrading their linear counterparts. 41 CircRNAs demonstrated long half-lives (over 48 hours) compared to their associated linear transcripts (less than 20 hours) when Hs68 cells were treated with actinomycin D. 5

| Cell, tissue, developmental stage and diseasespecific expression
Multiple studies have demonstrated the cell-type-specific expression of circRNA. 40,42 CircRNAs are substantially up-regulated in mouse brain tissue compared to heart, liver, lung and testis tissues.
Explanations could be that the parent genes of many circRNAs are exclusively expressed in the brain or that the ratio between the relative circRNA transcript abundance and the total transcriptional output of the parent gene locus is higher in the brain compared to other tissues. 42 CircRNA expression profiles were also different for distinct cardiac developmental stages in humans, demonstrating stage-specific expression of circRNA. 43 In mice, a global trend of cir-cRNA up-regulation was identified during the ageing process of neural tissues; however, no general trend was observed for linear RNA up-regulation. 30 The age-dependent neural accumulation of circR-NAs was also observed in Drosophila, where circRNA expression was far greater in adult heads compared to other larval, pupal and adult tissues. 44 CircRNAs are found to be differentially expressed in diseases, such as diabetes mellitus, cardiovascular diseases, neurological disorders and cancer, compared to healthy controls. 45,46 The expression of many circRNAs is also correlated with the stages of cancer, such as in CRC. [47][48][49]  down-regulated circRNAs in four CRC tissue samples compared to paired adjacent normal tissue. 47 The discrepancy in the number of differentially expressed circRNAs between these studies could have been attributed to differences in the number of samples, method of circRNA detection and statistical analysis. Furthermore, the data were based on a limited number of circRNA junction reads, which could have impacted accuracy. Nonetheless, several circRNAs have been found to be consistently differentially expressed between CRC and adjacent healthy tissues. These include up-regulated circRNAs such as ciRS-7, 48,55,56 circ-HIPK3 56,57 and down-regulated circRNAs such as circ-ITGA7. 56,58,59 Overall, a significant number of circRNAs are found to be differentially expressed between CRC and normal tissue, and their specific expression patterns may point to their potential involvement in CRC pathways.

| Primary tumour versus metastatic tissues
Metastasis is a major concern in CRC with approximately 20% of CRC patients already having metastasis at diagnosis. 60 Lymph nodes are often the first metastatic site for CRC and other solid tumours.
The liver is a common site for distant metastasis and is a leading cause of CRC mortality. 61,62 There have been 92 up-regulated and 21 down-regulated circRNAs identified in metastatic CRC tissue compared to primary CRC tissue. 63 Differentially expressed circR-NAs originated from all chromosomes except for the Y chromosome.
The up-regulated circ_0001178 and circ_0000826 in CRC metastasis were predicted to bind to multiple miRNAs and demonstrated high diagnostic potential for CRC metastasis. 63 In a meta-analysis of circRNA expression profiles in 1,430 CRC patients, differentially expressed circRNA in CRC correlated with tumour diameter, differentiation, TNM stage, invasion, and lymph node and distant metastasis. In particular, circRNAs up-regulated in CRC correlated with worse overall survival, whereas circRNAs down-regulated in CRC correlated with prolonged overall survival. 64 Additionally, the comparison between primary SW480 cells and metastatic SW620 cells revealed 623 differentially expressed circRNA, of which 275 were up-regulated and 348 were down-regulated in SW620 cells. 65 Many circRNAs, such as those derived from the GLI3 and RAPGEF5 genes, were down-regulated to undetectable levels in SW620 cells despite high expression in SW480 cells. Meanwhile, circRNAs derived from the organic cation transporter SLC22A3 locus were detected in SW620 cells but absent in SW480 cells. 65

