Role And Mechanism Of Circ-0615 Downregulation In Protecting Doxorubicin Induced Cardiotoxicity

Background:Cardiotoxicity is the main side effect of anthracycline antitumor drugs represented by doxorubicin.Currently,corresponding clinical treatment for doxorubicin induced cardiotoxicity is still lacking.Circular RNA is an endogenous noncoding RNA,which has been proved to play an important role in cardiac pathophysiology.In previous studies,we have clarified that a circular RNA,circ-0615,plays an important regulatory role in myocardial injury.However,its role in regulating doxorubicin induced cardiotoxicity remains unknown.In this research,we aim to clarify the function of circ-0615 in regulating doxorubicin-induced cardiotoxicity and its underly mechanism via functional assay in vivo and in vitro,Our project will reveal the circular RNA mechanism in the prevention and treatment of doxorubicin-induced cardiotoxicity and provide novel methods for the precise treatment of patients with doxorubicin cardiomyopathy.Methods:Firstly,the expression of circ-0615 in the cell and animal model of doxorubicin induced cardiotoxicity was detected by RT-q PCR.Then,doxorubicin treatment was used for establishing doxorubicin induced cardiotoxicity cell model in primary isolated neonatal rat cardiomyocytes.The effects of circ-0615 on cardiomyocyte apoptosis and intercellular oxidative stress induced by doxorubicin were tested via TUNEL staining,western blot,DHE staining and mito SOX staining.Meanwhile,intraperitoneal injection of doxorubicin was used for establishing doxorubicin induced cardiotoxicity mice model.The effects of intervening circ-0615on cardiomyocyte apoptosis and mitochondrial dysfunction induced by doxorubicin were tested via echocardiography,TUNEL staining,Masson staining and western blot.Next,we explored the downstream mechanism of circ-0615 regulating doxorubicin induced cardiotoxicity in vitro and clarified the effects of its downstream effector FTO on doxorubicin induced cardiotoxicity in vivo and vitro.Finally,function reversal assays in vitro were used to clarified whether FTO was the downstream of circ-0615 in regulating doxorubicin induced cardiotoxicity.Results:The expression of circ-0615 was significantly increased both in the doxorubicin-induced mice heart tissue sample and doxorubicin-treated cardiomyocytes.In NRCM in vitro,knockdown of circ-0615 could reduced doxorubicin-induced cardiomyocyte apoptosis,preserved mitochondrial DNA,inhibited oxidative stress and decreased accumulation of non-heme iron.On the contrary,overexpression of circ-0615 could aggravate the above pathological changes caused by doxorubicin.In animal experiment,knockdown of circ-0615 via tail vein injection of AAV9-sh-circ-0615 improved cardiac function,inhibited cardiac fibrosis,reduced the level of cardiomyocyte apoptosis,preserved mitochondrial DNA,inhibited oxidative stress and decreased accumulation of non-heme iron under condition of doxorubicin.The level of m~6A was increased significantly in the doxorubicin-induced mice heart tissue sample.Knocking down the expression of circ-0615 in the heart significantly reduced the level of m~6A in the heart tissue.M~6A demethylation transferase FTO is negatively regulated by circ-0615.In NRCM in vitro,overexpression of FTO reduced the doxorubicin-induced cardiomyocyte apoptosis while knockdown of FTO aggravated doxorubicin-induced cardiomyocyte apoptosis,reinforced mitochondrial DNA damage,elevated oxidative stress and increased iron accumulation.In animal experiment,knockdown of FTO via tail vein injection of AAV9-sh FTO aggravated cardiac dysfunction,promoted cardiac fibrosis,increased the level of cardiomyocyte apoptosis,reinforced mitochondrial DNA,elevated oxidative stress and increased accumulation of iron under condition of doxorubicin.Further studies found that knockdown of FTO in NRCM partially abolished the effects of circ-0615 knockdown on doxorubicin-induced cardiomyocyte apoptosis,mitochondrial DNA damage,oxidative stress and accumulation of iron,which indicating that FTO is the downstream molecule of circ-0615 in regulating doxorubicin-induced cardiotoxicity.Conclusion:This study found that circ-0615 is involved in the regulation of doxorubicin induced cardiotoxicity.Knockdown of circ-0615 could protect doxorubicin induced cardiotoxicity by inhibiting cardiomyocyte apoptosis,reducing the level of reactive oxygen species and improving mitochondrial iron metabolism.M~6A demethyltransferase FTO was proved to be the downstream negative regulator of circ-0615 in regulating doxorubicin induced cardiotoxicity.