Cardiovascular and cerebrovascular diseases (CVDs) are general terms used to refer to all cardiac and cerebral diseases related to vasculopathy. Despite improvements in pharmacotherapy and surgical interventions, as well as lifestyle modifications, morbidity and mortality in patients with CVDs remain high in recent years (1). CVDs are still the leading cause of death worldwide (1). Current treatments primarily alleviate symptoms or slow down disease progression. To develop novel preventive and therapeutic strategies, we should elucidate and understand the underlying molecular mechanisms of these diseases.

Circular RNAs (circRNAs) were first discovered in plant viroids more than 40 years ago (2). A few years later, circRNAs were observed by electron microscopy in the cytoplasmic fractions of eukaryotic cells (3). However, they were mainly considered to be errors of the normal splicing process and did not receive much attention (4). In recent years, advances in the high-throughput sequencing technology and circRNA-specific bioinformatics algorithms have resulted in the discovery and identification of thousands of circRNAs (5–9). Indeed, circRNAs are diverse, abundant, and expressed in a tissue- and developmental stage-specific manner (5, 8). Although the functions of most circRNAs remain elusive, a select number of circRNAs are known to function as competitive endogenous RNAs (ceRNAs) by decoying miRNAs from other target transcripts, thereby controlling gene expression at the posttranscriptional level (10).

Recent studies have identified circRNAs as ceRNAs in many diseases including CVDs (11–13). Here, we reviewed the role of circRNAs as ceRNAs in atherosclerosis, myocardial infarction, cardiac fibrosis, heart failure, aneurysm, and stroke, with a special focus on the molecular mechanism, which provides a new direction for further research on the pathogenesis and treatment of CVDs.

Several years before the discovery of ceRNAs, or natural miRNA sponges, many studies had found that artificial miRNA sponges were able to specifically and effectively inhibit miRNA activity (14–16). These synthetic miRNA sponges are usually expressed from strong promoters, engineered to carry multiple binding sites for a miRNA or miRNA family of interest and have been shown to derepress miRNA targets both in vitro and in vivo (14). The sponging constructs are not only invaluable tools for miRNA loss-of-function studies in vitro and in vivo, but also critical for the development of RNA-based therapeutic applications (15, 16).

The theoretical and practical development of the artificial miRNA sponge laid an important foundation for the ceRNA hypothesis. In 2011, Salmena et al. postulated that all endogenous RNA transcripts sharing common miRNA response elements (MREs) can communicate with and regulate each other through competition for a limited pool of miRNAs (17). This hypothesis not only attributes new functions for non-coding RNA transcripts, but also implies coding-independent functions of protein-coding messenger RNAs (17). Given that any transcripts containing MREs can theoretically serve as ceRNAs, they may represent a widespread form of posttranscriptional gene regulation in a variety of physiological and pathophysiological processes.

In 2013, two independent groups firstly discovered that circRNAs can serve as ceRNAs (18, 19). Hansen et al. showed that ciRS-7 (also known as CDR1as) contains 73 conserved binding sites for miR-7 (19). ciRS-7 strongly binds to miR-7 as well as the miRNA effector Argonaute protein 2 (AGO2) and efficiently suppresses miR-7 activity, thereby attenuating the availability of miR-7 to bind to its target mRNAs. In situ hybridization showed that miR-7 and ciRS-7 displayed clear co-expression in mouse brain sections and in primary cells isolated from mouse brain, suggesting a high level of endogenous interactivity between them. Another research group demonstrated that ectopic expression of human ciRS-7 in zebrafish impairs midbrain development, consistent with the effect of miR-7 inhibition induced by morpholinos injection (18). The midbrain reduction induced by ciRS-7 expression in zebrafish could be partially rescued by expressing miR-7, indicating the interaction between ciRS-7 and miR-7. These two studies also serve as the first functional analysis of naturally expressed circRNAs. Acting as ceRNAs, circRNAs can bind to miRNAs and inhibit the function of miRNAs, thereby upregulating miRNA target gene expression (Figure 1). Because of the lack of free ends, circRNAs are more resistant to exonucleases and more stable than linear RNAs (18, 19). These natures of circRNAs are thought to facilitate their effectiveness as ceRNAs. Indeed, so far many circRNAs have been found to act as ceRNAs both under normal physiological conditions and diseased conditions (20).

To investigate the interaction between circRNAs and miRNAs, several bioinformatic tools can be used. For instance, different miRNA-target prediction algorithms, including TargetScan (68), miRanda (69), and PITA (70) have greatly facilitated the discovery of circRNA-miRNA interactions. In addition, some tools incorporate gene expression data including AGO crosslinking-immunprecipitation (CLIP) data, which improve the capability to predict physiologically relevant circRNA-miRNA interactions. starBase 2.0 (http://starbase.sysu.edu.cn/) is the first database containing circRNA-miRNA interactions based on Ago and RNA binding protein (RBP) binding sites (71). It provides comprehensive interaction networks of ncRNAs, mRNA and RBP from 108 CLIP-Seq datasets. CircInteractome (http://circinteractome.nia.nih.gov/) combines many features from other websites, such as circBase, TargetScan, StareBase (72). Similarly, it can be used to explore the interacting miRNAs of circRNAs. Since every database uses distinct rules of targeting and produces different prediction outcomes, a combination of various databases may provide information in a reliable way.

