The Regulatory Functions of Circular RNAs in Digestive System Cancers

Circular ribonucleic acids (circRNAs), which are a type of covalently closed circular RNA, are receiving increasing attention. An increasing amount of evidence suggests that circRNAs are involved in the biogenesis and development of multiple diseases such as digestive system cancers. Dysregulated circRNAs have been found to act as oncogenes or tumour suppressors in digestive system cancers. Moreover, circRNAs are related to ageing and a wide variety of processes in tumour cells, such as cell apoptosis, invasion, migration, and proliferation. Moreover, circRNAs can perform a remarkable multitude of biological functions, such as regulating splicing or transcription, binding RNA-binding proteins to enable function, acting as microRNA (miRNA) sponges, and undergoing translated into proteins. However, in digestive system cancers, circRNAs function mainly as miRNA sponges. Herein, we summarise the latest research progress on biological functions of circRNAs in digestive system cancers. This review serves as a synopsis of potential therapeutic targets and biological markers for digestive system cancer.


Introduction
Digestive system (oral cavity, oesophagus, stomach, colorectum, liver, and pancreas)-related cancers seriously affect human health. To date, multiple digestive system cancers have been identified. Oral cancer is a general term for malignant tumours that occur in the mouth, ninety percent of which are oral squamous cell carcinomas (OSCCs) [1]. Globally, a large number of new cases of OSCC are diagnosed every year [2]. Oesophageal cancer, mainly oesophageal squamous cell carcinoma (ESCC), is not only the eighth most common cancer but also the sixth most common cause of death from cancer throughout the world. There are geographical differences in the morbidity and mortality of ESCC. For example, China has a high incidence of oesophageal cancer [3]. Furthermore, gastric cancer (GC) is also commonly diagnosed and is identified as a cause of death from cancer [4]. Colorectal cancer (CRC) is the third and the second most frequently diagnosed cancer in males and females, respectively. In most parts of the world, the CRC incidence ratio is higher in men than in women [5]. Hepatocellular carcinoma (HCC), which is the most common malignant liver tumour, is the second primary cause of cancer-related death globally [6]. In addition, pancreatic cancer (PC) is a highly lethal disease in older adults, for which the mortality closely parallels the incidence [7]. Consequently, a more profound comprehension of the molecular mechanisms of cancer progression in the digestive system is indispensable, which provides new ideas for the identification of new prognostic and diagnostic biomarkers for their prevention and treatment.
The term "circular ribonucleic acids (circRNA)" generally refers to a newly identified class of endogenous noncoding RNAs (ncRNAs). CircRNAs are covalent closed-loop structures without the 5 caps or 3 poly (A) tails that linear RNAs possess, which makes them more resistant to RNA exonucleases [8]. Furthermore, as shown in Figure 1A, circRNAs can originate from exons, introns (including intron lariats), or both exons and introns [9]. Importantly, different subclasses of circRNA molecules are produced by distinct biogenesis mechanisms [10,11]. The formation mechanism of exonic circRNAs (ecircRNAs) and exon-intron circRNAs (EIciRNAs) mainly includes three models: exon-skipping or lariat-driven circularisation, direct back-splicing or intron-pairing-driven circularisation, and RNA-binding-protein-driven circularisation [10]. The formation of intronic circRNAs (ciRNAs) depends on 2',5'-phosphodiester formation from three different pathways [11]. In the 1970s, circRNAs were initially discovered as viroids in RNA viruses [12]. With the continuous improvement of high-throughput sequencing technology and bioinformatics, abundant and diverse circRNAs have been identified in various developmental stages and physiological conditions in numerous organisms, including humans [13], mice [13], and monkeys [14]. Many reports have revealed that circRNAs are indispensable in various physiological and pathological processes [15][16][17][18][19]. Recently, multiple possible biological functions of circRNAs have been discovered ( Figure 1B). Typically, circRNAs function as efficient microRNA sponges or competing endogenous RNAs (ceRNAs) to affect gene expression [20]. In addition, circRNAs can regulate transcription [21] and protein functions [22]. Importantly, circRNAs are considered ncRNAs and have coding abilities [23]. In addition, circRNAs have been gradually confirmed to be related to ageing processes and various human diseases, including nervous system disorders, cardiovascular disorders, and cancers (e.g., gastrointestinal cancers) [24,25]. In this article, we highlight recent research on the latest progress in circRNA research on digestive system cancer and gut ageing. Finally, the future of circRNA research progress and directions for the diagnosis and targeted therapy of digestive system cancers are discussed.

