Summary of identified circRNA in the cardiovascular disease.
Circular RNAs (circRNAs) belong to the family of long noncoding RNAs (lncRNA) that, unlike linear RNAs, are characterized by a covalently closed circular RNA structure lacking 5′ cap and 3′ poly-adenylated tails. circRNAs have a role in epigenetic regulation of downstream targets. circRNAs play a crucial role in regulating gene and protein expressions by acting as a microRNA (miRNA) sponge and RNA binding protein (RBP) sponge and interact with proteins to affect cell behavior. circRNA expression profiles differ between physiological and pathological states. Moreover, the expression patterns of circRNAs exhibit differences in a tissue-specific manner. Although investigations on circRNAs have been exploding nowadays, yet only a limited number of circRNAs are identified. Furthermore, further researches are needed to shed light on their functions and targets. Therefore, circRNAs are becoming vital as potential biomarkers that may be used for the diagnosis and treatment of diseases. In this chapter, we review the current advancement of cirRNAs with regard to their biogenesis, biological functions, gene regulatory mechanisms, and implications in human diseases and summarize the recent studies on circRNAs as potential diagnostic and prognostic biomarkers based on existing knowledge.
- circular RNAs
- cardiovascular diseases
- neurological disorders
- immune regulation
The ENCyclopedia Of DNA Elements (ENCODE) project reported that noncoding RNAs (ncRNAs) unexpectedly consist of more than 70% of the human genome . After the data released by ENCODE project consortium, numerous studies have focused on the identification and function of these transcripts . ncRNAs can be a group based on their different characteristic features . Long noncoding RNAs (lncRNAs) are subclass of ncRNAs that have been recently proved to have a role in physiological and pathological processes . lncRNAs are >200 nucleotides long, divergent class of RNA transcripts that coordinate expression of protein-coding genes. Yet, they have a lack of ability to encode proteins . Circular RNAs (circRNAs) are a special subtype of lncRNAs . circRNAs are characterized by a single-stranded covalently closed loop structure with neither a 5′ cap nor a 3′ poly(A) tail . Due to their circular structure, circRNAs are more stable than the linear mRNA counterpart and not susceptible to RNA exonuclease cleavage [6, 7]. The presence of circRNA was first demonstrated in the cytoplasm of eukaryotic cells in 1979 . It was thought that circRNAs were a by-product formed during splicing mechanism in the first year . Numerous circRNAs have been predicted with the technical developments in high-throughput RNA sequencing (RNA-seq) and methodological innovations in bioinformatics. The presence and function of the predicted circRNAs in different tissues and cell lines are widely studied nowadays. After the determination of their role in the control of gene expression, circRNAs have gained great attention by researchers in this field. In this chapter, we will focus on circRNAs and their biological functions in health and disease.
2. Biogenesis of circRNAs
According to the gene structure they contain, circRNAs can be divided into three groups: exonic circRNA (ecircRNA), circRNAs from introns (ciRNAs), and exon-intron circRNA (elciRNA) . To date, many studies have shown that circRNAs mainly emerged during pre-mRNA splicing process of protein coding genes. Unlike canonical mRNA splicing mechanism, down-stream donor splice site is covalently joined with an upstream acceptor splice sites during circRNA formation. This splicing mechanism is called “back-splicing” . The back-splicing mechanism is depicted in Figure 1 . circRNAs can also be formed through the hybridization of complementary inverted sequences (such as human Alu repeats) in introns . If Alu sequences are located in different introns of the same gene, this leads to generate circRNAs, which contains multiple exons . Exon circularization is facilitated by cis-acting inverted repeat sequences as well as by trans-acting RNA-binding proteins (RBPs), which interact with unique sequences in introns [11, 12]. ecircRNA or elciRNA formation is promoted by either cis-acting elements or trans-acting factors as in Figure 2A . ecircRNAs can also be formed from elciRNAs by removal of intronic sequences . Apart from the other circRNAs, ecircRNAs are transported into cytoplasm . In human cells, the existence of ciRNAs is demonstrated by Zhang et al. . ciRNAs are generated through a lariat-derived mechanism relying on mainly a consensus motif containing a 7-nt GU-rich element adjacent to the 5′ splice site and an 11-nt C-rich element adjacent to the branchpoint site. After cleavage of 3′ end, stable ciRNA is produced . The predicted biogenesis of ciRNA is shown Figure 2B .
3. Gene regulation and biologic functions of circRNAs
The expression patterns of circRNAs are specific to the cell type or phase of development . Although the all-biological functions of circRNAs are not entirely defined, some are well studied in the literature. Biological functions of circRNAs include micro RNA (miRNA) sponge, regulation of gene expression and properties of mRNA binding, scaffolding, and cellular translocation.
3.1 circRNAs can act as miRNA sponges
As a major component of gene regulators, competing/competetive endogenous RNA (ceRNA) contains a micro RNA response element (MRE-competitively binds miRNA) and can affect the regulatory functions of miRNAs . Growing evidence has indicated that circRNAs can act as ceRNA or miRNA sponge molecules. Because of containing plenty of MRE, circRNAs can competitively bind to miRNAs (generally several copies of miRNA) and adsorb them like a sponge . As a result, miRNAs can no longer act on their target mRNA . Therefore, circRNAs can regulate the gene expression and also give rise to decreasing of the functional miRNA [17, 18]. Compared to other ceRNAs, circRNA binds more effectively to miRNAs. Therefore, they are also called “super sponge” . The most characteristic miRNA sponge “antisense to the cerebellar degeneration related protein 1 transcript” (CDR1as)/ciRS-7 includes approximately 70 conserved binding sites for miRNA-7 (miR-7) and forms a complex with Argonaute (AGO) proteins . CDR1as-miR-7 complex co-localizes in the cytoplasm and supresses degradation of miR-7-target mRNAs . Interestingly, circRNA has been reported that it is also displayed to be abundant in exosomes in serum . Therefore, Li et al. suggested that sorting of circRNAs to exosomes was regulated by altering levels of associated miRNA in producer cells [16, 22]. In addition, researchers have found that CDR1as including exosomes inhibit miR-7-induced growth in recipient cells . Testes-specific circRNA/circSry [the circular transcript of sex determining region Y (Sry) gene] can also serve as a sponge for miRNA-138 . It contains 16 MREs of miRNA-138 and regulates the expression of miR-138-target genes, functioning similar to CDR1as . Additionaly, many other circRNAs have been identified as miRNA sponges such as hsa_circ_001569, heart-related circRNA (HRCR), itchy E3 ubiquitin protein ligase circRNA (circITCH), forkhead box O3 circRNA (circ-foxo3), homeodomain interacting protein kinase 3 circRNA (circHIPK3), mitochondrial tRNA translation optimization 1 circRNA (circMTO1), zinc finger protein 609 circRNA (cirZNF609), and baculoviral IAP repeat containing 6 circRNA (circBIRC6) . Among them, cirITCH regulates the expression of ITCH by acting as a sponge for miR-214, miR-17, and miR-7 .
