CircRNAs: role in human diseases and potential use as biomarkers

Circular RNAs (circRNAs) are a class of endogenous RNAs characterized by a covalent loop structure. In comparison to other types of RNAs, the abundance of circRNAs is relatively low but due to the circular configuration, their stability is very high. In addition, circRNAs display high degree of tissue specificity. The sponging activity of circRNAs toward microRNAs is the best-described mode of action of circRNAs. However, the ability of circRNAs to bind with specific proteins, as well as to encode short proteins, propose alternative functions. This review introduces the biogenesis of circRNAs and summarizes the roles played by circRNAs in human diseases. These include examples of their functional roles in several organ-specific cancers, such as head and neck and breast and lung cancers. In addition, we review potential functions of circRNAs in diabetes, cardiovascular, and neurodegenerative diseases. Recently, a growing number of studies have demonstrated involvement of circRNAs in a wide spectrum of signaling molecular pathways, but at the same time many different and controversial views on circRNAs role and function are emerging. We conclude by offering cellular homeostasis generated by networks comprising circular RNAs, other non-coding RNAs and RNA-binding proteins. Accordingly, it is predictable that circRNAs, due to their highly stable nature and remarkable tissue specificity, will emerge as reliable biomarkers of disease course and treatment efficacy.

To exemplify the biomedical potential of circRNAs, we briefly review the biogenesis of circRNAs and then describe the role of some circRNAs in head and neck squamous cell carcinoma, breast and lung cancer. Among neurological disorders, we focus herein on Alzheimer disease. In addition, we report examples of circRNAs playing functional roles in cardiovascular diseases and in diabetes. Although the sponging mechanism of circRNAs toward microRNAs has emerged as the most common mechanism of action, additional modes of action have been proposed. CircRNAs can interact with proteins and some are translated into novel polypeptides or act as transcriptional regulators 2, [14][15][16][17][18][19][20][21] . Presumably, as many circRNAs are being characterized, additional modes of action will be uncovered soon. CircRNAs are involved in many signaling pathways and some of these molecular pathways have been already characterized for their important roles in human diseases and they are subjects of clinical trials 22,23 . These characteristics, together with their presence in accessible body fluids, such as saliva, blood, and urine, make the circRNAs promising therapeutic targets and potential biomarkers for human diseases [24][25][26] .

Biogenesis and function of circRNAs
CircRNAs can derive from exons, introns, antisense, 5′ or 3′ untranslated and intergenic genomic regions 27 . Exonic circRNAs (ecircRNAs) represent the most abundant species and they are produced by a "back-splicing" mechanism. During the biogenesis process, a downstream 5′ splice site of an exon is joined to an upstream 3′ splice site of the same or another exon, involving single or multiple exons 1,[28][29][30][31][32] . The molecules derived from this mechanism form a closed circular transcript and an alternatively spliced linear RNA with skipped exons 31 . Thus, the mechanism that generates circRNAs uses the canonical spliceosomal machinery 31 . As a consequence, transcription of circRNAs competes with canonical pre-mRNA splicing and affects the rate of canonical gene expression 28 .
There are several examples of circRNA-protein interactions in the cancer context (Fig. 1). The tumor suppressor circ-Foxo3 interacts with CDK2 and p21 to form a ternary complex and inhibit cell cycle progression in cancer 10 . The oncogenic circRNA circ-Amotl1 promotes cell growth through an interaction with the protooncogene c-MYC. Circ-Amotl1 is able to increase the retention of nuclear c-MYC, promote c-myc stability, and up-regulating c-myc targets 15 .
There is also evidence showing that circRNAs can be translated into functional proteins (Fig. 1). Circ-ZNF609 is one of the first examples described of a circRNA that can be translated into a protein. Circ-ZNF609 is involved in the regulation of myoblast proliferation 17 . The circular form of the SNF2 histone linker PHD RING helicase (SHPRH) gene, which encodes the protein SHPRH-146aa represents an additional example 18 . Both circ-SHPRH and SHPRH-146aa are highly expressed in normal human brains and their expression was found to be down-regulated in glioblastoma, suggesting a tumor suppressor role 18 . In a similar way, Zheng et al. 19 identified circPPP1R12A, which is up-regulated in colon cancer (CC) and can be translated into a protein contributing to the rapid proliferation of CC cells via the Hippo-YAP pathway.