CircRNAs Dysregulated in Digestive System Cancers
With the continuous improvement of high-throughput sequencing technology, numerous differentially expressed circRNAs have been identified in digestive system cancers (Table 1). For example, Sun et al. performed a comparative analysis of the global circRNA expression profiles between the malignantly transformed oesophageal carcinoma cell line SHEEC and the immortalised human oesophageal epithelial cell line SHEE [26]. They identified 813 significantly upregulated and 445 downregulated circRNAs [26]. Through differential gene expression analysis of ESCC and adjacent non-neoplastic tissues, 267 circRNAs-92 upregulated and 175 downregulated-were found to be dysregulated in ESCC tissues compared with non-neoplastic tissues [27]. Similarly, Shi et al. unearthed 744 dysregulated circRNAs in ESCC tissues [28]. The greater number of differentially expressed circRNAs found in the cell lines than in corresponding cancer tissue samples may be due to the differences between the simulated in vitro environment and the in vivo environment. Furthermore, different numbers of dysregulated circRNAs were found in the same kind of samples, possibly because of differences in the RNA quality, sequencing equipment, sequencing reagents, etc. The final results of high-throughput sequencing are affected by several factors, including the sample type, quantity, and quality; the sequencing equipment and reagents; and the subsequent data processing methodology. In conclusion, circRNAs might be implicated in the oncogenesis and evolution of digestive system cancers and could serve as new clinical diagnostic markers and therapeutic targets.

The Functions of CircRNAs in Digestive Tract Cancers
Recently, increasing evidence has demonstrated that many circRNAs potentially exhibit biological functions in digestive system cancers. Via influencing cell migration, proliferation, apoptosis, and invasion in digestive system cancer, circRNAs act as either oncogenes or tumour suppressors. Some mechanisms have been identified, such as serving as miRNA sponges and regulating protein functions. Among these, the principal mechanism of circRNAs unearthed in digestive system cancers is miRNA sponging. The mechanisms and functions of dysregulated circRNAs in digestive system cancers are displayed in Table 2. Down --Proliferation (−), migration (−) [55] Note: (+) means promotion and (−) means suppression.

Gastric Cancer
To date, 11 circRNAs-five upregulated (cancer-promoting) and six downregulated (cancersuppressing)-have been found to be involved in GC (Figure 2c). The action mechanisms of six of these circRNAs have been described, revealing the pathological process of GC and providing new approaches for the prevention and therapy of GC.
3.3.1. CircRNAs that Act as Oncogenes in GC By sponging members of the miR-125 family, the oncogenic circRNA circPVT1 may facilitate GC cell proliferation [15]. In addition, circRNA_001569 overexpression promoted cell viability and decreased GC apoptosis by suppressing miR-145 expression, which is mediated by NR4A2 [18]. Furthermore, inhibition of 3 circRNAs (circRNA0047905, circRNA0138960 and circRNA7690-15) restrained the cell invasion and proliferation of GC in vitro [19]. Future research should focus on the mechanism of action of these newly discovered circRNAs in GC.