Apart from common phenomenon, in some cases, the binding of circRNAs to miRNAs may not always lead to inhibition of miRNAs. Linearization and AGO2-mediated cleavage of CDR1as can occur when CDR1as interacts with miR-671. Thus, bound miR-7 is released from CDR1as . On the other hand, in spite of the role of circRNA in gene regulation as a classical sponge effect, some recent studies have revealed that the number of circRNAs with miRNA sponge property is limited. Besides inhibition effect, it has been regarded that the interaction between circRNAs and miRNA is also related to sorting, storage and localization of miRNA .
3.2 circRNAs regulate gene expression and interact with protein
In addition to their miRNA sponge function, circRNAs can also act as sponges for other components as RBPs. There are many proteins known as RBP such as AGO protein, RNA quaking, muscleblind (MBL) protein, RNA polymerase II (Pol II), eukaryotic initiation factor 4A-III . RBPs bind specific sequences to their target genes and control all stages of mRNA lifecycle including splicing, nuclear export, stability and subcellular localisation . A number of circRNAs contain a large amount of binding sites for a single or multiple RNA-binding proteins. For example, circRNA protein sponge derived from the
circRNAs also inhibits parental gene transcription in target genes via invading RNA binding sites. Strongly binding of circRNA, derived from sepallata3 (SEP3) gene, to its cognate DNA locus blocks the binding of its linear isoform to the cognate DNA. The formation of R-loop (RNA:DNA hybrid) gives rise to termination of SEP3 gene transcription . Moreover, circRNAs can be paired with DNA to generate DNA-RNA triple helixes. Therefore, this pairing may affect DNA replication .
Some ciRNAs (e.g., ci-ankrd52, ci-sirt7) and eIciRNAs (e.g., circEIF3J, circPAIP2) regulate the transcription of their parental genes. eIciRNAs can regulate parental gene transcription in a cis-acting manner [18, 23, 24]. Recent studies have been indicated that nuclear elciRNAs (localized to the promoter of their parental genes) interact with U1 small nuclear ribonucleoproteins (snRNPs) and RNA pol II and promote the transcription initiation of their parental genes [28, 30]. For example, eukaryotic translation initiation factor 3J circRNA (circEIF3J) and poly(A)-binding protein-interacting protein 2 circRNA (circPAIP2) have been suggested to have cis-regulatory effects on parental genes and promote transcription of EIF3J and PAIP2. This cis-regulatory effect occurs by binding of its circRNAs to Pol II, U1 snRNP, and their parental gene promoters [18, 25]. When transcription is initiated, the production of eIciRNA can be increased so that this phenomenon generates a positive feedback loop . Moreover, they have a function as positive regulators through their interactions with the elongating Pol II complex [24, 25, 31]. In addition, exonic circular antisense noncoding RNA in the INK4 (a family of cyclin-dependent kinase inhibitors) locus (circANRIL/cANRIL) reduces ANRIL that inhibits transcription of INK4/ARF gene by binding to the Polycomb Gene (PcG) complex. Thus, cANRIL regulates the transcription of INK4/ARF .
3.3 Cellular translocation properties
Some circRNAs may affect nuclear translocation of other proteins to nucleus and regulation of gene transcription. For example, CircAmotl1 may increase nuclear translocation of signal transducer and activator of transcription 3 (STAT3) to regulate the expression of mitosis-related genes .
Another capability of circRNAs is to ensure that cellular proteins remain in their natural cellular position. It has been reported that circAmotl1 can enhance stability of c-myc by maintaining its nuclear retention and increase its binding affinity to several promoters. Therefore, it upregulates c-myc targets such as hypoxia inducible factor-1 alpha (HIF-1α), cell division cycle 25A (Cdc25a), ETS Like-1 (ELK-1) . In another example, cytoplasmic circ-foxo3 interacts with differentiation-1 (ID-1), HIF-1α, focal adhesion kinase (FAK), the transcription factor E2 (E2F1) and prevents their translocation from cytoplasm to other location .
3.4 Scaffolding properties
circRNAs also serve as scaffolding in the assembly of protein complexes . It has been reported that circ-foxo3 acts as an adaptor to bridge between cyclin-dependent kinase 2 (CDK2) and CDK inhibitor p21 (cyclin-dependent kinase inhibitor 1A). This interaction (circ-foxo3/CDK2/p21) inhibits cell-cycle progression within G1 to S-phase transition [18, 24]. However, downregulation of circ-foxo3 leads to the release of CDK2 from p21 and CDK2 phosphorylates cyclin E and cyclin A for cell cycle progression. On the other hand, the circ-foxo3 connects the murine double minute 2 (MDM2) to tumor protein p53 (p53), and induces the degradation of p53 by ubiquitination. However, circ-foxo3 weakly interacts with foxo3 and suppresses foxo3 from MDM2-mediated polyubiquitination and proteasome degradation .
3.5 mRNA binding properties
Most circRNAs are capable of interaction with mRNAs. It has been reported that they can be able to regulate the stability of mRNAs as well. In addition to its miRNA sponge function, CDR1as is also proposed to form a duplex structure with CDR1 mRNA and stabilizes it. Similarly, stabilization of mature intercellular adhesion molecule 1 mRNAs in macrophages was found to be facilitated by RasGEF domain family member 1B circRNA (circRasGEF1B) .
