RESUMEN
RATIONALE: Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is restricted because it poses a risk of severe cardiotoxicity. Previous work has established that CircITCH (circular RNA ITCH [E3 ubiquitin-protein ligase]) is a broad-spectrum tumor-suppressive circular RNA and that its host gene, ITCH (E3 ubiquitin protein ligase), is involved in doxorubicin-induced cardiotoxicity (DOXIC). Whether CircITCH plays a role in DOXIC remains unknown. OBJECTIVE: We aimed to dissect the role of CircITCH in DOXIC and further decipher its potential mechanisms. METHODS AND RESULTS: Circular RNA sequencing was performed to screen the potentially involved circRNAs in DOXI pathogenesis. Quantitative polymerase chain reaction and RNA in situ hybridization revealed that CircITCH was downregulated in doxorubicin-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in the autopsy specimens from cancer patients who suffered from doxorubicin-induced cardiomyopathy. Cell death/viability assays, detection of cardiomyocyte necrosis markers, microelectrode array, and cardiomyocyte functional assays revealed that CircITCH ameliorated doxorubicin-induced cardiomyocyte injury and dysfunction. Detection of cellular/mitochondrial oxidative stress and DNA damage markers verified that CircITCH alleviated cellular/mitochondrial oxidative stress and DNA damage induced by doxorubicin. RNA pull-down assays, Ago2 immunoprecipitation and double fluorescent in situ hybridization identified miR-330-5p as a direct target of CircITCH. Moreover, CircITCH was found to function by acting as an endogenous sponge that sequestered miR-330-5p. Bioinformatic analysis, luciferase reporter assays, and quantitative polymerase chain reaction showed that SIRT6 (sirtuin 6), BIRC5 (baculoviral IAP repeat containing 5, Survivin), and ATP2A2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, SERCA2a [SR Ca2+-ATPase 2]) were direct targets of miR-330-5p and that they were regulated by the CircITCH/miR-330-5p axis in DOXIC. Further experiments demonstrated that CircITCH-mediated alleviation of DOXIC was dependent on the interactions between miR-330-5p and the 3'-UTRs of SIRT6, BIRC5, and ATP2A2 mRNA. Finally, AAV9 (adeno-associated virus serotype 9) vector-based overexpression of the well-conserved CircITCH partly prevented DOXIC in mice. CONCLUSIONS: CircITCH represents a novel therapeutic target for DOXIC because it acts as a natural sponge of miR-330-5p, thereby upregulating SIRT6, Survivin and SERCA2a to alleviate doxorubicin-induced cardiomyocyte injury and dysfunction.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Doxorrubicina/efectos adversos , MicroARNs/metabolismo , ARN Circular/fisiología , Proteínas Represoras/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sirtuinas/metabolismo , Survivin/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regiones no Traducidas 3'/genética , Adenovirus Humanos , Animales , Proteínas Argonautas/análisis , Sitios de Unión , Biomarcadores , Cardiotoxicidad/genética , Cardiotoxicidad/metabolismo , Cardiotoxicidad/terapia , Muerte Celular , Supervivencia Celular , Daño del ADN , Regulación hacia Abajo , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Inmunoprecipitación/métodos , Hibridación Fluorescente in Situ/métodos , Ratones , MicroARNs/genética , Mitocondrias Cardíacas/metabolismo , Mutación , Contracción Miocárdica/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Necrosis , Estrés Oxidativo , ARN Circular/efectos de los fármacos , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Survivin/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
Myocardial ischemia/reperfusion (I/R) injury is a common cardiovascular disease with high morbidity and mortality globally, which derives from acute myocardial infarction and coronary artery disease. Elabela has been proved to bind to apelin receptors in the heart. The present study aimed to investigate the protective effects of Elabela in myocardial I/R injury and illustrating the potential mechanisms. In this study, the rat I/R model was established in vivo. Following treatment with Elabela, the histopathological changes of heart tissue were evaluated by the hematoxylin and eosin- or Masson's trichrome staining. Apoptosis of heart tissue was examined using TUNEL staining. The expression of type I or III collagen and apoptosis-associated proteins was measured using western blotting. Moreover, myocardial ultrastructure in myocardium was detected via electron microscopy analysis. H9c2 cells were treated with hypoxia/reoxygenation (H/R) to mimic the myocardial I/R injury in vitro. After treatment with Elabela or Elabela combined with LY294002, the levels of oxidative stress and apoptosis were examined. The results revealed that Elabela significantly improved the pathological changes of rat myocardial tissues induced by I/R. Additionally, Elabela treatment reduced cardiomyocyte I/R induced fibrosis and apoptosis as well as ameliorated mitochondrial dysfunction in animal and cells. Within inhibition of PI3K pathway, the protective effects of Elabela was reversed. Taken together, these findings demonstrated that Elabela could protect against fibrosis, apoptosis and oxidative stress via PI3K/ATK signaling pathway in cardiac ischemia reperfusion.