CircRNAs that Act as Tumour Suppressors in GC
Six circRNAs-circYAP1, circLARP4, circRNA_100269, circPVRL3, circ-ZFR, and circ-104916have been reported to act as tumour suppressors in GC (Table 2). Previous studies analysed the functional mechanisms of circYAP1, circLARP4, circRNA_100269, and circ-ZFR and found that they are ceRNAs that bind to miRNAs to inhibit GC. For example, circYAP1 restrained the invasion and proliferation of GC cells by binding miR-367-5p to inhibit p27Kip1 expression [40]. In addition, circLARP4 restrained the biological behaviours of GC cells through sponging miR-424 [41]. Furthermore, by targeting the LATS1 gene, miR-424 facilitated the cell invasion and proliferation of GC [41]. CircRNA_100269 was found to target miR-630, suppressing tumour cell growth [42]. In addition, miR-630 could obstruct the function of circRNA_100269 [42]. Circ-ZFR induced cell cycle arrest, suppressed GC cell proliferation, and promoted apoptosis through sponging miR-107/miR-130a and adjusting PTEN, and circ-ZFR regulated the expression level of p53 protein in vivo [43].
Moreover, these circRNAs act through other mechanisms. In particular, circPVRL3 may have protein-coding ability in view of the structure of its open reading frame, internal ribosomal entry sites, and m6A modification [44]. Knockdown of circPVRL3 promoted the cell migration and proliferation of GC [44]. Circ-104916 overexpression in vitro effectively suppressed the cell migration, proliferation and invasion abilities of GC [45]. In addition, circ-104916 downregulated N-cadherin, Vimentin, and Slug and upregulated E-cadherin, which suggests that circ-104916 may participate in epithelial-mesenchymal transition (EMT) [45]. Therefore, overexpression of circRNAs in GC could be an effective method to prevent the progression of GC.

Colorectal Cancer
Ten circRNAs-six upregulated in CRC (cancer-promoting) and four downregulated (cancer-suppressing) circRNAs-have been determined to be involved in the regulation of CRC (Table 2 and Figure 2d). These circRNAs are related to the cell migration, proliferation, and apoptosis of CRC and participate in the regulation of signalling pathways (e.g., the Ras signalling pathway and Wnt/β-catenin pathway). Knowledge about these circRNAs facilitates a deeper understanding of CRC pathogenesis.
The circRNA circCCDC66 was found to serve as a miRNA sponge to protect MYC mRNA from attack through miR-93 and miRNA-33b [46]. CircRNA-ACAP2 acted as a miRNA sponge to increase the expression of T lymphoma invasion and metastasis protein 1 (Tiam1) through abolishing the inhibitory function of miR-21-5p for Tiam1 expression, thereby promoting the invasion, migration, and proliferation of SW480 colon cancer cells [47]. Moreover, hsa_circ_0020397 inhibited apoptosis and promoted the cell invasion and viability of CRC through enhancing the expression of programmed death-ligand 1 (PD-L1) and telomerase reverse transcriptase (TERT), which are the target genes of miR-138, whereas miR-138 had the opposite effect [51]. Specifically, hsa_circ_0000069 knockdown caused G0/G1 period cell cycle detention in vitro [48]. These findings provide in-depth insights into CRC progression and a reference for the design of therapeutic targets.

CircRNAs that Act as Tumour Suppressors in CRC
Several circRNAs, including circITGA7, cir-ITCH, circ_0026344, and hsa_circ_0000567, have been found to be downregulated in CRC. CircITGA7 acted as a suppressor to repress the cell metastasis and proliferation of CRC by stimulating the transcription of its host gene ITGA7 and inhibiting the Ras signalling pathway [52]. Furthermore, through heightening the level of ITCH and restraining the Wnt/β-catenin pathway, cir-ITCH suppressed the proliferation of CRC cells [53]. In addition, circ_0026344 reduced the cell invasion and growth of CRC while accelerating apoptosis through sponging miR-31 and miR-21 in CRC [54]. Moreover, hsa_circ_0000567 knockdown promoted CRC cell migration and proliferation in vitro [55]. Therefore, overexpression of these circRNAs might regulate CRC.