3.6 The effect of circRNA as a translator
Recent studies have shown that some circRNAs can be entered to translational process in spite of considering noncoding RNA [33, 34]. A limited number of studies have indicated the potential protein coding properties of circRNAs until now but the translational efficiency might be low . circRNA containing internal ribosomal entry site (IRES) and open reading frame can be translated into protein or polypeptide. In eukaryotes, IRES is an alternative way of initiating translation, independent of 5′ cap structure and 3′ poly (A) tail recognition . It has been demonstrated that the 40S subunit of the eukaryotic ribosome can interact with circRNA-containing IRES and then begin translation in
A recent study reported that N6-methyladenosine (m6A), a most common base modification of RNA, can promote the protein translation of circRNA in human cells, even if one m6A motif can initiate circRNA translation [18, 19, 29]. m6A-driven circRNA translation is prevalent, and several endogenous circRNAs have the potential for translation and regulatory role in a cell against enviromental factors .
3.7 The effect of circRNAs on splicing
Recent studies have shown that there is a competition between backsplicing and linear splicing. Thus, the biogenesis of circRNAs leads to loss of protein-coding mRNA levels and inhibits parental gene expression . On the other hand, the level of circRNA is negatively correlated to the splicing efficiency of certain genes due to the competition between linear splicing and circRNA biogenesis .
4. circRNAs in cardiovascular diseases
Cardiovascular disease (CVD) is one of the most important health problems. It causes most of the deaths worldwide . According to recent studies, a number of circRNAs may play a significant role during development of CVD or pathological conditions such as cardiac hypertrophy, coronary artery disease, atherosclerotic vascular disease, cardiomyopathy, cardiac fibrosis, heart failure (HF), ischemia, and myocardial infarction (MI) [36, 37, 38]. However, in development of heart disease, the regulatory mechanisms and functional importance of several circRNAs are not clear . circRNAs are also concentrated in body fluids such as seminal fluid, saliva, and blood. Thus, their potential usage as clinical biomarkers may be possible in the future .
Some heart specific RNA-splicing regulators are also important players for heart development. One of the RNA-splicing regulators is RBM20 that is required for splicing of cardiac-related genes such as titin . Its mutation leads to exon retention in the region of I-band and results in larger titin isoforms . According to RNA-seq researches in tissues from dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy, 80 different circRNAs are derived from the titin gene (TTN) [30, 40].
circANRIL is generated as an antisense transcript from the INK4A/ARF gene locus by alternative splicing . SNPs localized within chromosome 9p21 are likely to affect the INK4/ARF locus. These SNPs can regulate ANRIL splicing and may lead to circANRIL production . Interestingly, there is an association between 9p21 SNPs and the susceptibility to atherosclerosis . circANRIL is also implicated in the pathogenesis of atherosclerosis . In another study, Burd et al. suggested that 9p21 SNPs affect the coordination of ANRIL expression and splicing via interaction of different PcG complexes. Furthermore, PcG complexes are targeted to the INK4/ARF locus and that leads to inhibition of INK4/ARF transcription. Moreover, they also indicated that their study is the first to provide evidence for relationship between circRNA and atherosclerotic vascular disease (ASVD) .
circANRIL also disrupts exonuclease-mediated pre-rRNA processing and ribosome biogenesis by binding to pescadillo homologue 1 (PES1). This leads to nuclear stress and p53 activation in cells [38, 39]. Therefore, it supresses cell proliferation and inhibits apoptosis in vascular smooth muscle cells and macrophages. Consequenly, circANRIL acts as a protective factor against atherosclerosis [41, 43]. On the other hand, it has been indicated that a novel circular RNA product of ANRIL, cANRIL (exon4-6) also regulates the expression of INK4/ARF .
In addition, circRNA serves as a protein scaffold such as circAmotl1 in cardiac dysfunction [35, 43]. circAmotl1 facilitates phosphorylation of protein kinase B (AKT) and nuclear translocation of pAKT by forming ternary complexes with AKT and phosphoinositide-dependent protein kinase (PDK) [43, 44, 45]. Zeng et al. have suggested that pAKT translocation may be responsible for protection of heart cells from cardiomyopathy caused by doxorubicin .
circ-foxo3 is another circRNA described to may have a role in the cardiovascular diseases. Stress-related proteins (HIF-1α and FAK) and senescence-related proteins [inhibitor of DNA-binding protein (ID1) and E2F1] are arrested in cytoplasm by circ-foxo3. Therefore, circ-foxo3 prevents translocation of these proteins into the nucleus. As a consequence, this mechanism promotes cardiac senecence through ectopic expression of circ-foxo3 [41, 46]. Besides these functions, circRNAs are reported to also show their effects as miRNA sponge in cardiovascular diseases. circRNAs and their function in cardiovascular diseases are indicated in Table 1 .
