Circular RNAs: Expression, localization, and therapeutic potentials
Authors: Qiwei Yang, Feiya Li, Alina T. He, Burton B. Yang
Journal: Molecular Therapy (2021)

Circular RNAs (circRNAs) are RNAs with a unique circular structure that is generated from back-splicing processes. These circular molecules were discovered more than 40 years ago but failed to raise scientific interest until lately. Increasing studies have found that these circular RNAs might not just be byproducts of the splicing process but possess important regulatory functions through different cellular events. Most circular RNAs are currently being studied in the field of cancer, and many of them have been confirmed to be involved in the process of tumorigenesis. However, many circular RNAs are implicated in the developmental stages of diseases other than cancer. In this review, we focus on discussing the role of circular RNAs in non-cancer diseases, especially in cardiovascular diseases. Following the summary of the life cycle of circRNAs, we provide input on studying circRNA-protein interactions based on our experience, which modulate protein translocation. Furthermore, we outline the potential of circRNAs to be potent biomarkers, effective therapeutic targets, and potential treatments in cardiovascular diseases as well as other non-cancer fields. Circular RNAs (circRNAs) are RNAs with a unique circular structure that is generated from back-splicing processes. These circular molecules were discovered more than 40 years ago but failed to raise scientific interest until lately. Increasing studies have found that these circular RNAs might not just be byproducts of the splicing process but possess important regulatory functions through different cellular events. Most circular RNAs are currently being studied in the field of cancer, and many of them have been confirmed to be involved in the process of tumorigenesis. However, many circular RNAs are implicated in the developmental stages of diseases other than cancer. In this review, we focus on discussing the role of circular RNAs in non-cancer diseases, especially in cardiovascular diseases. Following the summary of the life cycle of circRNAs, we provide input on studying circRNA-protein interactions based on our experience, which modulate protein translocation. Furthermore, we outline the potential of circRNAs to be potent biomarkers, effective therapeutic targets, and potential treatments in cardiovascular diseases as well as other non-cancer fields. Viroids were the first circular RNAs (circRNAs) discovered in pathogens. Sanger et al.1Sanger H.L. Klotz G. Riesner D. Gross H.J. Kleinschmidt A.K. Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures.Proc. Natl. Acad. Sci. USA. 1976; 73: 3852-3856Crossref PubMed Scopus (1057) Google Scholar first described viroids in 1976 as "single-stranded and covalently closed circular RNA molecules." Following this, another study conducted by Hsu and Coca-Prados2Hsu M.T. Coca-Prados M. Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells.Nature. 1979; 280: 339-340Crossref PubMed Scopus (463) Google Scholar in 1979 described some circRNAs with no free flanking ends. They raised the possibility that the circularity of these RNAs is not dependent on any protein interactions. During that period of time, studies failed to follow up on these unexpected findings due to an explosion of scientific interest in studying the functions of linear RNA, especially after the polymerase chain reaction was invented in 1985. There were some studies in the late 1990s that described transcript products generated from non-canonical splicing. These products were reported to be generated from the deletion in a colon cancer gene3Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Scrambled exons.Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (616) Google Scholar and human EST-1 gene,4Cocquerelle C. Daubersies P. Majérus M.A. Kerckaert J.P. Bailleul B. Splicing with inverted order of exons occurs proximal to large introns.EMBO J. 1992; 11: 1095-1098Crossref PubMed Google Scholar as well as their isoforms. Later, a few more studies proposed potential biogenesis mechanisms for these circular molecules. A well-known hypothesis proposed by Dubin et al.5Dubin R.A. Kazmi M.A. Ostrer H. Inverted repeats are necessary for circularization of the mouse testis Sry transcript.Gene. 1995; 167: 245-248Crossref PubMed Scopus (99) Google Scholar based on the circRNA sry is that complementary intronic sequences drive circularization. Another hypothesis proposed by Pasman et al.6Pasman Z. Been M.D. Garcia-Blanco M.A. Exon circularization in mammalian nuclear extracts.RNA. 1996; 2: 603-610PubMed Google Scholar is that these circular molecules could be produced from nuclear extracts. circRNAs were found to be mostly located in the cytoplasm and expressed in a stage and tissue-specific manner. Up until the beginning of the 21st century, the number of studies on circRNAs increased, but they were still sporadic. People classified circRNAs as non-linear mRNAs,7Dixon R.J. Eperon I.C. Hall L. Samani N.J. A genome-wide survey demonstrates widespread non-linear mRNA in expressed sequences from multiple species.Nucleic Acids Res. 2005; 33: 5904-5913Crossref PubMed Scopus (30) Google Scholar RNAs with scrambled exons, or RNAs with exon shuffling.8Al-Balool H.H. Weber D. Liu Y. Wade M. Guleria K. Nam P.L. Clayton J. Rowe W. Coxhead J. Irving J. et al.Post-transcriptional exon shuffling events in humans can be evolutionarily conserved and abundant.Genome Res. 2011; 21: 1788-1799Crossref PubMed Scopus (34) Google Scholar circRNAs were generally regarded as products of splicing errors that were rare and held little significance. Although the presence of circRNAs was documented decades ago, very little about their biogenesis and functions were understood until the past decade. Starting in 2010, advancements in RNA-sequencing technologies along with the buildup of bioinformatics computational pipelines allowed circRNA research to explode. Several bioinformatics studies reported hundreds and thousands of circRNAs that were highly conserved.9Salzman J. Gawad C. Wang P.L. Lacayo N. Brown P.O. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types.PLoS ONE. 2012; 7: e30733Crossref PubMed Scopus (1377) Google Scholar,10Hansen T.B. Jensen T.I. Clausen B.H. Bramsen J.B. Finsen B. Damgaard C.K. Kjems J. Natural RNA circles function as efficient microRNA sponges.Nature. 2013; 495: 384-388Crossref PubMed Scopus (3970) Google Scholar In addition to discovering new circRNA products, revised RNA-sequencing techniques allowed for specific detection of circular products without overlapping detection of linear products. These methods include RNase R treatment of the samples before sequencing, which digests linear RNAs without affecting circRNAs, resulting in an enrichment of circRNAs compared to linear RNAs. Since 2017, computational pipelines and databases for circRNA annotation and quantification have been established and developed over time. On top of powerful bioinformatics techniques, novel methods for circRNA verification and validation were also developed.11Jeck W.R. Sharpless N.E. Detecting and characterizing circular RNAs.Nat. Biotechnol. 2014; 32: 453-461Crossref PubMed Scopus (1368) Google Scholar Most of these methods were adapted from linear RNA detection techniques. These include quantitative reverse transcriptase PCR, northern blot, and, later on, other quantitative PCRs such as digital PCR were developed. These methods made it possible for wet laboratory scientists to validate data from dry laboratories. Following the identification and validation of circRNAs, methods including vector expression plasmids and siRNA silencing were used to overexpress and knock down circRNAs, respectively, to study their functions. Furthermore, methods such as in situ hybridization and pull-down assays were used to study the underlying molecular mechanisms of circRNAs. With the advent of all of these techniques, circRNA research was able to expand. More and more studies have contributed to the conclusion that circRNAs are not simply byproducts of splicing errors and actually hold critical physiological and pathological functions during biological processes. In this review, we provide a brief update on the life cycle of circRNAs. We discuss the localization and translocation of