Abstract
circRNAs (circular RNAs) are emerging as powerful regulators of cardiac development and disease, but their roles in cardiac regeneration are still unknown. This study used superenhancers to distinguish key circRNAs in the regulation of cardiac regeneration and explored the mechanisms underlying circRNA functions. We used integrated bioinformatics analysis of RNA sequencing data and superenhancer catalogs to identify superenhancer-associated circRNAs. Quantitative polymerase chain reactions and in situ hybridization 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 proliferation and cardiac repair after myocardial infarction. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays were used to determine the binding of Meis1 (Meis homeobox 1) on circNfix-associated superenhancers. RNA pulldown and luciferase reporter assays were used to study circRNA interactions with proteins and miRNAs (micro RNAs). We identified a circRNA, Nfix circRNA (circNfix), that was regulated by a superenhancer and overexpressed in the adult heart in humans, rats, and mice. The transcription factor Meis1 bound to the superenhancer at the circNfix locus, and increased its expression. In vitro and in vivo, cardiomyocyte proliferation was increased by knockdown of circNfix, whereas it was inhibited by circNfix overexpression. Moreover, circNfix downregulation promoted cardiomyocyte proliferation and angiogenesis and inhibited cardiomyocyte apoptosis after myocardial infarction, attenuating cardiac dysfunction and improving the prognosis. Mechanistically, circNfix reinforced the interaction of Ybx1 (Y-box binding protein 1) with Nedd4l (an E3 ubiquitin ligase), and induced Ybx1 degradation through ubiquitination, repressing cyclin A2 and cyclin B1 expression. In addition, circNfix acted as a sponge for miR-214 to promote Gsk3β (glycogen synthase kinase 3 β) expression and repress β-catenin activity. Loss of superenhancer-regulated circNfix promotes cardiac regenerative repair and functional recovery after myocardial infarction by suppressing Ybx1 ubiquitin-dependent degradation and increasing miR-214 activity and thus may be a promising strategy for improving the prognosis after MI.
Highlights
CircRNAs are emerging as powerful regulators of cardiac development and disease, but their roles in cardiac regeneration are still unknown
A gene ontology analysis of proximal genes around embryonic-specific superenhancers in the heart showed that they are related to biological processes characteristic of cardiogenesis and angiogenesis, whereas adult-specific superenhancers are linked to the downregulation of signaling pathways essential for cardiogenesis (Figure IF in the online-only Data Supplement)
These results suggested that superenhancers might be critical drivers or inhibitors of cardiac regeneration
Summary
We used integrated bioinformatics analysis of RNA sequencing data and superenhancer catalogs to identify superenhancerassociated circRNAs. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays were used to determine the binding of Meis[1] (Meis homeobox 1) on circNfix-associated superenhancers. RNA pulldown and luciferase reporter assays were used to study circRNA interactions with proteins and miRNAs (micro RNAs). The data, methods and materials related to this study are available to other researchers on reasonable request. C57BL/6 mice and SD rats were purchased from Guangdong Medical Laboratory Animal Center. The Cas[9] knock-in mouse model was obtained from Shanghai Model Organisms Center, Inc. Mouse MI was performed as described.[20] All animal experiments were approved by the Animal Research Committee of Southern Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Neonatal cardiomyocytes were isolated from 1 day-old (P1) and 7-day-old (P7) C57BL/6 mice or P1 SD rats, and adult cardiomyocytes were isolated from ≈8 week-old adult mouse as previously described.[2]
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