|circRNA||Possible target||CVD||Biological function or description||Ref|
|circANRIL (ex 5–7)||PES-1||AS and CAD||Impairs pre-rRNA maturation and ribosome biogenesis and increases nucleolar stress and apoptosis||[32, 47]|
|circANRIL (ex 4–6)||ASVD||Neighboring gene regulation such as INK4a|||
|Hsa_circ_0003575||miR-199-3p, mir-9-5p, mir-377-3p, and miR-141-3p||AS||Regulates endothelial cell proliferation and angiogenesis acting as a miRNA sponge|||
|Hsa_circ_0010729||mir-186||AS and CHD||Regulates vascular endothelial cell proliferation and apoptosis via targeting miR-186 and HIF-1α axis|||
|circACTA2||miR-548f-5p||AS and CHD||Maintains contractile phenotype of VSMC|
Mediates NRG-1-ICD regulation of α-SMA expression in HASMCs
|circWDR77||mir-124||AS||Regulates VSMC proliferation and migration via targeting miR-124 and FGF2|
Inhibits the expression of SM22a and STIM1 by acting as a miRNA sponge
|circ-SATB2||mir-939||AS||Inhibits the expression of SM22a and STIM1 by acting as a miRNA sponge|
Regulates cell phenotypic differentiation, proliferation, apoptosis, and migration in VSMC
|circR-284||miR-221||AS and carotid plaque rupture||Reduces the proliferation of VSMCs by circR-284/mir-221/p27Kipi axis|
Upregulated circR-284:miR-221 ratio in the early stage of carotid plaque rupture
|hsa_circ_0124644||CAD||Potential biomarker of coronary artery disease|||
|hsa_-circ_0001879||CAD||Significant upregulated expression levels in CAD patients|||
|hsa_circ_0004104||Dysregulation of atherosclerosis-related genes by overexpression of hsa_circ_0004104|||
|CDR1as||miR-7a||MI||Upregulates the expression of PARP and SP1 acting as a miRNA sponge and promotes apoptosis|||
|MICRA||Acute MI, HF, and LVD||Potential biomarker of left ventricular dysfunction in the patients with acute MI||[58, 59]|
|MFACR||miR-652-3p||MI||Upregulates apoptosis and mitochondrial fission|||
|HRCR||mir-223||HF and cardiac hypertrophy||Increases the expression of ARC by acting as a miRNA sponge. Suppresses cardiac hypertrophy|||
|circ-081881||mir-548||Acute MI||Positively regulates PPARγ acting as a miRNA sponge|||
|circRNA-010567||miR-141||MI||May mediate fibrosis-associated protein resection|||
|circNCX1||miR-133a-3p||Ischemic myocardial injury||Promotes cardiomyocyte apoptosis by acting as a miRNA sponge and increased in response to ROS|||
|circAmotl1||AKT and PDK||Cardiac repair and cardiomyopathy||Facilitates the nuclear translocation of AKT and PDK1|
Improves survival and decreases apoptosis
|circTTN||DCM||Dysregulated in disease model||[65, 66]|
|circZNF609||miR-615-5p and miR-150-5p||Hypertension and CAD||Inhibits cell proliferation, migration, and tube formation and promotes cell apoptosis|
Acts as a miRNA sponge and leads to upregulation of MEF2A expression
|Hsa-circ-0005870||hsa-miR-619-5p, hsa-miR-5095, hsa-miR-1273 g-3p, and hsa-miR-5096||Hypertension||Downregulated in hypertension patients|||
|rno_circRNA_016002||Hypertension||Upregulated in hypertensive rat strains compared to normotensive rats|||
|hsa_circ_0014243||hsa-miR-10a-5p||EH||Crucial role in the genesis and development of EH and presents a certain diagnostic capability for EH|||
|hsa_circ_0037911||miR-637||EH||Upregulated in hypertension patients||[65, 71]|
|hsa_circ_0126991||EH||May serve as a stable biomarker for early diagnosis of EH|||
|circ-foxo3||ID-1, E2F1, FAK, and HIF-1α||Cardiac senescence||Interacts with ID-1, E2F1, FAK, and HIF-1α and induces cellular senescence in aging hearts|||
Although there is limited number of studies until today, CVD-related studies for circRNA are in progress. Therefore, it is still required the identification of circRNA as candidate biomarkers for CVDs. Moreover, biologic functions of circRNA in vascular endothelial cell and heart tissue should be validated in further studies.
5. circRNAs in neurological disorders
Recent studies have shown that circRNAs are plentifully expressed in normal neuronal cells [73, 74, 75]. They may be found abundantly in neuronal cells for several reasons: (i) brain contains more host genes of circRNA such as neuronal genes, which play roles in neurogenesis, neuronal development, and neuronal differentiation [11, 74], (ii) the expression levels of circRNAs are higher in brain than other tissues [75, 76], (iii) due to the slow division rates of neurons, circRNAs may accumulate more in the brain than other tissues , (iv) neuronal genes contain long introns (>10 kb) with inverted repeat sequences, thereby simplifying formation of circRNAs , and (v) circRNAs due to the absence of 5′ and 3′ ends result in greater stability than linear RNAs, leading to a relatively longer half-life . The half-life of circRNAs is approximately 20 h, compared with corresponding linear isoforms (no more than 8 h) .
The latest studies have shown that circRNAs could attenuate cell senescence and cell survival and may be involved in the regulation of aging and age-related neurological diseases [80, 81, 82]. Thus, circRNAs are expected to be new potential biomarkers and target for aging and age-related neurological diseases ( Table 2 ). These studies have suggested that circRNAs may play an important role in pathological mammalian brain function, which is implicated in disorders in central nervous system (CNS) including Alzheimer’s disease (AD), Parkinson’s disease (PD), neuropsychiatric disorders, prion disease, and inflammatory neuropathy.
|CircRNA||Target||Neurological disease||Possible mechanisms||Ref|
|ciRS-7||miR-7||AD||ciRS-7 is reduced in AD, and miR-7 can downregulate AD relevant targets, such as ubiquitin conjugating enzyme UBE2A, which play an essential role in the clearance of amyloid peptides||[83, 84]|
|circSry||miR-138||AD||mir-138 participate in learning and memory ability and is increased in AD, and it promotes tau phosphorylation by targeting the RARA/GCK-3β pathway||[85, 86, 87]|
|ciRS-7||miR-7||PD||miR-7 may downregulate α-synuclein expression, promotes the degradation of α-synuclein mRNA levels, and protects cells against oxidative stress|||
|Neuropsychiatric disorders||miRNA deregulation and affects brain function||[78, 89, 90]|
|ciRS-7||miR-7||Prion disease||Prion protein PrPc can upregulate expression of ciRS-7||[91, 92]|
|hsa-circRNA 2149||—||Inflammatory neuropathy||Hsa-circRNA 2149 has been detected in CD19+ leukocytes|||
|circSry||miR-138||Inflammatory neuropathy||miR-138 can balance the ratio of Th1 and Th2 via suppressing the function of RTF3||[93, 94]|
|circRNA100783||CircRNA100783 may be involved in chronic CD28-associated CD8 (+) T cell aging|
CDR1as, a circRNA, is highly plentiful and specifically expressed in the mammalian brain . Some studies have indicated that ciRS-7 contains multiple anti-miR-7 sequences. This suggests that ciRS-7 may function as a sponge to sequester the normal functions of miR-7 [57, 95, 96, 97]. ciRS-7 can regulate the stability of mRNA targets in the brain by binding to miR-7 [78, 85]. Besides, ciRS-7 can interact with multiple protein subunits, thus acting as “scaffolding” for RBPs [7, 98]. Thereby, it facilitates the interaction by potentially increasing the stability of the circRNA transcripts. Due to its multiple functions in brain, researchers have suggested that ciRS-7 can be a potential biomarker for neurodegenerative disorders including AD and PD .