circRNAs. We then bring new insights into circRNA functions in the cardiovascular field and discuss the potential of circRNA-based diagnostic and therapeutic strategies in the cardiovascular field as well as other non-cancer diseases. circRNAs have been extensively reported as evolutionarily conserved, stable, and tissue-specific, as well as subcellular location- and developmental stage-specific. circRNAs can be generated through a direct back-splicing process where the 3′ terminal of the RNA is directly joined to the 5′ terminal during the splicing process of precursor mRNA and thereby proceed to form a circular structure. This back-splicing process could be driven by an intronic complementary sequence (Figure 1A) or RNA-binding protein (RBP) (Figure 1B). They can also be generated by a lariat-mediated back-splicing biogenesis model where the RNA sequence that is cut off after the splicing process of the precursor mRNA forms a lariat structure, and such structure is further spliced to form a circular structure (Figure 1C).12Kristensen L.S. Andersen M.S. Stagsted L.V.W. Ebbesen K.K. Hansen T.B. Kjems J. The biogenesis, biology and characterization of circular RNAs.Nat. Rev. Genet. 2019; 20: 675-691Crossref PubMed Scopus (841) Google Scholar DDX39A and DDX39B are proteins that export the formed circular structure to the cytoplasm from the nucleus (Figure 1D). While DDX39A usually transports short circRNAs, DDX39B assists in forming longer ones.13Huang C. Liang D. Tatomer D.C. Wilusz J.E. A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs.Genes Dev. 2018; 32: 639-644Crossref PubMed Scopus (116) Google Scholar Formed circRNAs are ordinarily stable owing to their exclusive circular structure, which defends them from cleavage of the exonuclease. However, circRNAs still undergo degradation. (A) In the intronic complementary sequence (ICS)-driven back-splicing biogenesis model, the intron regions on both sides of the exons contain complementary sequences, which are paired and tightly connected and promote back-splicing to form a circular structure. (B) In the RNA-binding protein (RBP)-driven back-splicing biogenesis model, under the bridging action of RBP, the splicing sites at both ends of the exon are directly connected and promote back-splicing to form a circular structure. (C) In the lariat-driven back-splicing biogenesis model, the RNA sequence that is cut off during linear RNA splicing forms a lariat structure, and then forms a circular structure through back-splicing. (D) DDX39A and DDX39B proteins transport mature circRNAs from the nucleus to the cytoplasm. (E–H) The degradation process of circRNAs. (E) The YTHDF2 protein recognizes the m6A modification site of circRNA. At the same time, HRSP12 can bridge YTHDF2 and RNase P/MRP to form a complex, thereby initiating the circRNA degradation via RNase P/MRP. (F) MicroRNA miR-671 mediates the binding of CDR1as to AGO2 and triggers the degradation process regulated by PKR activation. (G) Pathological exogenous double-stranded RNA can activate RNase L in cells to degrade circRNAs. (H) The complex of UPF1 and G3BP1 proteins can mediate the degradation of circRNAs. (I) In the process of gene transcription, circRNAs can block exons by binding to genomic DNA, resulting in exon deletion during mRNA splicing. (J) circRNAs can serve as a miRNA decoy, inhibiting miRNA functions by binding to the miRNAs. (K) circRNAs inhibit biological activity of proteins by binding to their functional domain. (L) circRNAs combine with multiple proteins to form complexes and change their biological activity. (M) circRNAs can be translated into polypeptides or proteins. (N) Exosomes or extracellular vesicles can wrap circRNAs and secrete them out of the cell. (O) Under certain conditions, cells can take up exosomes or extracellular vesicles, and the intake circRNAs can function in the recipient cells. Due to the unique circular structure of circRNAs, they cannot be eliminated via conventional RNA degradation pathways, and their half-life is longer compared to linear RNA. However, they are still degraded, although their degradation mechanisms are not fully understood. Theoretically, even though the circular structure