Circular RNA in cardiovascular disease: Expression, mechanisms and clinical prospects

Abstract Circular RNAs (circRNAs) are a group of covalently closed, endogenous, non‐coding RNAs, which exist widely in human tissues including the heart. Increasing evidence has shown that cardiac circRNAs play crucial regulatory roles in cardiovascular diseases (CVDs). In this review, we aimed to provide a systemic understanding of circRNAs with a special emphasis on the cardiovascular system. We have summarized the current research on the classification, biogenesis and properties of circRNAs as well as their participation in the pathogenesis of CVDs. CircRNAs are conserved, stable and have specific spatiotemporal expression; thus, they have been accepted as a potential diagnostic marker or an incremental prognostic biomarker for CVDs.


| Biogenesis of circRNAs
CircRNAs are generated through a unique mechanism known as backsplicing. Recent research has shown that back-splicing is catalysed by the canonical spliceosomal machinery and modulated by both RBPs and intronic complementary sequences (ICSs). 8,9 However, unlike canonical splicing, the biogenesis of a circRNA requires that a donor splice site of an exon is not connected to an acceptor splice site of a downstream exon, as observed in linear splicing, but instead to an upstream acceptor site. 10 As a result of the back-spliced junction event that forms a unique exon-exon junction not present in the linear transcript, the order of exons in sequencing reads is changed. Moreover, circRNA can contain any number of exons, from none, to a single exon or multiple exons. 11 Three models of circRNA biogenesis through back-splicing have been accepted: intron-pairing-driven circularization, RBP-driven circularization and lariat-driven circularization. (Supplementary file 1).

| Classification of circRNAs
The majority of circRNAs are ecircRNAs, which lack intronic sequence in their reads. ciRNAs are formed without exon-skipping events; therefore, they contain only intron sequences. EIcirRNAs contain both intronic and exonic sequences. 12 In general, ecircRNAs, ciRNAs and EIcirRNAs are all intragenic circRNA arising from the sequences within the parental gene locus. Different from the location of intragenic circRNAs in the genome, intergenic circRNAs arise from the genomic interval between two genes. Gao et al 13  Therefore, intergenic circRNA sequences can be both intronic and exonic.

| Spatiotemporal specific expression
Though circRNAs show conservation among species, there are also some circRNAs that have species-, cell-, tissue-and developmental stage-specific expression. A comparative analysis of the expression of circRNAs from humans and mice found that only a small portion of human circRNAs could be detected in parallel mouse samples, and these circRNAs were conserved. In total, 85% of circRNAs in humans and 60% of circRNAs in mice showed species-specific expression patterns. 15 CircRNAs also exhibit cell-and tissue-specific expression patterns. A circRNA catalog of 20 different tissues and cells from a single donor revealed that circRNA expression was highly cell-and tissue-specific. 3 In addition, circRNAs exhibit dynamic expression patterns during the cardiac differentiation process. Specifically, Lei et al 4 detected some circRNAs, including circSLC8A1, circCACNA1D, circSPHKAP and cir-cALPK2, that showed cardiac-specific expression in human induced pluripotent stem cells (hiPSCs). The expression level of these circRNAs increased during cardiac differentiation and as a result, they were highly enriched in hiPSC-derived cardiomyocytes. A previous study detected differential expression levels of 226 circRNAs during the differentiation of human umbilical cord-derived mesenchymal stem cells into cardiomyocyte-like cells. 16 These findings indicate the potential of certain circRNAs that have significantly different expression levels during the cardiac differentiation process to serve as biomarkers of cardiomyocytes.
Further, the expression of most cardiac circRNAs was triggered by cardiac progenitor differentiation. 17 Specifically, circRNAs coming from the TTN gene were upregulated from the mesoderm stage to the definitive cardiomyocytes. 18 Thus, it can be inferred that differential expression of certain circRNAs may play a critical role in pathways that are activated during the process of cardiac differentiation.