5.1 circRNA in Alzheimer’s disease
Alzheimer’s disease is a chronic neurological disease. Lukiw et al. showed that the expression level of ciRS-7 is decreased in hippocampal CA1 region in sporadic AD . Functional deficiency of ciRS-7 can lead to upregulation of miR-7 in AD brain and may cause the downregulation of several AD-relevant mRNA targets, including the ubiquitin conjugating enzyme E2A (UBE2A) [83, 84, 99, 100]. This autophagic protein, UBE2A, is a central effector in the ubiquitination cycle. UBE2A is crucial for clearing amyloid peptides via phagocytosis and contributes to amyloidogenesis . In contrast to the previous studies, Shi et al. have shown that ciRS-7 promotes the degradation of amyloid precursor protein (APP) and beta-secretase 1 (BACE1) in an nuclear factor kappa beta (NF-κB)-dependent manner . Hence, future studies are needed to reveal ciRS-7 function/functions and its exact role in AD pathology.
CircSry can serve as a miRNA sponge in neural cells. CircSry inhibits miR-138 [53, 85], which is a potential molecular regulator of human memory function . CircSry has multiple binding sites for miR-138 and promotes tau phosphorylation by targeting the “retinoic acid receptor alpha/glycogen synthase kinase-3β” (RARA/GCK-3β) pathway . Some studies have indicated that miR-138 influences learning and memory abilities by regulating acyl protein thioesterase 1 [87, 102]. Therefore, association of circSry and miR-138 in AD should be further investigated.
5.2 circRNA in Parkinson’s disease
Parkinson disease, progressive age-related neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta [103, 104]. To date, five genes have been determined to cause PD, such as α-synuclein (SNCA), parkin, dj-1, PTEN-induced kinase 1 (pink1), and leucine-rich repeat kinase 2 (lrrk2) . SNCA is the key player in the pathogenesis of PD based on neuropathologic, genetic, and cellular evidence . The over-expression and aggregation of SNCA, a target gene of miR-7, is considered as a distinctive marker in PD [107, 108]. miR-7 has been proposed to play a role in PD by reducing expression of SNCA . ciRS-7 plays a protective role by inhibiting miR-7 that directly regulates the expression of SNCA . miR-7 alleviates SNCA expression dose-dependently and induces the degradation of SNCA mRNA levels . These results suggest that ciRS-7 serves as a miR-7 sponge
5.3 circRNA and inflammatory neuropathy
circRNAs may participate in inflammatory reactions that induce neuropathy. Some circRNAs may affect immune responses due to the fact that they contain virus miRNA binding sites. For instance, hsa-circRNA 2149 contains 13 unique, head to tail spanning reads. Researchers discovered hsa-circRNA 2149 in CD19+ leukocytes, but not CD341 leukocytes or neutrophils. On the other hand, circRNA100783 may be involved in chronic CD28-related CD8(+) T cell aging and for this reason could be a novel biomarker for this conditions . Furthermore, circSry, another circRNA, can repress miR-138 activity, which could balance T helper 1 (Th1) and T helper 2 (Th2) expressions through suppressing the function of runt-related transcription factor 3 (RUNX3) .
5.4 circRNA and prion diseases
Most prion diseases are infectious via transmissible particles composed of prion protein in scrapie (PrPSc), an isomer of noninfectious cellular prion protein (PrPc). Studies have discovered that ciRS-7 expression is induced by PrPc overexpression [91, 92]. ciRS-7 may suppress miR-7 activity and therefore ciRS-7 may be involved in the prion disease pathogenesis.
5.5 circRNA and neuropsychiatric disorders
Apart from in brain tumors, ciRS-7 may also play a role in neuropsychiatric disorders. Increased miR-7 levels have been determined in neuropsychiatric disorders, serving as a proof for ciRS-7-mediated deregulation of dendritic spine density via a miR-7-SHANK3 (SH3 and multiple ankyrin repeat domains 3) axis [89, 90]. In recent study, Piwecka et al. showed that ciRS-7 knockout mice display behavioural phenotypes related to neuropsychiatric disorders. Deleting of ciRS-7 locus in mice leads to synaptic transmission function disorder and unusual neuropsychiatric-like behavior . Other than miR-7, ciRS-7 also has a binding site to miR-671, which is deregulated in all brain regions in ciRS-7 deficient mice; however, the direction of changes was opposite. It is designated that the binding site on ciRS-7 is completely complementary to miR-671, and the interaction of these two molecules could lead to AGO-mediated ciRS-7 slicing and miR-671 deterioration. On the contrary, the binding sites on ciRS-7 are partial complementaries to miR-7. For this reason, it is likely that circRNAs can serve as a platform to store and transport certain miRNAs [78, 89, 90].
Currently, circRNA studies in the CNS are in progress. To date, there is a limited number of circRNA identified in neurological disorders. Moreover, previous studies mainly focus on ciRS-7 function. Therefore, it is still needed to identify candidate circRNAs as a potential biomarker in neurological disease. In addition, their functional properties in neuronal cells should be also validated in further studies.
6. The role of circRNAs in immune regulation
Although many circRNAs are under survey, their roles in autoimmune diseases remain incomprehensible, and there are insufficient data to determine their exact role of circRNAs in such diseases [24, 114].
