A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p

Non-coding RNAs (ncRNAs) are considered major players in physiological and pathological processes based on their versatile regulatory roles in different diseases including cardiovascular disease. Circular RNAs (circRNAs), a newly discovered class of RNAs, constitute a substantial fraction of the mammalian transcriptome and are abundantly expressed in the cardiovascular system. However, the regulatory functions of these circRNAs in ischemic cardiac disease remain largely unknown. Here, we investigated the role of a circRNA transcribed from the sodium/calcium exchanger 1 (ncx1) gene, named circNCX1, in oxidative stress-induced cardiomyocyte apoptosis during ischemic myocardial injury. Methods: Divergent polymerase chain reaction (PCR) was conducted to amplify the circRNA. The circular structure of circNCX1 was verified by Sanger sequencing and RNase R digestion. The subcellular localization of circNCX1 was detected by fluorescence in situ hybridization (FISH). To test the expression pattern and function of circNCX1 during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with H(2)O(2) or hypoxia-reoxygenation (H/R). Mechanistically, the interaction of circNCX1 with miRNA was examined by AGO2-IP and RNA pull-down assays. The regulatory role of circNCX1 in target gene expression was tested by western blot and luciferase reporter assays. At the animal level, we constructed a myocardial ischemia-reperfusion (I/R) mouse model to analyze the effect of circNCX1 on heart function, cardiomyocyte apoptosis and cardiac remodeling. Results: circNCX1 was increased in response to reactive oxygen species (ROS) and promotes cardiomyocyte apoptosis by acting as an endogenous miR-133a-3p sponge. Due to competitive binding of circNCX1 to miR-133a-3p, the suppressive activity of pro-apoptotic gene cell death-inducing protein (CDIP1) by miR-133a-3p was reduced. Knockdown of circNCX1 in murine cardiomyocytes and heart tissues reduced the levels of CDIP1 and attenuated the apoptosis and I/R injury. Conclusions: Our findings reveal a novel regulatory pathway that comprises circNCX1, miR-133a-3p and CDIP1, that is involved in cardiomyocyte apoptosis. This pathway may serve as a potential therapeutic avenue for ischemic heart diseases.