| Tumour cell lines versus non-malignant cells
Comparing the circRNA expression profile of the normal colon cell line NCM640 and the CRC cell lines SW480 and SW620, NCM460 contained a significantly higher number of circRNAs. Furthermore, 13,410 circRNAs were exclusively found in the NCM640 cell line, and 8,633 circRNAs were exclusively found in either SW480 or SW620 cells. There were 2,919 differentially expressed circRNAs between the NCM640 and CRC cell lines, of which 2,056 were between the NCM640 and SW480 cell lines and 1,758 were between the NCM640 and SW620 cell lines. 65 Many studies confirmed differential expression of particular circRNAs between normal colon and CRC cell lines. Circ_000984 was highly expressed in CRC cell lines compared to normal colon cells and intestinal epithelial cells. 66 Furthermore, circCCDC66 was detected in CRC cell lines Caco-2, HCT116, HT-29 and LS123, but absent in the normal colon-derived cell line CCD 841 CoN. 67 Yuan et al demonstrated that expression of tumour suppressor circ_0026344 was lower in CRC cell lines HCT116, SW480, SW620 and HT-29, compared to normal colon cell lines FHC and NCM460. 68

| Potential therapeutic targets for CRC
CircRNAs participate in a multitude of ways in CRC pathogenesis.   Altogether, this evidence strongly suggests that CDR1as and circH-IPK3 potentially serve as significant therapeutic targets. Other circR-NAs, such as circRNA-ACAP2, 69 circ_000984, 66 circ_0001955 70 and circ_0055625, 49 have also been discovered as potential therapeutic targets for CRC and function through sponging miRNAs ( Table 1).
The knockdown of these circRNAs produced similar results of CRC growth and proliferation suppression while promoting apoptosis.  involvement of these circRNAs in tumour pathways provides insight into their potential use to limit the growth and proliferation of CRC. Understanding the functions of circRNAs in CRC pathogenesis provides valuable insight into the potential for targeting circRNA in CRC therapeutics. Generally, the practicality of targeting upregulated circRNAs exceeds that of down-regulated circRNAs due to their ease of detection and knockdown. Future therapeutic methods may also consider engineering exogenous circRNAs to target specific miRNAs or proteins to inhibit oncogenic pathways.

| CircRNA AND DRUG RE S IS TAN CE IN CRC
Chemoresistance of CRC has become increasingly challenging and prevalent, being a burden on effective treatment and associated with a poor prognosis. 83 By looking at circRNA expression profiles of drug-resistant CRC tissue and determining circRNAs mechanisms in tumour invasion and signalling, dysregulated circRNAs could serve as biotargets to increase chemosensitivity.

| 5-fluorouracil resistance in CRC
Several circRNAs have been identified to confer drug resistance to CRC when dysregulated. 5-fluorouracil (5-FU), a pyrimidine analog, is the primary therapeutic component of regiments being offered to CRC patients. 84,85 However, with near 50% of CRC patients estimated to develop a level of resistance towards 5-FU in late stages of treatment, the risk of mortality and the risk of recurrence both increase considerably due to limited remaining effective therapeutic options. 85,86 CircRNA expression profiles identified 71 circRNAs that were differentially expressed between 5-FU resistant CRC cells and parental control cells. 86 Three circRNAs in particular, circ_0007031, circ_0000504 and circ_0007006, were significantly up-regulated. This dramatic modulation suggests their utility to predict the development of chemoresistance in CRC. Of these, circ_0007031 was up-regulated 116-fold in chemoresistant CRC cells and acted as a sponge for miR-885-3p, which is linked to the development of 5-FU resistance. 87 This overexpression of circ_0007031 has been suggested to play an important role in the development of 5-FU resistance. 88,89 Thus, circ_0007031 has the potential to serve as both a therapeutic target to be down-regulated and as a prognostic biomarker for treatment efficiency.
Circ_0000504 is highly up-regulated in chemoresistant CRC cells. 86 This circRNA sponges miR-485-5p, which negatively regulates STAT3, a transcription factor protein. 34 The silencing of STAT3 has been found to significantly decrease the survival of 5-FU resistant cells following treatment by 5-FU and irradiation. 90 Given that STAT3 is shown to increase chemosensitivity of 5-FU resistant CRC cells, targeted down-regulation of circ_0000504 would allow for the down-regulation of STAT3, potentially allowing for 5-FU resistance to be overcome.

