The effective function of circular RNA in colorectal cancer

Colorectal cancer (CRC) is the 3rd most common type of cancer worldwide. Late detection plays role in one-third of annual mortality due to CRC. Therefore, it is essential to find a precise and optimal diagnostic and prognostic biomarker for the identification and treatment of colorectal tumorigenesis. Covalently closed, circular RNAs (circRNAs) are a class of non-coding RNAs, which can have the same function as microRNA (miRNA) sponges, as regulators of splicing and transcription, and as interactors with RNA-binding proteins (RBPs). Therefore, circRNAs have been investigated as specific targets for diagnostic and prognostic detection of CRC. These non-coding RNAs are also linked to metastasis, proliferation, differentiation, migration, angiogenesis, apoptosis, and drug resistance, illustrating the importance of understanding their involvement in the molecular mechanisms of development and progression of CRC. In this review, we present a detailed summary of recent findings relating to the dysregulation of circRNAs and their potential role in CRC.


Background
Colorectal cancer (CRC) is one of the most common malignancies ranking third in the incidence and second in mortality among other cancers in the world. The global incidence of CRC is increasing, with approximately 3640 deaths and 17,930 new cases in 2020 [1,2]. The exact mechanisms underlying CRC development remain unknown, however, risk factors that are strongly related to CRC include genetics, diet, tobacco smoking, heavy alcohol consumption, inactive lifestyle and age, where > 50 is a significant risk factor for CRC. However, recent evidence has also detected an increased risk for young adults [3]. Clearly the disorder is multifactorial in nature, with no common identifiable predictor of pre-disposition [4]. Here, we will review the molecular evidence to date.
Genetic and epigenetic alterations have both been found in CRC patients; changes in chromosomal copy number, aberrant gene methylation, and dysregulated gene expression, including tumor suppressor genes such as APC, BRAF, DCC, TP53, SMAD4, SMAD2, oncogenes such as KRAS and NRAS, and DNA repair genes including MLH1 and MSH6 [5,6].
Dividing these mutation types into functional pathways broadly identifies three separate mechanisms: Chromosomal instability, which is the most common cause of genomic instability in CRC, significantly linked to alterations in APC and KRAS genes [7,8]. In hereditary and sporadic colorectal cancer, microsatellite instability (MSI) is another key pathway. Germline mutation in one of the DNA mismatch repair genes, MLH1, MSH2, MSH6, or PMS2 leads to hereditary nonpolyposis colorectal cancer (HNPCC), while MSI in sporadic colorectal cancer is predominantly due to hypermethylation of the MLH1 promoter and sometimes sporadic mutations [9]. Defects in the mismatch repair mechanisms can also lead to MSI status [10]. A third pathway is via epigenetic alteration. CpG island methylator phenotype (CIMP) differences can result in changes in gene expression or function without changing the DNA sequence of that particular gene [11]. Taken together; these three pathways indicate the genetic heterogeneity of CRC.
CRCs are classified into 4 subtypes: CMS1-CMS4 with different clinical and biological characterizations [12]. Despite recent advances in our knowledge of signaling pathways involved in CRC, chemo-and radiotherapy resistance remains the most significant hurdle in CRC treatment. Therefore, a novel methodology for improved early diagnosis is essential. Non-coding RNAs (ncRNAs) play important roles in the regulation of chemo-and radio resistance of CRC [13]. Thus, ncRNAs could serve as targets for the development of new therapeutic strategies for drug and radiation resistance in CRC [14,15]. circRNAs are a significant facet in ncRNAs biology, thus understanding of the role of circRNAs in CRC progression is pivotal to identifying new diagnostic, prognostic and predictive biomarkers for CRC [16]. In this review, we summarize the potential clinical implications of human circRNAs in CRC, for use as predictive biomarkers and/or therapeutic targets.