CircRNAs Play Important Roles in Digestive Gland Cancers
Digestive gland cancers comprise HCC and PC. Previous studies have reported that circRNAs participate in the regulation of HCC cell migration, invasion, and proliferation and in pancreatic ductal adenocarcinoma (PDAC) cell invasion and proliferation [56][57][58] (Table 3). These circRNAs play a very important role in promoting or inhibiting the growth of digestive gland cancers.

Hepatocellular Carcinoma
A total of 18 circRNAs, 11 of which are upregulated in HCC (cancer-promoting) and the other 7 of which are downregulated (cancer-suppressing), have been found to be involved in HCC (Table 3 and Figure 2e).

CircRNAs that Act as Oncogenes in HCC
Eleven circRNAs have been found to participate in the regulation of HCC cell migration, proliferation, and invasion by two action mechanisms (as ceRNAs and by binding to proteins) ( Table 3). CircFBLIM1 [59] and hsa_circ_0078710 [58] promoted HCC cell proliferation, invasion, and migration as ceRNA sponges. CircFBLIM1 was shown to regulate FBLIM1 expression by sponging miR-346 [59]. In addition, in HCC, hsa_circ_0078710 increased HDAC and CDK2 levels by sponging miR-31 and promoted HCC cell cycle progression, invasion, cell proliferation, and migration in vitro, as well as tumour formation by HCC cells in vivo [58]. Cullin 2 (Cul2) circular RNA (circ-10720) and circRNA_104075 have a functional mechanism similar to that of their upstream regulator. A recent study reported that through binding to the Cul2 promoter, Twist1 stimulates its transcription and selectively upregulates Cul2 circular RNA expression. These circRNAs can sponge miRNAs that target Vimentin, thus increasing the expression of Vimentin during EMT [60]. In addition, Cul2 circRNA stimulated the cell invasion, migration and proliferation of HCC [60], and circ_104075 acted as a ceRNA to encourage tumorigenesis by increasing the expression of YAP through sponging miR-582-3p [61].
Mechanistically, HNF4a binds to the -1409 to -1401 region of the circ_104075 promoter to increase the expression of circ_104075.
The remaining circRNAs act as oncogenes in HCC by binding to different miRNAs and influencing different signalling pathways (Table 3), which differentially affect the progression of HCC. Hsa_circ_101280 (hsa_circ_SLAIN1 and hsa_circ_0100929) greatly facilitated HCC tumorigenesis through upregulating JAK2 and adsorbing miR-375, which restrained apoptosis and enhanced proliferation of HCC cells [62]. Hsa_circ_0103809 promoted cell migration and proliferation and inhibited apoptosis in HCC through regulating the miR-490-5p/SOX2 signalling pathway [63]. These findings suggest that circRNAs play a pivotal role in HCC and could help us screen potential biomarkers and targets for the prevention and diagnosis of HCC. Note: (+) means promotion and (−) means suppression.

CircRNAs that Act as Tumour Suppressors in HCC
Seven circRNAs have been found to serve as antitumour factors via multiple action mechanisms in HCC. CircARSP91 (hsa_circ_0085154) expression was influenced by the upstream regulator AR-ADAR1 [69]. The androgen receptor (AR) contributes to sex disparity in HCC. AR suppressed circARSP91 expression by upregulating ADAR1 p110 in HCC. In addition, circARSP91 repressed HCC tumour growth both in vivo and in vitro [69]. Similarly, DHX9 could induce downregulation of cSMARCA5 (hsa_circ_0001445) in HCC. cSMARCA5 overexpression facilitated apoptosis and repressed the invasion, migration, and proliferation of HCC cells, and this circRNA upregulated TIMP3 by adsorbing miR-181b-5p and miR-17-3p [70,71]. Furthermore, circMTO1 [72], circADAMTS14 [73], the circRNA SMAD2 [74], and circC3P1 (miR-4641/PCK1) [75] could serve as miRNA sponges to restrain HCC cell tumorigenic behaviours. Moreover, overexpression of both circZKSCAN1 and ZKSCAN1 mRNA repressed the invasion, migration, and proliferation of HCC cells; however, the mechanism of action of circZKSCAN1 was not related to its parental gene ZKSCAN1 (a zinc finger family gene). Overall, circARSP91 and miR-4641/PCK1 could be the most promising biomarkers because of their inhibitory effects on HCC cell migration, invasion, and proliferation and their clear mechanism of action.