Finally, it is well accepted that intronic circRNAs (ciR-NAs) and exon-intron circRNAs (ElciRNAs) can act as transcriptional regulators (Fig. 1) 20,21 . The intronic cir-cRNA ci-ankrd52 is able to regulate its parental gene expression by modulating RNA polymerase II's elongation activity 21 . Similarly, two ElciRNAs, circEIF3J and cir-cPAIP2, are able to regulate the expression of their parental genes through a specific RNA-RNA interaction between U1 snRNA and the circRNA 20 . More recently Stoll et al. 40 showed that the intronic circle ci-Ins2, located mainly in the nucleus of pancreatic β cells, is able to regulate insulin secretion through interaction with the TAR DNA-binding protein 43 kDa (TDP-43).

CircRNAs in head and neck squamous cell carcinoma
Head and neck cancers represent the sixth most common cancer worldwide 41,42 . This cancer usually initiates in the squamous cells that line the mucosal surfaces inside the head and neck and can arise from the mucosal surfaces of the oral cavity (OSCC), oropharynx (OPSCC), and larynx. Head and neck cancers can also begin in the salivary glands and in paranasal sinuses and nasal cavities 41,42 . We showed that the circRNA circPVT1 acts as an oncogene in head and neck squamous cell carcinoma (HNSCC) 39 . CircPVT1 expression is regulated through the mut-p53/YAP/TEAD complex binding its own promoter, which is independent from the host gene PVT1 promoter 39 . CircPVT1 is overexpressed in tumors compared to matched-non-tumoral tissues and its expression is particularly high in patients with TP53 mutations 39 . This is an example of a circRNA acting as an oncogene and modulating the expression of miR-497-5p and some of its targets, such as aurka, mki67, and bub1, all genes involved in the control of cell proliferation. This is in line with the known role of miR-497-5p as a tumor suppressor in several cancers 39,[43][44][45][46] .
Xuan et al. 8 analyzed the circRNA expression in a cohort of Laryngeal squamous cell carcinoma (LSCC) tissues. They found that two circRNAs, hsa_-circRNA_100855 and hsa_circRNA_104912, were respectively up-and down-regulated in cancer tissues in comparison to the corresponding adjacent non-neoplastic tissues 8 . Patients with T3-4 stage, neck nodal metastasis, or advanced clinical stage had higher hsa_-circRNA_100855 expression and a lower hsa_-circRNA_104912 expression 8,48 .
CircHIPK3 is highly expressed in nasopharyngeal carcinoma (NPC) 9 . The silencing of circHIPK3 can reduce cell proliferation, migration, and invasion in vitro and it can repress tumor growth and metastasis in vivo 9 . Cir-cHIPK3 functions in NPC by sponging the miR-4288, which in turn targets the E74-like ETS transcription factor 3 (ELF3) 9 . Studying the circHIPK3-miR-4288-ELF3 molecular pathway could instruct ways to identify new therapeutic strategies focused on this regulatory loop.

CircRNAs in breast cancer
Breast cancer is the most common cancer in females and it can be classified in three major cancer subtypes according to estrogen or progesterone receptor expression and ERBB2 gene amplification: hormone receptor positive/ERBB2 negative (HR+/ERBB2-), ERBB2 positive (ERBB2+), and triple-negative 49 .
Galasso et al. performed a pilot study in which they described one of the first panels of circRNAs expressed in breast cancer by analyzing RNA sequencing data from five paired breast cancer samples 50 . At the same time, Nair et al. 51 developed an automated workflow called Circ-Seq to identify circRNAs in breast tumors and breast cancer cell lines. A recent work identified 235 differentially expressed circRNAs in breast cancer through highthroughput circRNA microarray analysis 52 . Among all the modulated circRNAs, circTADA2A-E6 (hsa_-circ_0006220) and circTADA2A-E5/E6 (hsa_-circ_0043278) were ranked in the top five down-regulated circRNAs by microarray analysis 52 . In particular, circTADA2A-E6 sponges miR-203a-3p and functions as a tumor suppressor by inhibiting cell proliferation, migration, and metastasis. The SOCS3 gene was predicted as a downstream target gene of the circTADA2A-E6/miR-203a-3p axis 52 , and a previous study reported that miR-203a-3p promotes cell proliferation by targeting SOCS3 in MCF-7 cells 53 . These results show that the circTADA2A-E6/miR-203a-3p/SOCS3 axis plays an important role in the inhibition of breast cancer progression.