| MECHANIS MS OF CIRCRNA S IN THE O CCURREN CE AND DE VELOPMENT OF C VDS
Some highly expressed circRNAs corresponded to key cardiac genes including TTN, RYR2 and DMD, suggesting the essential role of circR-NAs in maintaining normal heart function. 18 Further, differential expression of cardiac circRNAs is strongly related to CVDs. For example, in the heart of an adult mouse, Jakobi et al 19 reported that the majority of circRNAs could be matched to CVDs-related host genes including Ppp2r3a, Hectd1 and RYR2. Similarly, Maass et al 3 analysed an assembly of circRNA in the vena cava and right atrium separated from patients with diverse cardiac defects and reported that the altered levels of certain circRNAs contribute to multiple cardiovascular symptoms. For instance, circRNA isoforms derived from the RYR2 gene were linked to the pathogenesis of atrial fibrillation. 3 Another study confirmed that 6,234 circRNAs were differentially expressed between a foetal group with congenital ventricular septal defect (VSD) and a normal group. 20 Abnormal expression profiles of circRNAs were also associated with coronary heart disease and cardiomyocyte hypertrophy. 5,6 Specially, circSLC8A1, which was found to be the most abundant human cardiac circRNA, abnormally increased in cardiac tissues from individuals with dilated cardiomyopathy. 4 Collectively, these studies revealed that altered types and quantities of circRNAs are closely correlated with a number of CVDs. Detecting disease-associated circRNAs may be a potential method to determine pathological status.
Cardiac circRNAs mediate the pathogenesis of CVDs mainly through the following two major mechanisms: circRNA-miRNA-mRNA axis and interaction with proteins. CircRNAs can act as a miRNA 'sponge', and miRNA captured by circRNAs lose the ability to bind with downstream mRNA. In addition to the circRNA-miRNA-mRNA axis, circRNA can also interact with proteins to exert effects on CVDs. (Table 1).

| circRNA-miRNA-mRNA axis
circRNAs have various miRNA binding sites in their loop structure. 21 Therefore, circRNAs can bind with miRNAs; through this sponge effect, the cytoplasmic free miRNAs are decreased, resulting in the downstream target mRNAs of miRNAs being upregulated. This chain of interaction is known as a circRNA-miRNA-mRNA axis, which exert pivotal effects on diverse CVDs including atherosclerosis, coronary heart disease, myocardial infarction and heart failure.

Vascular endothelial cells and vascular smooth muscle cells (VSMCs)
are the two primary cell types in the pathogenesis of atherosclerosis.
Multiple circRNA-miRNA-mRNA axes effectively regulate the proliferation and migration of VECs and VSMCs, and subsequently change the functional state of blood vessels, modulating the progression of atherosclerosis.
Vascular endothelial cells, which comprise the inner layer of blood vessels, are considered to function as a significant barrier for the vascular wall. 22

| Heart failure (HF)
MI and pathological hypertrophy promote heart failure (HF). The most abundant human cardiac circRNA, circSLC8A1, is elevated while miR-133a is downregulated in cardiac hypertrophy. 4,41 Moreover, upregulated circSLC8A1 sequestered miR-133a to increase multiple target mRNAs of miR-133a, 42 indicating that the circSLC8A1-miR-133a-mRNAs axis serves as a pivotal mechanism in the pathogenesis of cardiac hypertrophy and the inhibition of circ-SLC8A1 may be a potential method to treat cardiac hypertrophy. In

| circRNA-miRNA-mRNA axis in other CVDs
Recent studies found that hsa-circ-0037909 and hsa-circ-0037911 were significantly upregulated in patients with essential hypertension compared to healthy group. 44,45 Subsequent analysis revealed that hsa-circ-0037909 and hsa-circ-0037911 could contribute to the pathogenesis of essential hypertension via acting as a sponge to inhibit miR-637 activity. 44,45 Moreover, Lu et al 46 found that circ-Nr1h4 was significantly downregulated in the injured kidney of mice with hypertension. CircNr1h4 protected kidney from hypertensive injury via acting as a sponge to inhibit miR-155-5p activity and upregulating its target gene fatty acid reductase 1. 46 Besides, 11 significantly dysregulated circRNAs were identified in septic shock-induced cardiac depression. 47 Further analysis found the possible relationship between these circRNAs and their target miRNAs/mRNAs, indicating the important role of circRNA-miR-NA-mRNA axis in the pathological process of myocardial depression in septic shock. 47 Moreover, differentially expressed circRNAs were discovered in both mice and human with abdominal aortic aneurysm (AAA). 48,49 Specially, circCCDC66 was upregulated in AAA. 48 Further research corroborated that depletion of circCCDC66 reduced apoptosis of VSMC and promoted proliferation of VSMC, indicating a suppressed AAA formation trend. 50 Besides, circCCDC66 induced the progression of AAA via acting as a miR-342-3p sponge to promote the effect of CCDC66 on the proliferation and apoptosis of VSMC. 50