of circRNAs protects them from exonuclease cleavage, the decay process could still be initiated by endonucleases and completed by a cascade of exonucleases or endonucleases. A recent study indicated that the modification N6-methylation of adenosine (m6A) of circRNA could be recognized by HRSP12, an m6A reader protein, which can interact with the RNase P/MRP endonuclease complex to trigger circRNA degradation14Park O.H. Ha H. Lee Y. Boo S.H. Kwon D.H. Song H.K. Kim Y.K. Endoribonucleolytic cleavage of m6A-containing RNAs by RNase P/MRP complex.Mol. Cell. 2019; 74: 494-507.e8Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar (Figure 1E). The degradation of circRNAs mediated by synthetic short hairpin RNAs (shRNAs)/small interfering RNAs (siRNAs) is a conventionalmethod used to study the functions of circRNAs. The only example of naturally occurring small RNA-mediated circRNA degradation involves miR-671, which has high complementarity to conserved binding sites on CDR1as and induces AGO2-mediated degradation15Hansen T.B. Wiklund E.D. Bramsen J.B. Villadsen S.B. Statham A.L. Clark S.J. Kjems J. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA.EMBO J. 2011; 30: 4414-4422Crossref PubMed Scopus (602) Google Scholar (Figure 1F). Another study found that poly(I:C) stimulation or encephalomyocarditis virus (EMCV) infection introduces pathological exogenous double-stranded RNA (dsRNA) into HeLa cells and activates endoribonuclease RNase L16Liu C.X. Li X. Nan F. Jiang S. Gao X. Guo S.K. Xue W. Cui Y. Dong K. Ding H. et al.Structure and degradation of circular RNAs regulate PKR activation in innate immunity.Cell. 2019; 177: 865-880.e21Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar (Figure 1G). In addition, there was spontaneous RNase L activation in peripheral blood mononuclear cells (PBMCs) that were derived from patients with systemic lupus erythematosus (SLE).16Liu C.X. Li X. Nan F. Jiang S. Gao X. Guo S.K. Xue W. Cui Y. Dong K. Ding H. et al.Structure and degradation of circular RNAs regulate PKR activation in innate immunity.Cell. 2019; 177: 865-880.e21Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar Such RNase L-mediated circRNA degradation requires protein kinase R (PKR) activation that is regulated by the formation of intra-dsRNA duplexes, a specific structure formed by many circRNAs. This activated RNase L can cause global degradation of circRNA. For circRNAs with complex secondary structures, the highly structured regions can be recognized by ATP-dependent RNA helicase upstream frameshift 1 (UPF1) as well as endonuclease G3BP1, which subsequently induce circRNA degradation17Fischer J.W. Busa V.F. Shao Y. Leung A.K.L. Structure-mediated RNA decay by UPF1 and G3BP1.Mol. Cell. 2020; 78: 70-84.e6Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar (Figure 1H). In addition to degradation inside the cells, exocytosis or endocytosis might occur to discharge circRNAs from cells or take them up to cells through exosomes18Li Y. Zheng Q. Bao C. Li S. Guo W. Zhao J. Chen D. Gu J. He X. Huang S. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.Cell Res. 2015; 25: 981-984Crossref PubMed Scopus (1107) Google Scholar,19Wang Y. Liu J. Ma J. Sun T. Zhou Q. Wang W. Wang G. Wu P. Wang H. Jiang L. et al.Exosomal circRNAs: biogenesis, effect and application in human diseases.Mol. Cancer. 2019; 18: 116Crossref PubMed Scopus (63) Google Scholar (Figures 1N and 1O). Increasing evidence has shown that circRNAs are present in exosomes.19Wang Y. Liu J. Ma J. Sun T. Zhou Q. Wang W. Wang G. Wu P. Wang H. Jiang L. et al.Exosomal circRNAs: biogenesis, effect and application in human diseases.Mol. Cancer. 2019; 18: 116Crossref PubMed Scopus (63) Google Scholar,20Preußer C. Hung L.H. Schneider T. Schreiner S. Hardt M. Moebus A. Santoso S. Bindereif A. Selective release of circRNAs in platelet-derived extracellular vesicles.J. Extracell. Vesicles. 2018; 7: 1424473Crossref PubMed Scopus (98) Google Scholar In some cases, circRNA levels in exosomes could be far more than those in cells.18Li Y. Zheng Q. Bao C. Li S. Guo W. Zhao J. Chen D. Gu J. He X. Huang S. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.Cell Res. 2015; 25: 981-984Crossref PubMed Scopus (1107) Google Scholar,21Dou Y. Cha D.J. Franklin J.L. Higginbotham J.N. Jeppesen D.K. Weaver A.M. Prasad N. Levy S. Coffey R.J. Patton J.G. Zhang B. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.Sci. Rep. 2016; 6: 37982Crossref PubMed Scopus (198) Google Scholar Exosomal circRNAs can bind to proteins and become more involved in cell-to-cell communication.22Bao C. Lyu D. Huang S. Circular RNA expands its territory.Mol. Cell. Oncol. 2015; 3: e1084443Crossref PubMed Scopus (28) Google Scholar In sum, a lot remains unknown about circRNA degradation and mediation of signal communication, and this awaits to be addressed in future investigations. circRNAs have been found to function through some proposed mechanisms: regulating transcription (Figure 1I), acting as microRNA (miRNA) sponges (Figure 1J), regulating other protein activities through binding them or scaffolding them (Figures 1K and 1L), and encoding proteins (Figure 1M). Beyond circRNAs in cancer fields, the functions of circRNAs in other non-cancer diseases, especially cardiovascular diseases, have been reviewed as well.23Lim T.B. Lavenniah A. Foo R.S. Circles in the heart and cardiovascular system.Cardiovasc. Res. 2020; 116: 269-278PubMed Google Scholar, 24Aufiero S. Reckman Y.J. Pinto Y.M. Creemers E.E. Circular RNAs open a new chapter in cardiovascular biology.Nat. Rev. Cardiol. 2019; 16: 503-514Crossref PubMed Scopus (125) Google Scholar, 25Lu D. Thum T. RNA-based diagnostic and therapeutic strategies for cardiovascular disease.Nat. Rev. Cardiol. 2019; 16: 661-674Crossref PubMed Scopus (86) Google Scholar, 26Gomes C.P.C. Schroen B. Kuster G.M. Robinson E.L. Ford K. Squire I.B. Heymans S. Martelli F. Emanueli C. Devaux Y. EU-CardioRNA COST Action (CA17129)Regulatory RNAs in heart failure.Circulation. 2020; 141: 313-328Crossref PubMed Scopus (45) Google Scholar In this review, we classify some new and interesting developments based on functional mechanisms of circRNAs in the cardiovascular field (Figure 2). The diagram shows relevant circRNA functions in the cardiovascular system. The left column in pink specifies the cardiovascular dysfunction of interest. The middle column in orange indicates circRNAs that could lead to cardiovascular diseases through the miRNA sponge mechanism. The right column in green indicates circRNAs that could lead to cardiovascular diseases through the protein-binding mechanism. The most abundant circRNA in cardiomyocytes is circSLC8A1, which plays a role in myocardial hypertrophy caused by pressure overload27Lim T.B. Aliwarga E. Luu T.D.A. Li Y.P. Ng S.L. Annadoray L. Sian S. Ackers-Johnson M.A. Foo R.S. Targeting the highly abundant circular RNA circSlc8a1 in cardiomyocytes attenuates pressure overload induced hypertrophy.Cardiovasc. Res. 2019; 115: 1998-2007Crossref PubMed Scopus (47) Google Scholar,28Chen L.L. The expanding regulatory mechanisms and cellular functions of circular RNAs.Nat. Rev. Mol. Cell Biol. 2020; 21: 475-490Crossref PubMed Scopus (157) Google Scholar and myocardial damage caused by ischemia-reperfusion29Li M. Ding W. Tariq M.A. Chang W. Zhang X. Xu W. Hou L. Wang Y. Wang J. A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p.Theranostics. 2018; 8: 5855-5869Crossref PubMed Scopus (108) Google Scholar by sponging miR-133a. In an angiotensin II-induced cardiac hypertrophy mouse model, circRNA_000203 was upregulated in cardiomyocytes and acts as a sponge for miR-26b-5p and miR-140-3p, leading to increased expression of Gata4 and aggravated cardiac hypertrophy.30Li H. Xu J.D. Fang X.H. Zhu J.N. Yang J. Pan R. Yuan S.J. Zeng N. Yang Z.Z. Yang H. et al.Circular RNA circRNA_000203 aggravates cardiac hypertrophy via suppressing miR-26b-5p and miR-140-3p binding to Gata4.Cardiovasc. Res. 2020; 116: 1323-1334Crossref PubMed Scopus (12) Google Scholar A recent study showed that in calcific aortic valve disease (CAVD), circRIC3 is significantly upregulated, leading to osteogenic trans-differentiation of valvular interstitial cells through the circRIC3/miR-204-5p/DPP4 pathway, an indicator of accelerated valvular calcification. It was demonstrated that melatonin inhibits circRIC3 expression, thereby reducing