Pancreatic Cancer
CircRNA_100782 and circ-PDE8A, which are upregulated in PC, are involved in the regulation of PDAC as miRNA sponges. Chen et al. found that circRNA_100782 knockdown inhibited the proliferation of PDAC cells by decreasing the expression of signal transducer and activator of transcription 3 (STAT3) and interleukin-6 receptor (IL6R), which are the target genes of microRNA-124 (miR-124) [57]. In addition, a study revealed that tumour-released exosomal circ-PDE8A serves as a miRNA sponge for miR-338 to adjust MACC1 and promotes the invasion of PDAC cells through the MACC/MET/ERK or AKT pathways [56]. These findings suggest that circRNA_100782 and circ-PDE8A act as oncogenes in PDAC and could be biomarkers for PDAC (Figure 2f).

CiRS-7
CiRS-7, also called Cdr1as, serves as a super sponge for miR-7 and possesses over 60 common binding sites [20]. The overlapping co-expression of ciRS-7 and miR-7 was observed in the mouse brain, suggesting a high degree of endogenous interaction [91]. A study has found that ciRS-7 may affect the progress of sporadic Alzheimer's disease (AD) through the miRNA-7/UBE2A signalling circuit [92]. In addition, ciRS-7 has been found to participate in the pathogenesis of multiple tumours ( Figure 3A), which demonstrates that ciRS-7 may be a novel therapeutic target and prognostic marker for these deadly diseases.
Recent research revealed that ciRS-7 overexpression in ESCC encouraged cell invasion, migration and proliferation in vitro, as well as lung metastasis and tumour growth in vivo, by abolishing the tumour-suppressive effects of miR-7. Importantly, ciRS-7 served as a sponge for miR-7, resulting in the activation of the NF-κB/p65 pathway mediated by HOXB13 [77]. In addition, knockdown of the stem cell marker Kruppel-like factor-4 (KLF-4), which is a target gene of miR-7, attenuated cell invasion induced by ciRS-7 overexpression. Moreover, BAY 11-7082, an NF-κB inhibitor, also counteracted, to some extent, ciRS-7-mediated cell invasion [78]. Furthermore, ciRS-7 overexpression could block miR-7-induced tumour suppression, including reductions in cell migration and apoptosis, by counteracting the PTEN/PI3K/AKT pathway mediated by miR-7 in GC [79]. CiRS-7 overexpression in CRC cells dramatically reduced the tumour-suppressive activity (repression of cell invasion, migration and proliferation, and acceleration of apoptosis) of miR-7, and ciRS-7 regulated the EGFR/RAF1/MAPK pathway through suppressing the activity of miR-7 [80]. Intriguingly, ciRS-7 silencing inhibited CRC cell invasion and proliferation and decreased IGF-1R and EGFR expression, which could be rescued, in part, through a miR-7 inhibitor [81]. In HCC, ciRS-7 knockdown reduced the expression of PIK3CD and CCNE1, the direct targets of miR-7. In addition, ciRS-7 knockdown restrained HCC cell invasion and proliferation by adsorbing miR-7 [82]. Thus, ciRS-7 has a complex action mechanism in four digestive system cancers and could thus have great potential among the abovementioned six circRNAs as a therapeutic target for digestive system cancers.

CircRNA_100290
CircRNA_100290 (hsa_circRNA_100290) may serve as a ceRNA to adjust the expression of CDK6 by adsorbing miR-29b in OSCC [83] (Figure 3B). Knockdown of circRNA_100290 inhibited the proliferation of OSCC cells in vitro and in vivo [83]. Furthermore, silencing circRNA_100290 greatly decreased the rate of proliferation, restrained the invasion and migration abilities, and encouraged the apoptosis of CRC cells in vitro [84]. CircRNA_100290 is also a ceRNA for FZD4 that sponges miR-516b, resulting in Wnt/β-catenin pathway activation in CRC [84]. Therefore, circRNA_100290 may be an ideal biomarker for OSCC and CRC.