CircRNA-000911 is another circRNA acting in breast cancer as a tumor suppressor 54 . Wang et al. 54 showed that circRNA-000911 suppresses the proliferative, migratory, and invasive capacities of breast cancer cells by sponging miR-449a and releasing Notch1, a functional target of miR-449a. This mechanism includes the involvement of Ago2, an essential protein for circRNA sponge activity 4,55 . The consequence of circRNA-000911 down-regulation in breast cancer is the up-regulation of miR-449a and downregulation of Notch1. Importantly, one downstream effector of Notch1 is the nuclear factor-kB (kB), which normally promotes breast cancer tumorigenesis and progression 54 .
The circRNA circEPSTI1 (hsa_ circRNA_000479) is upregulated in breast cancer and it is a prognostic marker and mediator of triple-negative breast cancer (TNBC) progression 56 . CircEPSTI1 behaves as an oncogene promoting TNBC cell proliferation in vitro and in vivo, and it is able to sponge both miR-4753 and miR-6809 (ref. 56 ). BCL11A is a direct target gene of both miRNAs, and it is inhibited as a consequence of circEPSTI1 knockdown. It follows that the circEPSTI1-miR-4753/6809-BCL11A axis could be an interesting pathway to investigate in order to identify new therapeutic strategies for the treatment of TNBC 56 .
CircANKS1B is another circRNA up-regulated in TNBC and its expression is associated with both lymph node metastasis and advanced clinical stage 57 . Cir-cANKS1B is able to sponge miR-148a-3p and miR-152-3p, thereby increases the expression of transcription factor USF1, which in turn up-regulates TGF-β1 expression 57 . The up-regulation of TGF-β1 results in activation of the TGF-β1/Smad signaling pathway, promoting epithelial-to-mesenchymal transition (EMT) 57 . The results suggest that circANKS1B is an interesting cir-cRNAs to study in order to find alternative therapeutic strategies for inhibiting breast cancer metastasis.

CircRNAs in lung cancer
Lung cancer is one of the most common cancers in the world with 5-year survival rates varying from 92 to 0%, depending on disease stage at diagnosis 58 .
Circ-ITCH is generated from several exons of the ITCH E3 ubiquitin protein ligase (ITCH) and it shares the miR-7 and miR-214 binding sites with the three-prime untranslated regions (3′-UTR) of its parental gene ITCH 59 . Circ-ITCH plays an inhibitory role in lung cancer progression by sponging miR-7 and miR-214 and regulating the expression of ITCH 59,60 . ITCH negatively regulates canonical Wnt signaling by targeting the dishevelled-2 (Dvl2) protein 61 . In lung cancer the downregulation of circ-ITCH brings to an increase of miR-7 and miR-214, thereby to a decrease of their target gene, ITCH. As a consequence, the Wnt/β-catenin pathway is enhanced, thereby promoting the development and progression of cancer 59,60 . Another circRNA that indirectly affects ITCH expression is hsa_circ_0043256. The circRNA hsa_circ_0043256 is able to sponge miR-1252, which binds the ITCH 3′-UTR 62 . Both circRNAs, circ-ITCH and hsa-circ_0043256, behave as tumor suppressors in lung cancer and their combined action could be used to design new strategies for the treatment of this malignancy.
In contrast to circ-ITCH, hsa_circ_0012673 is overexpressed in lung adenocarcinoma and promotes cell proliferation through the miR-22/ErbB3 pathway 63 . Hsa_circ_0012673 is able to sponge miR-22, which targets ERBB3/HER3, an important receptor tyrosine kinase in lung adenocarcinoma. ERBB3/HER3 is a member of the epidermal growth factor receptor (EGFR/ERBB) family 64 and EGFR mutations were characterized for their important role in lung cancer 65 .

CircRNA in Alzheimer's disease
Alzheimer's disease (AD) is the most prevalent cause of dementia affecting millions of people worldwide 66 . AD is a progressive and neurodegenerative disorder characterized by widespread neuronal atrophy and two histopathological hallmarks: extracellular senile plaques consisting of amyloid-β peptides, and intracellular neurofibrillary tangles composed of abnormally hyperphosphorylated Tau protein 66 .
