The Role Of CircRNA-4087 In Modulating Myocardial Ischemia/Reperfusion Injury And Its Mechanism

BackgroundMyocardial ischemia/reperfusion injury(I/R)refers to the aggravation of ischemic myocardial injury after reperfusion.It mainly includes arrhythmia and myocardial contractile dysfunction and irreversible damage of reperfusion myocardium.In recent years,I/R injury has become more common with thrombolytic therapy,PCI,cardiovascular surgery,and the widespread use of cardiopulmonary bypass technique.The mechanism of I/R injury include excessive production of reactive oxygen species,calcium overload,excessive apoptosis of cardiomyocytes,and inflammatory responses.How to reduce ischemia/reperfusion myocardial injury is of great significance in improving the clinical prognosis of patients with I/R.Circular RNA(circRNA)is a type of single-stranded,closed RNA formed by back splices.Its formation mainly involves intron-paired splicing,RNA-binding protein driven loop formation and lasso-driven loop formation.Most of the circRNA located in the cytoplasm,and some circRNAs containing introns can also located in the nucleus.CircRNA has been considered to have no physiological function due to the low expression level.Until recent years,the second generation of sequencing has found tens of thousands of circRNAs,which are widely found in multi-cell organisms such as drosophila,mice and humans.Some circRNAs are found to be highly expressed in specific cell types or tissues.RNA sequencing of adult mouse hearts revealed the enrichment of 575 circRNAs.The sequencing of myocardial tissue in normal adults revealed the enrichment of more than 300 circRNAs,suggesting that circRNA is present in myocardial tissue with high expression.The circRNA is very stable compared to the corresponding linear RNA,so it can accumulate in tissues to perform some specific functions.The functions of circRNA include regulation of transcription,binding proteins and miRNA sponges,which are closely related to the occurrence and development of various diseases such as tumors and cardiovascular diseases.CircRNAs contain numerous miRNA binding sites,therefore it can function as miRNA sponge.Recent studies have found that circRNA000203 can bind miR-26 b,causing the increase of Col1a2 and CTGF expression,which promotes the occurrence of myocardial fibrosis;CircRNA Cdr1 as can bind miR-7 and increase the expression of its downstream target genes SP1 and PARP,which promote the occurrenceof myocardial infarction;Overexpression of circRNA HRCR inhibit the pathological cardiac hypertrophy in mice;MFACR can bind miR-652-3p,up-regulate the expression level of MTP18,reduce myocardial cell apoptosis and reduce myocardial infarct size.These studies have shown that circRNA can interfere with the function of miRNA through miRNA sponge,thus affecting the development of cardiovascular disease.However,there are few reports of circRNA in myocardial I/R injury,and the role and function of circRNA needs further exploration.Objective1.To explore differentially expressed myocardial ischemia/reperfusion-related circRNA.2.To study the role of circRNA4087 in myocardial ischemia-reperfusion injury and its function on apoptosis.3.To explore the potential mechanism of circRNA4087 to regulate cardiomyocyte function.Methods1.We constructing ischemia/reperfusion injury model in adult SD rats,collecting myocardial tissue samples,and verifying the circRNAs expression by qPCR.The circular structure of the circRNA4087 sequence was subsequently verified by RNase R digestion assay and PCR experiments.The over-expression and down-regulation adenovirus were constructed by seamless cloning technology.The obtained adenovirus was amplified and purified.2.The primary cardiomyocytes of neonatal rats were isolated,and the model of hypoxia-reoxygenation injury of cardiomyocytes in vitro was constructed by hypoxia workstation.The viability of cardiomyocytes was detected by cck8 method,ATP content in cardiomyocytes was detected by ATP detection kit,and the ROS was detected by mitoSOX red reagent.Tunel kit and Annexin V/PI apoptosis kit were used to detect the apoptosis state of cardiomyocytes.The overexpression adenovirus of circRNA4087 was injected in the mouse heart,and the apoptosis state was detected by Tunel kit.3.We use the miRanda and miRbase database to find potential target miRNAs that circRNA4087 may bind.The ability of circRNA4087 to bind miRNA was detected by Ago2 pull-down assay;luciferase-4087 luciferase reporter plasmid was constructed bymolecular cloning,and luciferase reporter assay was used to screen for possible target miRNA.Fluorescence in situ hybridization experiments were used to validate the location of miRNA and circRNA4087.Results1.By qPCR validation,we found that circRNA4087 was significantly up-regulated in myocardium/ischemia reperfusion model.We selected circRNA4087 for subsequent experiments.2.Circular RNA4087 can resist the digestion of RNase R enzyme.The results of forward and reverse PCR showed that only the reverse primer of circRNA4087 can amplify a single band in cDNA,which proved that the sequence of circRNA4087 was a ring structure.We constructed circRNA4087 over-expression and down-regulation adenovirus,and verified in cardiomyocytes successfully.The adenovirus had no significant effect on the host gene;2.Overexpression of circRNA4087 inhibited cardiomyocyte viability,reduced intracellular ATP content,and promoted cardiomyocyte apoptosis;However,down-regulation of circRNA4087 can increase intracellular ATP content and inhibit cardiomyocytes apoptosis.Compared with the mice injected with vector adenovirus,the circRNA4087 group showed a significant increase in apoptosis;3.The sequencing results were analyzed and we found 52 miRNAs candidates that may bind to circRNA4087.After examining 52 possible binding miRNAs,we found that circRNA4087 can bind to miR-16 and play an important role in regulating myocardial function.Fluorescence in situ hybridization experiments revealed that miR-16 and circRNA4087 have a high degree of colocalization in the cytoplasm of cardiomyocytes,suggesting the combination of miR-16 and circRNA4087.Conclusion1.We found I/R related circRNA4087 was significantly upregulated during myocardial ischemia/reperfusion injury.We use seamless cloning technology to construct over-expression and down-regulation adenovirus of circRNA4087.The amplified and purified adenovirus has high specificity and efficiency,and can be used in subsequent experimental research.2.Overexpression of circRNA4087 inhibited cell viability,reduced ATP content in cardiomyocytes and promoted cardiomyocyte apoptosis.In hypoxia/reoxygenationmodel,down-regulation of circRNA4087 protected cardiomyocytes and inhibited cardiomyocyte poptosis.CircRNA4087 may bind to miR-16 and play an important role in regulating myocardial function.