CircHIPK3
CircHIPK3 (hsa_circ_0000284) promoted cell proliferation through serving as a miRNA sponge for miR-124/miR-29b in GC. COL1A1, COL4A1 and CDK6 might play important functions mediated by the circHIPK3-miR-29b/miR-124 axes in GC [85] (Figure 3C). In addition, circHIPK3 knockdown markedly curbed cell invasion, migration, and proliferation, boosted the apoptosis of CRC in vitro, and inhibited CRC metastasis and growth in vivo. Furthermore, circHIPK3 overexpression significantly counteracted the miR-7-mediated repression of malignant phenotypes of CRC cells through upregulating the proto-oncogenes (FAK, IGF1R, EGFR, and YY1) targeted by miR-7. An upstream regulator of circHIPK3 expression in CRC is c-Myb, which is a transcription factor [86]. These findings suggest that circHIPK3 may be a therapeutic biomarker for GC and CRC. In addition, circHIPK3 regulates the proliferation, migration, invasion and apoptosis of non-small-cell lung cancer (NSCLC) cells through miR-149-mediated FOXM1 expression regulation, potentially providing novel insight into the pathogenesis of NSCLC [93].

CircAGO2
CircAGO2 (hsa_circ_0135889), an intronic circRNA produced from the AGO2 gene, was upregulated in both GC and CRC and promoted the invasion, metastasis and cell growth of cancer in vitro and in vivo [87]. In addition, circAGO2 facilitated its enrichment and activation on the 3'-untranslated region of target genes by interacting with human antigen R (HuR) protein, reducing argonaute 2 (AGO2) binding and repressing the silencing of AGO2/miRNA-mediated genes related to cancer progression [87]. Overall, circAGO2 might regulate cancer progression by facilitating the human antigen R (HuR)-inhibited effects of argonaute 2 (AGO2)-miRNA complexes. These results demonstrate that circAGO2 may be a potential therapeutic and diagnostic target for GC and CRC.

Hsa_circ_0014717
The overexpression of hsa_circ_0014717 induced G0/G1 period cell cycle arrest, restrained colony formation and CRC cell proliferation in vitro and inhibited xenograft tumour growth in vivo. Furthermore, hsa_circ_0014717 upregulated the expression of the cell cycle inhibitory protein p16 [94], and the knockdown of p16 reversed the inhibition of hsa_circ_0014717 on CRC cell growth [94]. Shao et al. discovered that hsa_circ_0014717 is stable in human gastric juice and that its characteristics make it suitable for use in clinical detection methodologies [88]. These results suggest that hsa_circ_0014717 may be a useful diagnostic marker for CRC.

Hsa_circ_0001649
Hsa_circ_0001649 overexpression inhibited cell invasion, migration, and proliferation and stimulated apoptosis in HCC [89]. In addition, the expression of hsa_circ_0001649 was heightened in serum samples from patients with CRC after surgery [90]. However, the concrete mechanism of action of hsa_circ_0001649 in HCC and CRC is not clear. Future studies should emphasise the function and mechanism of hsa_circ_0001649 and its potential as a biomarker in HCC and CRC.