aortic valve calcification.31Wang Y. Han D. Zhou T. Zhang J. Liu C. Cao F. Dong N. Melatonin ameliorates aortic valve calcification via the regulation of circular RNA CircRIC3/miR-204-5p/DPP4 signaling in valvular interstitial cells.J. Pineal Res. 2020; 69: e12666Crossref PubMed Scopus (3) Google Scholar Similarly, in vascular smooth muscle cells, overexpression of circLRP6 promotes atherosclerosis development by sponging miR-145.32Hall I.F. Climent M. Quintavalle M. Farina F.M. Schorn T. Zani S. Carullo P. Kunderfranco P. Civilini E. Condorelli G. Elia L. circ_Lrp6, a circular RNA enriched in vascular smooth muscle cells, acts as a sponge regulating miRNA-145 function.Circ. Res. 2019; 124: 498-510Crossref PubMed Scopus (72) Google Scholar,33Heumüller A.W. Dimmeler S. Circular RNA control of vascular smooth muscle cell functions.Circ. Res. 2019; 124: 456-458Crossref PubMed Scopus (7) Google Scholar In aortic smooth muscle, circCHFR promotes the migration and proliferation of the vascular smooth muscle through the miR-370/FOXO1/cyclin D1 pathway;34Yang L. Yang F. Zhao H. Wang M. Zhang Y. Circular RNA circCHFR facilitates the proliferation and migration of vascular smooth muscle via miR-370/FOXO1/cyclin D1 pathway.Mol. Ther. Nucleic Acids. 2019; 16: 434-441Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar meanwhile, circNRG-1 promotes smooth muscle cell apoptosis through miR-193b-5p/NRG-1.35Sun Y. Zhang S. Yue M. Li Y. Bi J. Liu H. Angiotensin II inhibits apoptosis of mouse aortic smooth muscle cells through regulating the circNRG-1/miR-193b-5p/NRG-1 axis.Cell Death Dis. 2019; 10: 362Crossref PubMed Scopus (15) Google Scholar Not only can circRNA exert regulatory control over miRNA, but miRNA can also regulate circRNA activities. In doxorubicin (Dox)-induced cardiotoxicity, miR-31-5p is upregulated, which suppresses Quaking (QKI), an RBP known to influence circRNA production. As a result, circPAN3 synthesis is suppressed, which leads to cardiomyocyte apoptosis.36Ji X. Ding W. Xu T. Zheng X. Zhang J. Liu M. Liu G. Wang J. MicroRNA-31-5p attenuates doxorubicin-induced cardiotoxicity via quaking and circular RNA Pan3.J. Mol. Cell. Cardiol. 2020; 140: 56-67Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Besides regulating gene expression via the classic miRNA sponging mechanism, circRNAs play roles in cardiac disease via other modes of action. In cardiomyocytes, circFoxo3 expression is significantly higher in older heart tissues compared to younger heart tissues.37Du W.W. Yang W. Chen Y. Wu Z.K. Foster F.S. Yang Z. Li X. Yang B.B. Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses.Eur. Heart J. 2017; 38: 1402-1412Crossref PubMed Google Scholar We found that circFoxo3 interacts with ID-1 and E2F1, FAK, and HIF-1α, which are anti-senescence proteins and anti-stress proteins, respectively, preventing their transfer from cytoplasm to nucleus and mitochondria, which blocks their downstream activity. In a Dox-induced mouse model of cardiomyopathy, circFoxo3 promotes cellular senescence and aggravates Dox-induced cardiomyopathy.37Du W.W. Yang W. Chen Y. Wu Z.K. Foster F.S. Yang Z. Li X. Yang B.B. Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses.Eur. Heart J. 2017; 38: 1402-1412Crossref PubMed Google Scholar Similarly, the expression of super-enhancer-regulated circNfix is also higher in adult hearts than neonatal hearts. circNfix regulates Gsk3β signaling activity by sponging miR-214, and also binds to Y-box binding protein 1 (Ybx1) and an E3 ubiquitin ligase (Nedd4l) to promote the interaction between them and induce Ybx1 degradation. As a result of these two modes of action, cardiomyocyte proliferation is suppressed. It was shown that inhibiting circNfix led to promotion of cardiomyocyte proliferation and angiogenesis, reduced myocardial dysfunction, and protected the heart after myocardial infarction (MI).38Huang S. Li X. Zheng H. Si X. Li B. Wei G. Li C. Chen Y. Chen Y. Liao W. et al.Loss of super-enhancer-regulated circRNA Nfix induces cardiac regeneration after myocardial infarction in adult mice.Circulation. 