Super-enhancer-associated CircRNA Nfix Regulates Endogenous Cardiac Regeneration In Adult Mice

BackgroundActivation of cardiomyocyte(CM)proliferation in situ is a promising approach to replace lost CMs after injury or diseases,such as myocardial infarction(MI)and heart failure.Circular RNAs(circRNAs)are a novel class of non-coding RNAs that are circularized by joining the 3’ end of the RNA to the 5’ end.The circular structure maintains their stability and strengthens their miRNA/protein binding capacity.Thus,circRNAs may be more effective than noncircular RNAs in regulating the cardiac gene regulator network and inducing CM proliferation.Moreover,emerging evidence indicates circRNAs may play fundamental roles in regulation of cardiac regeneration.Certain kinds of circRNAs have been found to control the regulation of cell proliferation,differentiation,development,and even tissue regeneration.CircRNAs also participate in biological processes and pathological settings of the cardiovascular system,including the development of cardiomyopathy,CM hypertrophy,senescence and apoptosis.Therefore,targeting circRNAs might be an attractive strategy in promoting cardiac regenerative response for its important roles in cell proliferation and CM functions.However,the role of circRNAs in cardiac regeneration has not been previously investigated.Identification and validation of the key circRNA function in the acquisition of regenerative capability is also critical to seek the possible therapeutic targets.Recently,differentially expressed circRNAs between neonatal and adult hearts have been uncovered by RNA sequencing,but it is difficult to further recognize the subgroups essential for cardiac regeneration from the fairly large number of candidates.Super-enhancers(SEs)are large clusters of active enhancers that are enriched for the binding of key master transcriptional factors.These clusters of enhancers are developmental and cell type specific,and act as causal mechanisms for cell differentiation,specification and development by controlling cell-identity genes or ncRNA expression.Because SEs are frequently identified near genes or ncRNAs that are important for controlling cell identity and differentiation,such SEs may be used to quickly identify key nodes regulating cell differentiation and development.Importantly,SE-associated ncRNAs have been found to drive cardiogenesis and CMs differentiation,both of which are the key links during CM proliferation.Here,we postulated that SEs are likely to marks circRNAs that control CM differentiation and development,thereby helping to identify key circRNAs regulating cardiac regeneration.In line with this hypothesis,we first performed a comprehensive analysis of the relationship between SEs and circRNA networks in hearts.We further identified circRNA Nfix as a CM-enriched circRNA that was regulated by the binding of Meis1 on its associated SE in the adult heart.Of crucial importance,downregulation of circNfix promoted adult CM proliferation and cardiac regeneration by rescuing Ybx1 and miR-214,which significantly decreased the fibrotic area and promoted functional recovery after MI.MethodsWe used integrated bioinformatics analysis of RNA sequencing data and SE catalogs to identify SE-associated circRNAs.Quantitative PCR and in situ hybridizations were performed to determine the circRNA expression patterns in hearts.Gain-and loss-of-function assays were conducted to detect the role of circRNAs in cardiomyocyte(CM)proliferation and cardiac repair after myocardial infarction(MI).Chromatin immunoprecipitation(ChIP)and electrophoretic mobility shift assays(EMSA)were used to determine the binding of Meis1 on circNfix-associated SE.RNA pulldown and luciferase reporter assays were used to study circRNA interactions with proteins and miRNAs.ResultsWe identified a circRNA,circNfix,which was regulated by a SE and was overexpressed in adult hearts of rats and mice.The transcriptional factor Meis1 bound to the SE in the circNfix locus and increased its expression.Knockdown of circNfix increased while circNfix overexpression inhibited CM proliferation in vitro and in vivo.Moreover,circNfix downregulation promoted CM proliferation and angiogenesis and inhibited CM apoptosis after MI,which attenuated cardiac dysfunction and improved the prognosis.Mechanistically,circNfix reinforced the interaction of Ybx1 with Nedd41,an E3 ubiquitin ligase,and induced Ybx1 degradation through ubiquitination,which repressed cyclin A2 and cyclin B1 expression.In addition,circNfix acted as a sponge of miR-214 to promote Gsk3βexpression and repress β-catenin activity.ConclusionLoss of SE-regulated circNfix promotes cardiac regenerative repair and functional recovery after MI by suppressing Ybx1 ubiquitin-dependent degradation and increasing miR-214 activity,and thus may be a promising strategy to improve the prognosis after MI.