The non-coding RNAs
The majority of the human genome (~ 90%) is transcribed as ncRNAs, which contain multiple classes of RNAs with various lengths [17]. Many studies have identified functional roles for ncRNAs, in various physiological and pathological processes, such as diabetes, cardiovascular disease, and cancer [18][19][20]. Classes of short ncRNAs include microRNAs (miRNAs), small interfering RNAs (siRNAs) and short piwi-interacting RNAs (piRNAs), meanwhile, linear lncRNAs (long non-coding RNAs) and circular RNAs are both classed as long noncoding RNAs [21]. circRNAs, however, are a new class of long ncRNAs, processing largely from exotic or intronic sequences, and are remarkably unique in structure and chemical characteristics compared with linear RNAs. circRNA biogenesis is based on the back-splicing process, and closed 5-3ʹ ends negate degradation by RNA exonuclease or RNase R [22]. Classification of circRNAs is largely based on sequence origin, where subgroups include the circular intronic RNAs (ciRNAs), the exonic circRNAs (EcircRNAs), and exon-intron circRNAs (EIciRNAs) [23]. EcircRNAs, which predominantly exist in the cytoplasm, comprise the majority of all circRNAs. EcircRNAs can be formed by three different mechanisms, including lariat-driven circularization, RNA-binding protein (RBP)driven circularization, and back splicing. EIciRNAs however, are formed only by back splicing of ciRNAs, which depends on a 7-nt GU-rich element and an 11-nt C-rich element, important in escaping debranching and exonucleolytic degradation [23,24]. circRNAs have relatively stable structure and show tissue-specific expression, also displaying developmental stage regulation, with evolutionary conservation among species [25].

Functions of circRNAs
circRNAs have regulatory roles in gene expression by sponging miRNAs, competing with other RNAs for binding to miRNAs and RNA binding proteins (RBPs) to modulate the local concentration of RBPs and RNAs as part of the competing endogenous RNA (ceRNA) network [26]. circRNAcircRNACDR1as (ciRS-7), for example, which has more than 70 conserved binding sites for miR-7, and is highly expressed in human and mouse brains [27,28]. SRY, which encodes both linear and circular RNAs, is involved in sex determination in testis development. cir-cRNA SRY can control metastasis and invasion of tumor cells via sponging miR-138 [29,30]. Another circRNA, known as CircITCH, plays similar roles as a miRNA sponge, via miR-7, miR-17, and miR214, to inhibit proliferation through the Wnt/β-catenin signaling pathway [31], which is illustrated in Fig. 1A.
Although circRNAs are considered to be non-coding RNAs due to lack of 5'-cap structure and 3'-polyadenylation tail, circRNAs have been shown to generate protein products in a cap-independent manner [32]. Interestingly, many circRNAs are sometimes translated, indeed using high-content genomic screening, Legnini et al. found Circ-ZNF609 can translate into a protein in a splicing-dependent and cap-independent manner [33]. Yang Y et al. discovered CircFBXW7, produced from the FBXW7 gene, encoding a novel 21-kDa protein FBXW7-185aa, which reduced the half-life of c-Myc by antagonizing USP28-induced c-Myc stabilization [34].
The overall activities of circRNAs are intricately intertwined with RNA binding proteins, modulating the stability of mRNAs, regulating gene transcription, and translating proteins [35] and are involved in the regulation of cell proliferation, pluripotency and early lineage differentiation, epithelial-mesenchymal transition (EMT), cancer progression and chemoradiotherapy resistance, as shown in Fig. 2.

Upregulation of circRNAs in CRC
Among all the validated aberrantly expressed circRNAs in colorectal cancer, upregulation of circRNAs more often associates with oncogenesis. Xia et al. found abnormally expressed circRNAs through CircRNA high-throughput sequencing, identifying Circ-0053277 as having the ability to sponge miR-2467-3p, and as being significantly upregulated in CRC tissues, where it facilitated CRC cell migration, proliferation, and epithelial-mesenchymal transition [36]. Similarly, Li et al. identified CircVAPA as being upregulated in tissues and plasma, serving as a sponge for miR-101. Furthermore, they showed that the expression level of miR-125a was decreased in CRC cells, and CircVAPA knockdown repressed CRC cells cycle progression, invasion, and migration [37]. Knockdown of CircVAPA can also suppress CRC cell cycle progression, invasion, and migration by sponging miR-125a [38].
Yahang et al. found that Hsa_Circ_0026416 which was upregulated in CRC tissues and plasma, and has a key role in promoting the progression of CRC both in vitro and in vivo, may function as a ceRNA to sponge miR-346 [39].
Knockdown of another upregulated circRNA, Cir-cACAP2 (hsa_circ_0007331), which was reported to be significantly upregulated in CRC tissues and colon cancer cells lines, suppressed proliferation and invasion by downregulating T lymphoma invasion and metastasis protein 1 (Tiam1) expression, through upregulated miR-21-5p expression (40). Another highly overexpressed circRNA in CRC is Hsa_ circ_0136666, derived from the PRKDC gene, which can regulate proliferation and migration of CRC cells by sponging miR-136 [41].