Diagnostic Value Assessment
To date, circRNAs have been reported as potential diagnostic targets based on several efficient indicators in five digestive system cancers, such as receiver operating characteristic (ROC) curves, sensitivity and specificity ( Table 5).
The diagnostic value of the combined circRNAs is higher than that of the single circRNAs in OSCC and PC. Three circRNAs have been identified as potential biomarkers for the diagnosis of OSCC, including hsa_circ_001242 [95], hsa_circ_0001874 and hsa_circ_0001971 [96]. Notably, the AUC (0.922) of the combination of hsa_circ_0001874 and hsa_circ_0001971 was larger than that of a single circRNA. A similar result was found in PC. Recent research revealed that circ-LDLRAD3 may be a new biomarker in the diagnosis of PC [97]. The expression of circ-LDLRAD3 in PC tissues was correlated with clinical stage, T classification, venous invasion, and lymphatic invasion, and in the plasma of patients with PC, it was significantly associated with CA19-9, N classification, venous invasion, clinical stage, metastasis, and lymphatic invasion [97]. Moreover, the AUC of circ-LDLRAD3 was 0.67. When combined with CA19-9, the AUC was increased to 0.87. These results suggest that the higher diagnostic value of the combination circRNAs could be an important choice for digestive system cancers.

Therapeutic Potential of CircRNAs
CircRNAs could have several advantages as potential targets for the treatment of digestive system cancers. First, circRNAs are stable molecules that may be relatively abundant in cells, tissues, and developmental stages. In addition, circRNAs are resistant to RNase R activity and exist in tissue [101], blood [104], saliva [96], and exosomes [105]. Moreover, circRNAs are highly conserved among species [106]. To date, multiple circRNAs involved in the regulation of digestive system cancer progression have been by in vivo and in vitro experiments [68,107]. A previous study suggested that circRNAs offer new opportunities to design innovative therapeutic strategies for cancer treatment [106]. Thus, circRNAs are very promising as treatment targets. For example, CiRS-7/Cdr1as is a miR sponge that is upregulated and involved in the regulation of ESCC [77], GC [78], CRC [79], and HCC [80] by different pathways, which suggests that the downregulation of ciRS-7/cdr1as expression might provide an effective treatment method. CircRNAs can act as sponges for miRNAs. Artificial circRNA sponges resistant to nuclease digestion could be synthesised using simple enzymatic ligation steps [108]. The synthetic circular miR-21 (scRNA21) sponge inhibited cancer cell proliferation and suppressed the activity of miR-21 on downstream protein targets, including the cancer protein DAXX [108]. Therefore, by studying the structure of endogenous circular RNA sponges to design and develop effective artificial sponges, we could eventually regulate the function of miRNAs in diseases.

Human Disease-Related CircRNA Databases
Following the application of high-throughput RNA sequencing and various modern computational approaches, a large number of human circRNA-related datasets have been established ( Table 6). The circRNA basic information integration and analysis databases are circBase [111], CIRCpedia v2 [112], circView [113], and circBank [114]. It is worth noting that circBase and circBank provide the system nomenclature for circRNAs. However, circBase may fail to achieve accurate classification and positioning. CIRCpedia v2, circView, and circBank achieve more comprehensive and expert basic information integration regarding human circRNA. There are seven databases for the functional annotation of circRNAs ( Table 6). The DeepBase v2.0 database focuses on the identification, expression, evolution, and function of circRNAs [115]. The CircInteractome and circNet databases emphasise circRNA-miRNA-gene and circRNA-protein regulatory networks [116]. The circFunBase database specifically provides the experimentally validated and computationally predicted functions of circRNA and circRNA-miRNA interaction networks [117]. The system nomenclature, function, and regulatory networks of human circRNAs (including digestive system cancer-related circRNAs) could be obtained from the above databases.
In summary, these databases help researchers understand the essential information on circRNAs in digestive system cancers and other human diseases and promote understanding of the occurrence and development of disease as well as its diagnosis and prognosis.

Identification of CircRNAs
Several techniques have been used to detect circRNAs in digestive system cancers from primary tumours and control tissues or cells [49,60], including microarray analysis [49] and high-throughput RNA sequencing [60]. Microarray analysis is suitable for screening known human circRNAs. High-throughput RNA sequencing is suitable for identifying new circRNAs and splice sites of circRNAs with sufficient underlying sequencing depth. Total RNA was treated with RNase R to degrade the linear RNA in library construction for screening circRNAs [43]. Then, circRNAs were experimentally validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with GAPDH as the reference gene [43]. Covalent loci of circRNA can be verified through Sanger sequencing. Furthermore, bioinformatics analysis is an accurate and convenient approach that can rapidly generate helpful information for further verification [127]. Thus, human disease-related circRNA databases could help us to analyse nomenclature, expression patterns, and evolution and predict the functions of human circRNAs.