The connection between miRNAs and immunity has been well-studied, which has led to the hypothesis that circRNAs may contribute to immune regulation by interacting with miRNAs. In particular, due to their abilities to serve as miRNA and protein sponges, they can regulate gene expression and encode proteins. Therefore, circRNAs can participate in the development and progression of different immune responses and immune diseases [23, 24, 114]. On the basis of the current studies, the majority of circRNAs defined in autoimmune diseases are ecircRNAs, and a few are ciRNAs and eIciRNAs [23, 24, 114, 115, 116]. The circRNAs identified to date, their functions, and roles in immunological diseases are shown in Table 3 .
|Disease||CircRNA||Regulation||miRNA sponge targets||Potential functions||Ref|
|It may be improved as novel noninvasive biomarkers for SLE|||
|CDR1as/ciRS-7||↓||—||It functions as the miR-7 sponge to increase expression of PTEN and restricts hyper-responsiveness of B cells||[100, 118]|
|RA||Hsa_circ_104871||↑||—||It serves as potential biomarkers for diagnosis and performs severity or pathological course of RA|||
|Hsa_circ_0057980||↓||miR-181d||It functions as the miR-181d sponge to suppress the development of RA||[86, 120]|
|Hsa_circ_0088088||↓||miR-16||It functions as the miR-16 sponge to suppress the development of RA||[121, 122, 123]|
|Hsa_circ_0001045||↑||miR-30a||It functions as the miR-30a sponge to promote the biogenesis of RA|
|MS||Hsa_circ_0005402||↓||—||It can be improved as MS biomarkers|||
|Hsa_circ_0035560||↓||—||It arranges negatively the biogenesis of MS|
|GSDMB ecircRNA||↑||miR-1275||It functions as the miR-1275 and miR-149 sponges to induce MS||[124, 125]|
|miR-149||Both circRNAs are derived from the ANXA2|
|PBC||Hsa_circ_402458||↑||miR-522-3p||It may be appropriate for PBC diagnosis||[121, 126]|
|miR-943||It functions as the miR-522 and miR-943 sponges to counter chronic inflammation and aberrant TGF-β signalling of PBC|||
|SCID||Circ-CDC42BPA||↑||—||It disrupts transduction of B cell signalling to induce formation of SCID||[128, 129]|
|Circ-TNFRSF11A||↑||—||It attends in the SCID-mediated alteration of different signalling pathways||[128, 130]|
|WAS||Circ-ROBO1||↑||—||It activates the pathogenesis of WAS||[128, 131]|
|Circ-CDC42BPA||↑||—||It disrupts transduction of B cell signalling to induce formation of WAS|||
It will be important in future studies to determine biological functions of circRNAs in immune cells. circRNAs may serve as both potential biomarkers and immune regulators [23, 24, 114, 115, 116]. Hence, it may be helpful to improve our understanding of the molecular biological basis of autoimmune diseases.
7. circRNAs in cancer
Cancer is one of the most common causes of death in worldwide. As stated in world cancer report (2014), 10 million people of the world develop all types of cancer each year. Moreover, over 6 million patients around the world die from this disease annually . Unfortunately, the number of patients diagnosed with cancer is increasing and is estimated to increase in future in worldwide [133, 134]. Even if, a functional improvement in the treatment approach is established, and new therapeutic strategies are still needed for therapy of cancer. Therefore, the identification of the altered pathways and gene transcripts has been the subject of researches recently. miRNAs have a role in gene regulation and affect various molecular biological processes such as cell growth, development, differentiation, proliferation, and cell death . As circRNAs interact with miRNAs and then influence the mRNA expression levels of target genes, the identification of circRNA-miRNA-mRNA network has become the objective of cancer researches.
There are numerous investigations on circRNAs and their functions in cancer as compared with other diseases. To date, most of the studies have focused on miRNA sponge function of circRNAs. miRNAs have been classified depending on the effect of miRNAs on downstream target/targets . miRNAs can act as oncogenes or tumor suppressors during carcinogenesis . Likewise, circRNAs are also named according to their behaviour during tumorigenesis. While some circRNAs contribute to tumor progression and metastasis, the others suppress oncogenesis.
Studies on altered expression of circRNAs in (lung and breast cancer) tumor samples are summarized in Tables 4 and 5 . Moreover, in these selected studies, the circRNA-miRNA-mRNA interaction network is well defined.
|CircRNA||circRNA expression status||Target miRNA||miRNA expression status||Target mRNA/signaling pathway||mRNA expression status||Main findings of the studies||Ref|
|hsa_circ_0001946||↓||hsa-miR-7-5p, hsa-miR-671-5p, hsa-miR-1270, hsa-miR-3156-5p||↑||NER signaling pathway||Activated||Compared to pairs of adjacent nontumor tissues, expression of hsa_circ_0001946 is downregulated in 43 NSCLC tissues|
There was a decrease in hsa_circ_0001946 expression on the cisplatin-resistant A549/CDDP cells compared with the parental A549 cells
|↑||miR-135a-5p||↓||SIRT1||↑||Compared to pairs of adjacent nontumor tissues, expression of circ_0001946 is upregulated in 72 lung adenocarcinoma tissues|
The circ_0001946 expression is upregulated in the four lung adenocarcinoma cell lines compared with the nonmalignant human lung epithelial cell line
The increase in circ_0001946 expression in tumor samples is an independent prognostic factor for the patients with lung adenocarcinoma as well as advanced TNM stages
|circAGFG1||↑||miR-203||↓||ZNF281||↑||Compared to pairs of adjacent nontumor tissues, expression of circAGFG1 is upregulated in 20 NSCLC tissues|
circAGFG1 enhances ZNF281-mediated migration and proliferation of NSCLC
|hsa_circRNA_102984 (circPTPRA)||↓||miR-96-5p||↑||RASSF8/e-cadherin||↑||Compared to pairs of adjacent nontumor tissues, expression of hsa_circRNA_102984 (circPTPRA) is downregulated in 34 NSCLC tissues|
circPTPRA acts as a miR-96-5p sponge, and it leads to upregulation of RASSF8 levels in both
|circ_0020123||↑||miR-488e3p||↓||ADAM9||↑||Compared to pairs of adjacent nontumor tissues, expression of circ_0020123 is upregulated in 55 NSCLC tissues|