Downregulated circRNAs in CRC
As well as being overexpressed, other circRNAs are downregulated in CRC. Wang X et al. showed hsa_ Circ_001988 was significantly downregulated in 31 matched colorectal cancer tissue samples, proposing this circRNA as a novel diagnosis potential biomarker in the CRC [42]. Geng Y reported hsa_Circ_0009361 to be significantly downregulated in both CRC tissues and derived cells. circRNA promoting the proliferation, epithelial-mesenchymal transition, migration, and invasion of CRC cells by sponging of miR-582. Conversely, overexpression of hsa_Circ_0009361 caused upregulation in the expression of adenomatous polyposis coli 2 (APC2) and blocked the activity of the Wnt/β-catenin pathway [43]. Circ-ITGA7, which sponges' miR-370-3p to increase ITGA7 transcription-, through inhibition of RREB1 via oncogenic Ras has been shown to be down-regulated in CRC tissue samples [44]. Indeed, Circ-ITGA7 has also been shown to directly act as a tumor suppressor in CRC, with clinical features including cancer differentiation, lymph node metastasis, distant metastasis, and alterations in the TNM stage [45]. circRNA Circ-FBXW7 silencing was previously reported to enhance the proliferation, cell migration, and invasion of CRC cells in culture.
In contrast, overexpression of Circ-FBXW7 significantly suppressed CRC cell proliferation, migration, and invasion. Similarly, Circ-FBXW7 silencing was also shown to stimulate tumor growth in SW480 and SW620 tumor models, whereas Circ-FBXW7 overexpression repressed tumor progression in the same system. This suggests that Circ-FBXW7 could serve as a target biomarker of CRC. Potential mechanisms have been proposed, including upregulated mRNA and protein expressions of NEK2 and mTOR, and diminished the PTEN expression (46). circR-NACirc_021977 is another circRNA found to be downregulated in CRC. Circ_021977 was shown to sponge miR-10b-5p, with a regulatory axis inhibiting proliferation, migration, and invasion in CRC via p21 and p53 [47]. Dysregulated circRNA expression in CRC is summarized in Table 1.

circRNAs in predicting response to chemoradiotherapy
Targeted therapy, chemotherapy, and multiagent regimens, for example, FOLFIRI (5-FU and irinotecan) and FOLFOX (5-FU oxaliplatin) can be applied as the standard treatment of CRC. However, chemotherapy has its limitations, including toxicity, low response rates, unpredictable innate and acquired resistance mechanisms, and low tumor-specific selectivity [137]. Recent studies have shown that different ncRNAs such as circRNAs, may play important roles in the regulation of chemoresistance and affect the sensitivity of tumors to chemotherapy and radiotherapy through modification of various signaling pathways, including cell cycle, proliferation, apoptosis, and DNA damage repair [84,112]. hsa_circRNA_0001313 is one of the upregulated circRNAs in radio-resistant CRC tissues. Inhibition of hsa_circRNA_0001313 induces radio-sensitivity, reduced cell viability, and increases caspase-3 activity and colony formation by negatively modifying miR-338-3p in CRC cells, which has shown in Fig. 1B [124]. Another recent study reported that CircDDX17 was down-regulated in CRC, and its overexpression induced inhibition of 5-Fu resistance, blocked tumor growth, and CRC progression via sponging miR-31-5p [131]. Interestingly, Circ-32883 was upregulated in CRC tissues and its overexpression was positively associated with chemoresistance through its potential action as a sponge for miR-501-5p. This miRNA binds to EML5 mRNA, inhibiting its expression. Thus, promoting resistance to FOLFOX therapy [112]. Other circRNAs related to chemotherapy resistance are summarized in Table 2.