Function and Mechanism of CircRNA
To provide the foundation for research on circRNA function, the subcellular location (nucleus or cytoplasm) of circRNAs first need to be analysed based on efficient nuclear/cytoplasmic RNA separation [44] and fluorescence in situ hybridisation (FISH) [48,52]. Then, gain-of-function (GOF) and loss-of-function (LOF) are employed to study circRNA functions in digestive system cancer cell lines and xenograft nude mouse models [46,67,72,75]. Human circRNA can be synthesised and cloned into the overexpression vector pcDNA3.1 [45]. The circRNA-pcDNA3.1 overexpression vector can then be transfected into digestive system cancer cell lines [40]. Next, those transfected cell lines are injected into a xenograft nude mouse model to test the GOF of circRNAs in an animal model [46,67,68,72,73,75]. On the other hand, small interfering RNAs (siRNAs) have been designed for the junction of circRNAs and transfected into digestive system cancer cell lines to examine the LOF of circRNAs [23]. Thus, multiple pathological processes can be researched with the aid of GOF and LOF of circRNAs in digestive system cancer, such as cell migration, proliferation, invasion, and anchorage-independent growth.
CircRNAs can act as a sponge binding to miRNA, thereby reducing the ability of miRNA to target mRNAs. Multiple databases can be used to analyse circRNA-miRNA-mRNA networks, such as circNet [123] and circFunBase [117] for known circRNAs and Targetscan [128] and RNAhybrid [129] for new circRNAs. A dual-luciferase assay (psiCHECK-2 vector) has been used to validate the relationship between circRNA and miRNA [42,51]. RNA immunoprecipitation (RIP) assays for AGO2 (argonaute 2) in digestive system cancer cell lines have been used to investigate the expression levels of endogenous circRNAs and miRNAs pulled down from AGO2-expressing cells by qPCR analysis [41]. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to annotate genes targeted with the purpose of studying how circRNAs regulate parental gene expression [130]. On the other hand, circRNAs can bind to proteins and sequester them to particular subcellular locations [60,69]. Several techniques targeting this mechanism have been developed. To date, RNA pull-down assays and RIPs have been utilised to detect circRNA-protein interactions [131]. Following the discovery of new circRNA mechanisms, the development of new technology will help us to employ the function and regulatory mechanisms of circRNAs.

Conclusions
CircRNAs are a type of endogenous RNA that are stable and resist RNA nuclease digestion. CircRNAs have emerged as important regulators in multiple digestive system cancers. Dysregulated circRNAs were found to act as oncogenes or tumour suppressors in digestive system cancers ( Figure 3). In the complex tumour microenvironment, overexpression of the downregulated circRNAs or depletion of the upregulated circRNAs in patients are the main approaches for circRNA-based therapy. According to current research data, the regulatory mechanism of circRNAs in digestive system cancers is very complex, and the underlying molecular mechanisms by which circRNAs cause and inhibit these cancers remain largely ambiguous. CircRNA transcriptome databases of OSCC, ESCC, GC, CRC, HCC, and PDAC have been reported and are convenient sources for selecting targets and biomarkers. The dysregulated circRNAs between digestive system cancer and normal cells/tissues may participate in regulating the inhibition and promotion of these digestive system cancers, suggesting that circRNAs could serve as therapeutic targets and biomarkers for clinical diagnosis. However, the above assumptions continue to present substantial difficulties and enormous challenges in clinical practice, and more studies on the significance, efficiency, generality, security, and reliability of these approaches are needed. Thus, we anticipate that this review will increase the comprehension of the principal functions of many circRNAs and their multiple regulatory hubs in the progression of digestive system cancers.