Dube et al. 67 generated RNA-seq data from individuals with and without AD to quantify cortical circRNA expression. The results showed that there are significant associations between circRNA expression and AD diagnosis, clinical dementia severity, and neuropathological severity 67 . Interestingly, circRNA expression changes can be observed early on, in pre-symptomatic AD and in autosomal dominant AD 67 . The microtubule-associated Tau protein plays a central role in AD since it is responsible for amyloid-beta induced neuronal cell death 68 . The MAPT gene generates the Tau protein.
Using a PCR screen of RNA from human brain tissues, Welden et al. showed that the MAPT locus generates circRNAs through a backsplicing mechanism, but the role of these circRNAs is still unclear 69 .
Similarly, CDR1as has been one of the first circRNAs that were characterized. It derives from the cerebellar degeneration-related protein 1 antisense transcript (CDR1AS) and contains over 70 conventional binding sites for miR-7 4,33,70 . Down-regulation of CDR1as causes up-regulation of miR-7 and, consequently, negative regulation of its respective targets, such as ubiquitin protein ligase A (UBE2A) [71][72][73] . UBE2A is important for clearing amyloid peptides and it was found depleted in the AD brain [71][72][73] .

CircRNAs in cardiovascular diseases
RNA-Seq analysis of ribosome-depleted libraries from hearts of human, mouse, and rats origins, detected more than 9000 candidate circRNAs for each species 74 . A similar analysis listed more than 15,000 cardiac circRNAs in humans 75 . Although the study showed no statistically significant circRNA that was differentially expressed in diseased hearts compared to healthy hearts, other studies are needed to elucidate the role of circRNAs in cardiac diseases 75 . On the other hand, the analysis found significant differential expressed circRNAs during cardiomyocyte differentiation 75 .
Many of the identified cardiac circRNAs are yet uncharacterized in terms of their specific function. Nevertheless, the identification of cardiac circRNAs represents a potential strategy to use circRNAs as target molecules in the prevention and treatment of cardiovascular diseases.
The first circRNA described with a cardioprotective role was the heart-related circRNA, HRCR. This circRNA acts as a miR-223 sponge to inhibit cardiac hypertrophy and heart failure 76 . MiR-223 is able to suppress the expression level of its target, ARC, the apoptosis repressor with CARD domain protein. HRCR acts as an antihypertrophic molecule due to its sponging mechanism toward miR-223, which causes up-regulation of ARC 76 .
More recently, circFndc3b was identified as another circRNA involved in cardioprotection. CircFndc3b interacts with the RNA-binding protein Fused in Sarcoma (FUS) to regulate VEGF expression and signaling 12 . Acting on the FUS/VEGF-A axis, circFBDc3b is able to enhance angiogenesis and retard cardiomyocytes and endothelial cell apoptosis 12 .
Yet another circRNA, Cdr1as (ciRS-7), acts as a miR-7a sponge in myocardial cells 77 . It was shown that ciRS-7 induces apoptosis in myocardial infarction (MI) in mice by means of increasing caspase-3 activity. CiRS-7 is upregulated in infarcted hearts, and it is able to inhibit the miR-7a mediated cardiomyocyte protection against MI injury acting as a miRNA sponge 77,78 . CiRS-7's sponge mechanism toward miR-7a determines the up-regulation of two miR-7a targets, PARP and SP1. These proteins play pro-apoptotic roles during MI 77 .
MFACR (mitochondrial fission and apoptosis-related circRNA) regulates mitochondrial fission and apoptosis in the heart, while acting as a miRNA sponge for miR-652-3p 79 . MiR-652-3p down-regulates its target, MTP18, a nuclear-encoded mitochondrial membrane protein that contributes to mitochondrial fission in mammalian cells 79,80 . As a result, the MFACR-activated pathway instigates cardiomyocyte death through miR-652-3pdependent up-regulation of MTP18 expression 79 .
Another circRNA involved in cardiomyocyte apoptotic events is circNCX1, which is generated from the sodium/ calcium exchanger 1 (ncx1) gene 81 . circNCX1 acts as a miRNA sponge for miR-133a-3p, which is able to target the pro-apoptotic gene called Cell Death-Inducing p53-target Protein 1 (CDIP1). Importantly, miR-133a-3p plays a cardioprotective role and it is suppressed by the circNCX1 sponge mechanism 81 . This is an example of circRNAs that enhances damage following a MI event, primarily by promoting apoptosis of cardiomyocytes 81 .