The circ_0020123 expression is upregulated in the four NSCLC cell lines compared with the nonmalignant human bronchial epithelial cells
The increase in circ_0020123 expression in tumor samples has been correlated with short overall survival rate in NSCLC patients
|↑||Compared to pairs of adjacent nontumor tissues, expression of hsa_circ_0020123 is upregulated in 80 NSCLC tissues|
Upregulation of hsa_circ_0020123 expression in tumor samples has been correlated with short overall survival in NSCLC patients
The hsa_circ_0020123 expression is upregulated in the six lung cancer cell lines
|circVANGL1||↑||miR-195||↓||Bcl2||↑||Compared to pairs of adjacent nontumor tissues, expression of circVANGL1 is upregulated in 95 NSCLC tissues|
The circVANGL1 expression is upregulated in the five NSCLC cell lines compared with the nonmalignant human bronchial epithelial cells
Upregulation of circVANGL1 expression leads to higher stage, bigger tumor size, and shorter overall survival in NSCLC patients
(named as circP4HB)
|↑||miR-133a-5p||↓||Vimentin||↑||Compared to pairs of adjacent nontumor tissues, expression of circP4HB is upregulated in 80 NSCLC tissues|
Upregulation of circP4HB expression leads to higher metastatic capacity and shorter survival in NSCLC patients
|circ_0026134||↑||miR-1256||↓||TCTN1 and GAGE1||↑||Compared to pairs of adjacent nontumor tissues, expression of Circ_0026134 is upregulated in 30 NSCLC tissues|
The Circ_0026134 expression is upregulated in the four NSCLC cell lines compared with the nonmalignant human bronchial epithelial cells
|Circ-FOXM1 (hsa_circ_0025033)||↑||miR-1304-5p||↓||PPDPF and MACC1||↑||Compared to pairs of adjacent nontumor tissues, expression of Circ-FOXM1 is upregulated in 80 NSCLC tissues|
The Circ-FOXM1 expression is upregulated in the four NSCLC cell lines compared with the nonmalignant human bronchial epithelial cells
The increase in circ-FOXM1 expression in tumor samples was correlated with short overall survival rate in NSCLC patients
|circ_0003645||↑||miR-1179||↓||TMEM14A||↑||Compared to pairs of adjacent nontumor tissues, expression of circ_0003645 is upregulated in 59 NSCLC tissues|
The circ_0003645 expression is upregulated in the four NSCLC cell lines compared with the nonmalignant human bronchial epithelial cells
The increase in circ_0003645 expression in tumor samples is an independent prognostic factor for the patients with NSCLC as well as advanced TNM stages
|hsa_circ_0002360||↑||hsa-mir-3620-5p||↓||PHF19||↑||Compared to pairs of adjacent nontumor tissues, expression of hsa_circ_0002360 is upregulated in 18 lung adenocarcinoma tissues|||
|↓||SF3B3||↑||Compared to pairs of adjacent nontumor tissues, expression of circRNA 100146 is upregulated in 40 NSCLC tissues|||
|circFGFR3||↑||miR-22-3p||↓||Gal-1||↑||Compared to pairs of adjacent nontumor tissues, expression of circFGFR3 is upregulated in 63 NSCLC tissues|
The increase in circFGFR3 expression in tumor samples is correlated with the poor prognosis of NSCLC patients
|Akt and Erk 1/2 signaling pathway||Activated|
Wnt/b-catenin signaling pathway
|Compared to pairs of adjacent nontumor tissues, expression of circ_0006427 is downregulated in 94 lung adenocarcinoma|
The circ_0006427 expression is downregulated in the four lung adenocarcinoma cell lines compared with the nonmalignant human lung epithelial cell line
The decrease in circFGFR3 expression in tumor samples is correlated with the poor prognosis of lung adenocarcinoma patients
|↓||miR-429 miR-200b-3p||↑||TIF1γ||↓||circPTK2 has an important role in regulating TGF-β-induced EMT and tumor metastasis|||
|hsa_circ_100395||↓||miR-1228||↑||TCF21||Compared to pairs of adjacent nontumor tissues, expression of hsa_circ_100395 is downregulated in 69 NSCLC|
The hsa_circ_100395 expression is downregulated in the six lung cancer cell lines compared with the the nonmalignant human bronchial epithelial cells
Downregulation of hsa_circ_100395 expression in tumor samples is correlated with TNM stage and lymphoid node metastases
|circ-BANP||↑||miR-503||↓||LARP1||↑||Compared to pairs of adjacent nontumor tissues, expression of circ-BANP is upregulated in 59 NSCLC|
The circ-BANP expression is upregulated in the four lung cancer cell lines compared with the nonmalignant human bronchial epithelial cells
Upregulation of circ-BANP expression in tumor samples predicted lower Survival rate
|↑||miR-1275||↓||FOXK1||↑||Compared to pairs of adjacent nontumor tissues, expression of circMAN2B2 is upregulated in 41 NSCLC|
The circMAN2B2 expression is upregulated in the four lung cancer cell lines compared with the nonmalignant human lung epithelial cells
|circ_0016760||↑||miR-1287||↓||GAGE1||↑||Compared to pairs of adjacent nontumor tissues, expression of circ_0016760 is upregulated in 83 NSCLC|
The circ_0016760 expression is upregulated in the four lung cancer cell lines compared with the nonmalignant human bronchial epithelial cells
Upregulation of circ_0016760 expression in tumor samples predicted short overall survival in NSCLC patients
|CircRNA||circRNA expression status||Target miRNA||miRNA expression status||Target mRNA/signaling pathway||mRNA expression status||Main findings of the studies||Ref|
|circ_0006528||↑||miR-7-5p||↓||Raf1||↑||Compared to adjacent nontumor tissues, expression of circ_0006528 is upregulated in BCa tissues|
The increase in circ_0006528 expression in tumor samples has been correlated with advanced TNM stage and poor prognosis
|MAPK/ERK signaling pathway||Activated|
|circKIF4a (hsa_circ_0007255)||↑||miR-375||↓||KIF4A||↑||Compared to pairs of adjacent nontumor tissues, expression of circKIF4A is upregulated in 57 TNBC tissues|
circKIF4A expression increased in the five TNBC cell lines compared with the four NTNBC and nonmalignant breast epithelial cell line
The increase in circKIF4A expression in tumor samples has been correlated with worse outcome of TNBC patients
|hsa_circ_0004771||↑||miR-653||↓||ZEB2||↑||Compared to pairs of adjacent nontumor tissues, expression of hsa circ 0004771 is upregulated in BCa tissues|
hsa circ 0004771 expression increased in the five BCa cell lines compared with nonmalignant breast epithelial cell line