In addition to the above mentioned prediction methods, experimental validation is required to prove that a circRNA has a bona fide miRNA sponging function. First, dual-luciferase reporter assays can be utilized to screen the potential interacting miRNAs. The circRNA sequences containing wild-type (WT) or mutated miRNA binding sites are inserted into luciferase reporter vectors. Transfection of a miRNA mimic can reduce the luciferase activities in the WT group, but not in the mutated group. Second, RNA fluorescence in situ hybridization (RNA-FISH) can be used to assess the colocalization of circRNAs with miRNAs in cells and tissues. RNA-FISH probe should target circRNA backspliced junctions in order to avoid recognition of their cognate linear RNAs. It is important to note that RNA-FISH signals for circRNAs and miRNAs appear as distinct puncta in the cell, not as the diffuse signals typically seen when detecting protein. Third, AGO2 RNA immunoprecipitation (RIP) can be employed to confirm a physical interaction between the circRNA, miRNA and AGO2. It is a more direct evidence supporting the miRNA sponging role of a circRNA. Fourth, RNA pull-down assay can be performed using biotinylated probes designed specifically for the circRNA/miRNA of interest. After confirming that the circRNA/miRNA is successfully enriched in the pull-down material, the interacting miRNA/circRNA molecules can be studied by RT-qPCR analysis.

As discussed in this review, many circRNAs serve as ceRNAs to mediate miRNA functions in CVDs (Table 1). These circRNA-miRNA-mRNA competitive endogenous RNA regulatory networks represent a novel section in the pathogenesis and development of CVDs. As lots of circRNAs are deregulated or show a disease-specific profile in CVDs and might play a role in the progression of CVDs, they might serve as promising biomarkers for early diagnosis or therapeutic targets in the future. However, how to effectively regulate circRNA levels in target cells is now still unresolved. Extracellular vesicles are naturally occurring RNA carriers that can induce physiological changes in target cells through the transfer of numerous molecules. These vesicles can be adapted for the delivery of circRNAs or siRNAs targeting the specific backspliced sequence of circRNA. Therefore, exogenous upregulation or downregulation of circRNAs to regulate miRNAs could become a novel therapy for CVD treatment. In addition, a recent study found that artificial circRNA sponges can competitively inhibit pro-hypertrophic miR-132 and−212, thereby attenuating pressure overload-induced cardiac hypertrophy (73). Thus, introducing synthetic circRNAs sponges into target cells might also serve as new efficient approaches for future CVD therapy. Indeed, the superior ability of circRNA in sponging miRNAs and its unique cellular stability confer them great potential for use as therapeutic drugs. Nevertheless, the real clinical application of circRNAs as molecular drugs or targets is challenging. How to ensure therapeutic efficacy and safety, and avoid off-target adverse effects awaits additional and extensive investigation.

Despite growing evidence for the phenotypic effects of circRNA deregulation in CVDs, the current ceRNA hypothesis is still controversial regarding whether they are really active in the endogenous cellular context. Importantly, optimal ceRNA-mediated regulation is observed when miRNA and ceRNA levels are near equimolarity. The levels of circRNA need to be high enough to relieve the miRNA repression on mRNA. Thus, methods to quantitatively determine absolute expression levels of circRNA and miRNA will allow a more precise determination of ceRNA effectiveness of circRNAs in diverse physiological and pathological conditions. Another important factor is the number of miRNA-binding sites. CircRNAs that harbor multiple binding sites for the same miRNAs may serve as better ceRNA candidates than those with fewer sites. Among the circRNAs we discussed in this review, with the exception of MFACR (37), circSlc8a1 (52), circNCX1 (38), circLrp6 (22), HRCR (51), which contains 15, 10, 8, 7, 6 binding sites for miR-652-3p, miR-133a, miR-133a-3p, miR-145, miR-223, respectively, the majority of circRNAs harbor only a single or very few binding sites for the same miRNAs (Table 1). To function as efficient miRNA sponges, the copy numbers of these circRNAs that contain minimal numbers of miRNA-binding sites should attain levels comparable to the miRNAs. However, most of the researches are lack of assessments of absolute levels of circRNAs and miRNAs. Therefore, assessing the stoichiometry of a circRNA with the interacting miRNA is clearly needed in future research. In addition, factors such as miRNA/circRNA binding affinity and RBPs that could occupy MREs can also affect the ceRNA activity of circRNAs. The binding affinity of miRNA and its RNA target, to a great extent, is influenced by matching between MREs and seed sequences. In general, 8-mer seed match has higher affinity than those with 7-mer, and 6-mer has weakest miRNA target-site efficacy (74). As a competitor, circRNA should have a better affinity to miRNA than target mRNAs. RBP can interfere with circRNA-miRNA interactions by directly occupying RNA target sites, or by altering the circRNA secondary structure to influence its affinity for miRNAs. Therefore, technologies such as HITS-CLIP/iCLIP/PAR-CLIP, the mass-spectrometry based quantitative proteomic approaches for capturing RNA-interactome and measuring circRNA-binding protein abundance will help to assess the effect of competitive binding of RBP to MREs.

In summary, circRNAs acting as ceRNAs play important roles in CVDs. Research on the circRNA-mediated ceRNA regulatory network will open up new avenues for basic CVD research, as well as stimulate the development of novel diagnostic and therapeutic strategies for CVDs.

XM and D-lL: wrote the manuscript. X-dX: critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.