CircRNAs in diabetes
Diabetes is a group of metabolic disorders all characterized by hyperglycemia, namely high levels of sugar in the blood. This condition is associated with various pathological states, such as cardiovascular disease, retinopathy, nephropathy, and neuropathy 82 .
A recent work showed the human pancreatic islets express thousands of circRNAs 83 . The circRNA Cdr1as is already known for its miR-7 sponging activity in embryonic zebrafish brains and in infarcted hearts 4,34,84 . Moreover, Cdr1as is able to affect miR-7 function in adult islet cells 84 . Xu et al. 84 showed that miR-7 is highly expressed in islet cells, and its overexpression in transgenic mice β-cells causes diabetes due to impaired insulin secretion and β cell dedifferentiation. Cdr1as promotes insulin secretion by sponging miR-7 in islet cells 84 . Hence, the interaction between Cdr1as and miR-7 in insulin secretion may become a new therapeutic target for improving β cell function in diabetes 84 .
The circRNA circHIPK3 was found up-regulated in retinas and retinal endothelial cells of patients with diabetes. CircHIPK3 is able to regulate the retinal vascular endothelial function while sponging miR-30a-3p 85 . As a consequence of its action as miRNA sponge, circHIPK3 increases the expression of VEGFC, FZD4, and WNT2, leading to endothelial proliferation and vascular dysfunction 85 . It follows that circHIPK3 could serve as a valid target for diabetic retinopathy.

CircRNAs as potential disease biomarkers
Both prognostic biomarkers and markers that predict responses to a drug or other treatment modalities must bear high specificity for a given pathophysiological condition and a highly reproducible detection capacity. Accordingly, the renewed identification of circRNAs has opened a new potential strategy for diagnosis and for monitoring progression of different human diseases (Table 1). This is primarily due to the high tissue specificity of circRNAs, their relatively high stability in tissues and body fluids, as well as ease of detection using rather simple technologies, such as real-time PCR 1,86,87 . Cir-cRNAs are highly abundant in blood 25 and there are also evidences of circRNAs in urine samples 26 , for example to assist monitoring of patients who have undergone kidney transplantation, or for diagnosis of patients with bladder cancer 26,88 .
Importantly, several recent studies reported the presence of circRNAs in extracellular vesicles, mainly exosomes, which are targets for discovery of additional types of new biomarkers [89][90][91] . The abundance and diversity of circRNAs in human blood exosomes is already available in a database called exoRBase 92 . Likewise, cir-cRNAs with diagnostic potential have been found in urine exosomes 93,94 . Additionally, another database, MiOnco-Circ, was created based on sequencing of more than 2000 tumor samples, and many urine circRNAs were identified as possible biomarkers for prostate cancer 95 .
There are several papers that have shown correlations between expression of specific circRNAs and tumor grade, size, metastatic spread, and lymph node involvement. This is the case of hsa_circ_002059 and hsa_-circ_0000190, which were found to be decreased in plasma of patients with gastric cancer 96,97 . Likewise, it has been reported that circFARSA is elevated in plasma of patients with non-small-cell lung cancer (NSCLC), in direct association with tumor cell aggressiveness in vitro 98 . The circRNA F-circEA-2a is another candidate biomarker in NSCLC. Generated from the prevalent fusion gene in lung cancer, EML4-ALK, circRNA F-circEA-2a appears elevated in plasma samples 99 .
A screening seeking differentially expressed circRNAs in plasma of patients with hepatocellular carcinoma related to the hepatitis B virus, reported elevated expression of hsa_circ_0027089 and classified it as a potential biomarker 100 . Additionally, an atlas of Blood-Based Biomarkers for Early Diagnosis of Cancers (BBcancer) has recently been established 101 . It includes data from 5000 samples across 15 different types of cancer 101 .
A recent study evaluated the presence of circRNAs in cerebrospinal fluid of patients with Alzheimer's disease (AD) and found 112 up-regulated and 51 down-regulated circRNAs 102 . Some of these circRNAs were confirmed by real-time PCR, with circ-LPAR1, circ-AXL, and circ-GPHN elevated and circ-PCCA, circ-HAUS4, circ-KIF18B, and circ-TTC39C decreased in patients with AD 102 .