circRNAs as biomarkers for colorectal cancer
Through improvements in high-throughput sequencing, circRNA microarray, and chip analysis we now know circRNAs are differentially expressed in CRC, and certain circRNAs are involved in various biological processes such as proliferation, migration, invasion, and apoptosis. Due to the unique structure of circR-NAs, which confers resistance to RNase and longer half-lives, they can therefore be potential candidates for diagnostic biomarkers. However, the underlying biological function of circRNAs requires further investigation [138,139]. Several circRNAs have been proposed as useful therapeutic targets for CRC. For instance, hsa_circ_022382 which is derived from the human FADS2 gene is overexpressed in 200 CRC tissues, where CircFADS2 overexpression was positively associated with clinicopathological features. CircFADS2 expression may therefore be a promising biomarker for prognostic investigation in CRC patients [95]. In another study, hsa_circ_0026344 was shown to be significantly downregulated in 32 CRC patients compared to paired adjacent non-tumorous tissues. The expression of hsa_ circ_0026344 was correlated with tumor size and lymph metastasis. Functionally, circRNA-0026344 overexpression significantly suppressed CRC cell proliferation and colony formation as well as promoted apoptosis by     regulating miR-21 and miR-31 levels [45]. Other cir-cRNAs with biomarker potential are summarized in Table 3.

circRNAs as therapeutic targets in colorectal cancer
Targeted therapy has been widely used in the clinic due to its excellent efficacy, and it can work on cancerous cells by directly inhibiting cell proliferation, differentiation, and migration [50]. Indeed, monoclonal antibodies, for instance, are currently an important player in targeted therapies [51]. circRNAs moderate drug resistance by sponging microRNAs both in traditional chemotherapeutic drugs, advanced targeted drugs, and immunotherapeutic drugs. For example, therapeutic targeting of ciRS-7 may become a promising strategy for colorectal cancer patients, since higher expression of ciRS-7 correlated with multiple clinicopathologic factors, such as advanced T-stage, lymph node, and distant metastasis, and ciRS-7 overexpression promotes the EGFR/RAF1/ MAPK pathway by inhibiting miR-7 activity [121,155]. Yang et al. indicated that high expression of circPTK2 positively correlated with poorer survival, showing CircPTK2 can bind to vimentin and promote EMT growth and metastasis in CRC cells, therefore ciRS-7 may become a therapeutic target for CRC metastasis [51]. The relation between circPTK2 in CRC is shown in Fig. 3.
Another highly expressed circRNA in CRC tissue is Circ_001680 which was observed to enhance the proliferation and migration capacity of CRC cells. Fluorescence reporter assays confirmed that circ_001680 alters the expression of BMI1 by targeting miR-340. More importantly, Circ_001680 was found to promote the propogation of cancer stem cells in CRC and induce resistance against Irinote by modifying the miR-340 target gene BMI1 n [53]. Safe and effective delivery of ncRNAs is a significant therapeutic paradigm for all cancers. Since unmodified oligonucleotides are not stable in circulation, modifications of oligonucleotides are essential to increasing efficacy and stability. Most current oligonucleotide therapies need an additional delivery system to achieve these desired biological effects. Several options need to be considered in selecting a delivery system, including stability, evasion of the innate immune system, avoidance of non-specific interactions with serum proteins, and non-target cells. One of the common strategies to increase the circulation time for therapeutic oligonucleotides is shielding the exterior of delivery vehicles with polyethylene glycol (PEG). This strategy may prevent the non-specific function of particles with immune cells and other non-target tissues. Although a variety of delivery systems has been developed in the laboratory, challenges remain in bringing the full potential of RNAi to clinical approaches [156]. circRNAs however, offer significant increases in stability over current strategies.

Conclusions and perspectives
Following advancements in high-throughput sequencing, the field of circRNAs has attracted more attention and is currently an area of intense interest in the field of cancer research. circRNAs are an ideal biomarker in cancer, and are stably expressed in exosomes, blood, and saliva, where specific circRNAs have been indicated as promising prognostic or diagnostic biomarkers already. Abnormal expression of circRNAs has been observed in a wide range of human malignancies and their dysregulation can alter gene expression networks, leading to dramatic changes in cell fates, including cancer initiation and progression. circRNAs can be both oncogenic and anti-oncogenic, so could potentially be utilized in the treatment and prognosis of colorectal cancer. Although recent advances on circRNAs have highlighted some interesting insights, much work remains to be done to translate circRNAs into clinical application for clinical patient benefit. Major hurdles include the development of an efficient siRNAs delivery system, and the assessment of safety and side effects, yet, clearly circRNAs have significant potential for the treatment and diagnosis of CRC.