| Interaction between circRNAs and RBPs
CircRNA-protein interaction is another molecular mechanism relevant to human CVDs. Du et al 51

| Atherosclerosis
The interaction between circRNAs and RBPs could disturb ribosomal biogenesis, thus regulating the pathogenesis of atherosclerosis.
The PeBoW complex is essential for ribosomal biogenesis; Pes1 is a nucleolar protein that is a key component of the PeBoW complex. 52 A previous study has shown that the deletion of the C-terminal domain of Pes1 effectively inhibited rRNA maturation. 53 Additionally, it was found that the C-terminal domain of Pes1 contained binding sites of both pre-rRNA and circ-ANRIL. 54 These results indicate that the interaction between circ-ANRIL and the C-terminal domain of Pes1 can modulate the process of turning pre-rRNA into mature rRNA, subsequently affecting ribosomal biogenesis. Further experiments confirmed that the upregulation of circ-ANRIL reduced rRNA maturation. The accumulated pre-rRNA induced nucleolar stress and then stabilized the pro-apoptotic protein p53. 54 Consequently, circ-ANRIL promotes the apoptosis of different cells in the pathological process of atherosclerosis via interacting with Pes1; however, it is unknown whether circ-ANRIL is atheroprotective in atherosclerosis. For instance, the apoptosis of VECs is pro-atherosclerotic, whereas the apoptosis of VSMCs is anti-atherosclerotic. A recent study found a decreased expression level of circ-Fndc3b in post-MI heart tissue. 56 The overexpression of circ-Fndc3b could increase the level of VEGF, thus promoting angiogenesis and maintaining cardiac function. Detailed mechanisms revealed that the interaction between circ-Fndc3b and FUS, an RBP that participates in many cellular processes, including angiogenesis and apoptosis, played a crucial role in this process. 56 Therefore, the circ-Fndc3b-FUS-VEGF axis modulates cardiac repair after MI. Another study found that circ-Hipk3 could bind with Notch1 intracellular domain (N1ICD) and promoted N1ICD acetylation and nuclear translocation, which is essential for cardiomyocyte proliferation after MI. 40,57

| Translational regulation by circRNAs
CircRNAs can also regulate translation of their linear counterpart through binding with RBPs. 58,59 Several circRNAs that bind to RBP HuR were verified in HeLa cells. 60 Among these circRNAs, circ-PABPN1 was one of the most significantly enriched HuR targets and mechanistically suppressed HuR binding to PABPN1 mRNA to competitively inhibit translation of PABPN1. 60 Hu et al 61

| CLINI C AL PROS PEC TS
Compared with linear biomarkers, circRNAs are abundant, conserved and have a stable circular structure, which promotes their use as biomarkers for diagnosis and prognosis of CVDs.

| CON CLUS IONS
CircRNAs are a type of non-coding RNAs with a closed loop structure. Previously published studies have annotated multiple cardiac circRNAs and some circRNAs that show differential expression can be used as biomarkers for the diagnosis of CVDs; however, the specificity of the diagnosis remains a challenge due to the wide distribution of circRNAs in varied cells and tissues. Most studies have revealed the differential expression of certain circRNAs in the plasma or serum, but the origin of these RNAs in circulation has not been clearly clarified. Besides, majority of researches about circRNAs and CVDs just included limited number of patients; thus, meaningful effects of circRNAs in CVDs and the significant value of candidate circRNAs as biomarkers could be ignored. Further studies, especially the large-scale prospective clinical trials with well-stratified patients, should be performed to enhance the predictive and diagnostic value of certain circRNAs in CVDs. Moreover, although circRNAs act as miRNA sponge and interact with RNA-binding proteins, additional pathways remain to be explored in the pathogenesis of CVDs at the molecular level. As for the clinical application, a recent study found that artificial circRNA sponges (circmiRs) which target several miRNAs could extenuate pressure overload-induced cardiac hypertrophy, 68 indicating that circmiRs may provide a new therapy for CVDs, with its extended half-lives and lower requirement of dose compared to current alternatives.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.