Another study showed that it is possible to differentiate patients with AD and healthy individuals by testing the expression of circRNAs in peripheral blood mononuclear cells (PMBCs) 103 . Hsa_circRNA_405619 and hsa_-circRNA_000843 were shown to be elevated in PBMCs of patients with AD in comparison to healthy individuals, while hsa_circRNA_100861 and hsa_circRNA_102448 appear decreased in the same patients 103 .
Several published reports relate to circRNAs in different cardiovascular diseases 104,105 . The presence of hsa_-circRNA_025016 in the plasma of patients is able to predict postoperative atrial fibrillation, while MICRA (myocardial infarction-associated circRNA) can help predicting left ventricular dysfunction in patients with acute myocardial infarction 106,107 . Similarly, in addition to being much more expressed than its linear form, an isoform of circANRIL has been shown to be elevated in whole blood of cardiac patients and playing an atheroprotective role, unlike its linear counterpart, which appears to play a proatherogenic role 108 .

CircRNAs implicated in molecular pathways disclose their potential use as therapeutic molecules
Although several circRNAs have been found to be either up-or down-regulated in human tissues, not always their specific role in molecular pathways has been elucidated. Specific circRNAs act in the Wnt signal transduction pathway: circRNA ITCH is active in lung cancer 59 and cZNF292 is active in glioma 109 (Fig. 2). Silencing cZNF292 blocked glioma cell cycle progression by means of inhibiting the Wnt/ß-catenin signaling pathway 109 .
Wnt signaling can be divided into β-catenin-dependent (or canonical) and β-catenin-independent (or non-canonical) signaling 22,110,111 . This pathway plays a critical role during embryonic development, including cell fate specification, cell proliferation, and cell migration. Moreover, the role of Wnt signaling has been well characterized in several diseases, such as cancer, diabetes, and cardiovascular disorders [112][113][114] . Accordingly, clinical trials that tested Wnt signaling drugs have shown promising outcomes, and circRNAs affecting the Wnt pathway might serve as targets for new therapies 22,115 . CircANKS1B promotes the epithelial-to-mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) 57 (Fig. 3). EMT takes place in a diverse range of physiological and pathological conditions 116 . The molecular reprogramming occurring during EMT is orchestrated by a complex combination of factors, possibly including circRNAs. The biogenesis of numerous cir-cRNAs is promoted during EMT transition by the RNAbinding protein quaking-5 (QKI-5) 16 . Recently, several clinical trials have been launched based on the current knowledge of EMT heterogeneity and plasticity 117 . The next challenge will be to include circRNAs as biomarkers or pharmacological targets in the protocols of new clinical trials addressing EMT.
The circPVT1 and the circPPP1R12A act within the Hippo-YAP signaling pathway, respectively in head and neck squamous cell carcinoma and in colon cancer 19,39 (Fig. 4). The Hippo pathway is recognized as an evolutionarily conserved signal transduction pathway that controls proliferation, organ size, and shape during development 23 . Moreover, the Hippo pathway is involved in multiple physiological processes, such as tissue growth, regeneration, and repair, maintaining the tissue homeostasis 23 . Hippo signaling plays an important role as a tumor suppressor in cancer and its deregulation is a key feature for cancer development, progression, and resistance to cancer treatment 23,118,119 . We showed that the mutant form of p53 (mut-p53) physically interacts with the transcriptional cofactor Yes-Associated Protein (YAP) in breast cancer 120 . YAP and TAZ are the main effectors of the Hippo pathway 120 . Hippo pathway inactivation determines the translocation to the nucleus of YAP and TAZ that regulate transcriptional activation in collaboration with mut-p53. In this context, Hippo effectors YAP and TAZ can act either as tumor suppressors, when located in the cytoplasm, or as oncogenes in the nucleus. In its wild type conformation, p53 works as a tumor suppressor regulating the cellular homeostasis 23 . At the same time the "p53 status", wild type or mutant, can be considered a critical point in determining the tumor suppressor or oncogenic activity of the Hippo pathway 23 . Currently, there are several pathway modulators of the Hippo pathway that are subject of clinical development, such as the Verterpofin who inhibits the YAP-TEAD interaction 121 or the PRIMA1-MET that restores the proapoptotic function of p53 with consequent activation of downstream target genes 122 . Importantly, we showed that YAP binds circPVT1 in head and neck squamous cell carcinoma. Moreover, we demonstrated that mut-p53 stabilizes the YAP/circPVT1 complex 39 32 . Another regulatory mechanism for circRNA biogenesis uses the ADAR protein, which is capable of modifying nucleotides in intronic repeat sequences 29 . Ivanov et al. 29 demonstrated that ADAR antagonizes the expression of several circRNAs by editing intronic sites that flank exons and promote back-splicing. It is becoming clearer that introns are more important sequences for circRNAs biogenesis than initially anticipated, and that specific proteins might regulate backsplicing. Apparently, several different mechanisms control circRNA biogenesis, but it is yet unclear how do they work and which one, if any, is predominant over the others. The unique circular configuration confers to cirRNAs not only resistance to digestion by ribonucleases, but it also translates to a longer half-life compared to the respective mRNAs [124][125][126] . As a consequence, circRNA levels are typically reduced in rapidly proliferating cells, such as in cancer cells. Thus, the association between lower circRNA levels and cancer could be due, in some cases, to a simple dilution effect mediated by cell division, as in colorectal cancer 127 . Hence, the meaning of circRNA expression levels should be carefully evaluated based on the specific cellular context. In addition, many circRNAs are sensitive to RNase R treatments, thus contradicting claims related to high stability of these molecules 128 .