| Other drug resistances in CRC
Resistance to other drugs has been found to be conferred by various circRNAs as well. Several circRNAs have been identified to confer drug resistance to CRC when dysregulated. Microarray analysis of circRNA expression profiles identified 1,505 dysregulated circRNAs between chemosensitive and chemoresistant HCT116 CRC cells. Of these cir-cRNAs, 773 were up-regulated and 732 were down-regulated, and the authors identified circ_32883 in particular as a biotarget for CRC drug resistance since it was found considerably up-regulated in oxaliplatinresistant cells compared to chemosensitive CRC cells. 91 Another study found that circ_0000338 increased FOLFOX drug sensitivity in CRC cells, while its knockdown resulted in increased chemoresistance. 91 This suggests that circ_0000338 may increase the sensitivity of CRC cells to chemotherapy. However, when circ_0000338 was highly ex-

| CircRNA A S B I OMARKER S FOR CRC DE VELOPMENT
CircRNAs have several remarkable characteristics that provide tremendous potential for their use as biomarkers due to their abundance, stability, conservation across species, and disease-specific and dynamic expressions. Table 2 provides a summary of the following circRNAs as potential biomarkers.  95 Circ_0003906 is down-regulated in CRC, and its low expression was correlated with lymph metastasis and poor differentiation. 96 Finally, the AUC of the circ_001988 ROC curve was 0.788, suggesting acceptable accuracy as a diagnostic marker.

| CircRNA as diagnostic markers
Circ_001988 is down-regulated in CRC, and its expression was shown to correlate with differentiation and invasion. 98 The diagnostic potential of many other circRNAs have not been investigated using ROC curves, but their expressions are nonetheless closely related to the clinicopathological factors of CRC. It was found that the expression of circPPP1R12A was positively correlated with tumour proliferation, invasion and metastasis. 73 CiRS-7 is up-regulated in CRC, and its expression was positively correlated with tumour size, stage and metastasis. 48,55 One study identified 623 differentially expressed circRNAs between the metastatic SW620 cells and the primary SW480 cells. The top 15 circRNAs were proposed as potential diagnostic markers for metastasis. 65 CircRNAs are also abundant and highly stable in exosomes found in serum. 99 RNA-seq analysis demonstrated that circRNAs were enriched in secreted exosomes compared to the cells. Furthermore, circRNAs from cancer xenografts were shown to enter the circulation and were readily measured in the serum, suggesting the effectiveness and simplicity of diagnostic circRNA detection from the serum. 99 Plasma levels of circ-CCDC66, circ-ABCC1 and circ-STIL were significantly decreased in CRC compared to normal controls. 100 The AUC of the ROC curve of the three-circRNA panel was 0.78, which was higher than that of traditional protein biomarkers, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). The combination of the three-circRNA panel with CEA and CA19-9 demonstrated an AUC of 0.855, thus potentially improving diagnostic ability. 100 Though its specific diagnostic accuracy has not been confirmed through ROC curve analysis, the expression of circNSUN2 was significantly increased in the serum from CRC with liver metastasis compared to primary CRC, suggesting its diagnostic potential. 77 CircRNAs from serum have also been reported as potential diagnostic markers in other cancers, such as circ_0001785 for breast cancer. 101

| CON CLUS ION
The potential applications offered by circRNAs continue to grow as we gain a better understanding of them. With the level of ease that cir-cRNA can be collected from patients through saliva and blood, circRNA may one day be added to the arsenal of common clinical laboratory tests. Their ability to serve as biomarkers may even be used as screening tools for CRC prevention. The abilities to dampen miRNA function and to regulate protein function render circRNA not only as a master gene regulator in development and progression of CRC, but also as novel therapeutic targets with more robust effects for treating CRC. Results from in vitro cell culture models and preclinical animal models are providing us with a promising future where retuning circRNA expression in CRC patients will reduce CRC, DALY and YLL rates regardless of cancer stage, level of severity, or drug resistance. We recognize the complexity and numerous questions behind the many circRNAs that escape our understanding and hope that this is a priority for future research to elicit ground-breaking change.

ACK N OWLED G EM ENTS
This work was supported by the Canadian Institutes of Health

Research (PJT 162448) and the Natural Sciences and Engineering
Research Council of Canada (RGPIN-2019-04545).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.