The increase in hsa circ 0004771 expression in tumor samples has been correlated poorer survival prognosis
|circTADA2A-E6||↓||miR-203a-3p||↑||SOCS3||↓||Compared to adjacent nontumor tissues, expression of Hsa circTADA2A-E6 is downregulated in TNBC tissues|
The decline in Hsa circTADA2A-E6 expression in tumor samples was associated with poor patient survival for TNBC
|circAGFG1||↑||miR-195-5p||↓||CCNE1||↑||Compared to adjacent nontumor tissues, expression of circAGFG1 is upregulated in TNBC tissues|
circAGFG1 expression increased in the six TNBC cell lines compared with nonmalignant breast epithelial cell line
The expression levels of circAGFG1 were reversely correlated with overall survival of patients with TNBC
|hsa_circ_000479||↑||miR-4753 miR-6809||↓||BCL11A||↑||Compared to pairs of adjacent nontumor tissues, expression of circEPSTI1 is upregulated in 10 TNBC tissues|
The increase in circEPSTI1 expression in tumor samples was positively correlated with tumor size, lymph node infiltration and TNM stage, and associated with poor prognosis
|hsa_circ_0008039||↑||miR-432-5p||↓||E2F3||↑||Compared to pairs of adjacent nontumor tissues, expression of hsa_circ_0008039 is upregulated in 38 TNBC tissues|
hsa_circ_0008039 expression increased in the six BCa cell lines compared with nonmalignant breast epithelial cell line
|hsa_circ_0007534||↑||miR-593||↓||MUC19||↑||Compared to pairs of adjacent nontumor tissues, expression of hsa_circ_0007534 is upregulated in 40 BCa tissues|
hsa_circ_0007534 expression increased in the five BCa cell lines compared with nonmalignant breast epithelial cell line
|circRNA-000911||↓||miR-449a||↑||Notch1||↓||Compared to pairs of adjacent nontumor tissues, expression of circRNA-000911 is downregulated in 35 BCa tissues|
hsa_circRNA_000911 expression decreased in the six BCa cell lines compared with nonmalignant breast epithelial cell line
|↑||miRNA-661||↓||MTA1||↑||Compared to pairs of adjacent nontumor tissues, expression of circ-UBAP2 is upregulated in 78 TNBC tissues|
circ-UBAP2 expression increased in TNBC cell lines compared with nonTNBC cell lines
The increase in circ-UBAP2 expression in tumor samples has been correlated with reduced OS in TNBC patients
|↑||miR-3607||↓||FOXC1||↑||Compared to pairs of adjacent nontumor tissues, expression of CircIRAK3 is upregulated in 35 BCa tissues|
CircIRAK3 expression increased in TNBC cell lines compared with normal mammary epithelial or ER-positive cell lines
The increase in CircIRAK3 expression in tumor samples has been correlated worse recurrence-free survival in breast cancer patients
|circ_0005230||↑||miR-618||↓||CBX8||↑||Compared to pairs of adjacent nontumor tissues, expression of circ_0005230 is upregulated in 76 BCa tissues|
circ_0005230 expression increased in six BCa cell lines compared with nonmalignant mammary epithelial cell lines
The increase in circ_0005230 expression in tumor samples has been correlated worse overall survival in breast cancer patients
|↑||miR-148a-3p||↓||USF1||↑||Compared to pairs of adjacent nontumor tissues, expression of CircANKS1B is upregulated in 23 TNBC tissues|
CircANKS1B expression increased in TNBC cell lines compared with NTNBC cell lines
The increase in CircANKS1B expression in tumor samples has been correlated worse overall survival in breast cancer patients
|hsa_-circ_005239 circGFRA1||↑||miR-34a||↓||GFRA1||↑||Compared to pairs of adjacent nontumor tissues, expression of circGFRA1 is upregulated in 51 TNBC tissues|
The increase in circGFRA1 expression in tumor samples has been correlated short overall survival in TNBC patient
circGFRA1 expression increased in TNBC cell lines compared with NTNBC cell lines
By taking all studies together, circRNAs may be candidate surrogate molecular markers for cancer in different aspects, such as angiogenesis, metastasis, and drug resistance. Although to date some circRNA-miRNA-mRNA axis is predicted in cancer-associated pathways, the function and importance of dysregulated circRNAs still need to be supported in larger numbers of samples and patients, in various cancers.
8. Research databases of circRNA
With the increasing interest in circRNAs, comprehensive circRNA databases are required for prediction of circRNAs and their targets . To evaluate and simplify the properties and interaction of various circRNAs with other RNAs from different aspects, numerous databases have been published (circlncRNAnet, starBase v2. 0, circBase, circRNABase, circ2Traits, nc2Cancer, DeepBase v2. 0, CircInteractome, TSCD, CIRCpedia, circRNADb, CircNet, CircR2Disease, circBank, and so on) . Examples of circRNA databases and their usage in researches are shown.
starBase v2. 0 determines miRNA-circRNA interactome and includes miRNA, mRNA, and lncRNA information .
circ2Traits can be provided information about miRNA-circRNA interaction and its association with particular diseases .
CircInteractome can be used in coupling the circRNA with related RNA-binding proteins .
TSCD is helpful to describe tissue-specific circRNAs in mouse and human genomes .
CIRCpedia includes reverse and variable splicing sites of circRNAs from individuals and mouse samples .
circBank can be a resource to facilitate the research of function and regulation of circRNAs .
In summary, circRNAs, a new class of noncoding RNAs, are widely investigated by researchers due to their role in post transcriptional gene regulation. Recent studies have indicated their effects on the development of diverse diseases by acting as a miRNA sponge, RBP sponge, and transcriptional modulator or direct encoding proteins. Although the miRNA sponge function of circRNAs is currently investigated in the diseases, other mechanisms of circRNAs are still under investigation, and further studies are needed. After the interpretation of their function in disease pathogenesis, they may have a potential to become a drug target. Using circRNAs as biomarkers or therapeutic targets needs to be further investigated due to their complex roles. Based on these characteristics, circRNAs are likely to guide the development of new diagnostic and therapeutic strategies as well as prevention of diseases.
Conflict of interest
The authors declare no conflict of interest.