Although the microRNA sponging mechanism is the better-described role for circRNAs, many circRNAs putatively act as sponges toward only a single, or very few miRNA targets 129 . Notably, there are some prerequisites that should be fulfilled in order to identify a circRNA as a putative ceRNA: the presence of multiple binding sites for the miRNA target, relatively high abundance of the cir-cRNA, a miRNA target with fewer target genes, and a circRNA with a better affinity toward the miRNA than the mRNAs-miRNA affinity 130 . Moreover, a circRNA that triggers the degradation process of a target miRNA, and not only inhibits the interaction between miRNA and mRNA, might act as a better ceRNA candidate 129 .
The most common approach for assessing the sponging mechanism is the ectopic overexpression of binding sites for a specific miRNA. However, the result of this kind of experiment should be interpreted with caution since it could be biased by the introduction of sufficiently high numbers of binding sites able to inhibit the activity of the miRNA in question 126 . At the same time, one should also keep in mind that if a circRNA only binds with the cognate miRNA and inhibits its function without degrading it, the abundance of the miRNA would not be affected. Piwecka et al. 131 have shown that the choice between either degradation or functional inhibition depends on whether the binding sites connecting circRNAs and miRNAs are completely complementary or they only partially match each other. It follows that a reliable in silico analysis of putative binding sites for circRNAs on miRNA targets is an essential requirement. While most studies have shown repression of miRNAs, Hansen et al. 132 showed that the interaction between Cdr1 and miR-671 would actually lead to degradation of this cir-cRNA through AGO2 rather than by the expected miRNA degradation mode 132 . Therefore, it is possible that additional circRNA-miRNA interactions regulate RNA circles.
With a few exceptions, the majority of circRNAs are expressed at low levels in both normal and cancer cells; hence they are unlikely to have only secondary roles in cellular physiology. However, the cascade of events that a single circRNA might unleash can potentially be of great importance from the clinical point of view, as we have shown for the circRNA circPVT1 (ref. 39 ). The roles of circRNAs must be carefully assessed in light of the various processes of their biogenesis and degradation, in addition to their broad capabilities for interacting with miRNAs and proteins.

Conclusions
The biochemical and molecular characteristics of cir-cRNAs hold the promise that specific circles of RNA will be utilized in the future as disease biomarkers and pharmacological targets, thus opening new possibilities for early detection and treatment [133][134][135] . The large spectrum of mechanisms of action used by circRNAs makes the understanding of their role not only challenging but also promising in terms of resolving the complex molecular mechanisms activated in human disorders. Indeed, cir-cRNAs can act as tumor suppressors or as oncogenes in oncology 136,137 . Likewise, circRNAs are involved in cardioprotection against heart failure, as well as mediate cardiomyocyte death in myocardial infarction 12,[76][77][78][79] . Moreover, circRNAs are extensively expressed in the mammalian brain 138,139 . Networks of circRNAs, RNAbinding proteins and microRNAs play important roles in different human diseases, which reflects the complex regulatory potential of circRNAs. Hence, it is likely that the next few years will witness the discovery of more circRNAs and new modes of their action in human disorders.