2019; 139: 2857-2876Crossref PubMed Scopus (113) Google Scholar circFndc3b, whose expression is decreased after MI, has a similar protein binding function. Upregulation of circFndc3b results in the enhancement of angiogenesis, reduction of infarct size, and improvement of post-MI cardiac function by forming a complex with fused in sarcoma (FUS) and vascular endothelial growth factor-A (VEGF-A).39Garikipati V.N.S. Verma S.K. Cheng Z. Liang D. Truongcao M.M. Cimini M. Yue Y. Huang G. Wang C. Benedict C. et al.Circular RNA circFndc3b modulates cardiac repair after myocardial infarction via FUS/VEGF-A axis.Nat. Commun. 2019; 10: 4317Crossref PubMed Scopus (117) Google Scholar Lastly, autophagy-related circRNA (ACR) binds to Dnmt3b, which inhibits the methylation of Pink1 gene, thereby promoting Pink1 expression. Pink1 promotes the phosphorylation of FAM65B, which inhibits cell death and autophagy in the heart, ultimately protecting the heart from reperfusion injury.40Zhou L.Y. Zhai M. Huang Y. Xu S. An T. Wang Y.H. Zhang R.C. Liu C.Y. Dong Y.H. Wang M. et al.The circular RNA ACR attenuates myocardial ischemia/reperfusion injury by suppressing autophagy via modulation of the Pink1/FAM65B pathway.Cell Death Differ. 2019; 26: 1299-1315Crossref PubMed Scopus (89) Google Scholar van Heesch et al.'s41van Heesch S. Witte F. Schneider-Lunitz V. Schulz J.F. Adami E. Faber A.B. Kirchner M. Maatz H. Blachut S. Sandmann C.L. et al.The translational landscape of the human heart.Cell. 2019; 178: 242-260.e29Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar breakthrough discovery from 80 human heart translatomes found that at least 40 circRNAs produced from 39 genes have the ability to encode proteins, including newly detected ribosome-associated circRNAs, such as circCFLAR, circSLC8A1, circMYBPC3, circRYR2, and CDR1as. The authors first identified circRNAs bound to ribosomes to detect translational potential. Then, they compared the nucleic acid sequence of the ribosome binding region with the back-splicing junction region of circRNAs to confirm that the circRNAs bound by ribosomes were in a circular structure. Furthermore, they verified the polypeptide (or microprotein) encoded by the back-splicing junction region of six circRNAs by shotgun mass spectrometry. Translations of 5 out of 40 circRNAs were considered to be m6A-driven, while others were more likely to be IRES-driven.42Yang Y. Fan X. Mao M. Song X. Wu P. Zhang Y. Jin Y. Yang Y. Chen L.L. Wang Y. et al.Extensive translation of circular RNAs driven by N6-methyladenosine.Cell Res. 2017; 27: 626-641Crossref PubMed Scopus (722) Google Scholar These circRNAs may have translation potential in other organs and tissues besides the heart, such as the liver and kidney.41van Heesch S. Witte F. Schneider-Lunitz V. Schulz J.F. Adami E. Faber A.B. Kirchner M. Maatz H. Blachut S. Sandmann C.L. et al.The translational landscape of the human heart.Cell. 2019; 178: 242-260.e29Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Although the roles of peptides/microproteins encoded by circRNAs in cardiovascular disease have not been fully uncovered, these findings reveal increased functional diversity of circRNAs, which were previously thought to be non-coding. Elucidating the function of these peptides/microproteins will likely increase our understanding of the physiological and pathological states of the heart. Even though miRNA sponge mechanisms were applied for loads of circRNA functions in all disease fields, direct binding between circRNA-mRNA or circRNA-protein started to raise interest in their serving as mechanisms underlying circRNA functions. One important consequence of these interactions is the regulation of the localization of proteins. Cytoplasm localization of proteins can be facilitated by circRNAs. circFoxo3, for instance, a circRNA that is highly expressed in the heart tissues of aged mice and patients, as mentioned before, promoted cellular senescence in vitro and decreased cardiac functions in vivo. circFoxo3 is mainly localized in the cytoplasm, where the interaction between it and ID1 and E2F1 (the anti-senescence proteins), as well as FAK and HIF1α (the anti-stress proteins), led to retention of these proteins in the cytoplasm. As a result, the functions of these affected proteins that act a