The Molecular Mechanism Of CircRNA45524 Regulating Pyroptosis In Cardiomyocytes

Objective The prevalence and mortality of cardiovascular diseases in China are increasing year by year.With the rapid development of the times and the increasing degree of aging.Myocardial infarction is one of the cardiovascular diseases with rapid development and high mortality.At present,the effective treatment of myocardial infarction is through drugs and interventional therapy.Although these methods can recanalize the blocked blood vessels,they will cause ischemia-reperfusion injury to the heart and aggravate the development of the disease.In the development of most diseases,there is a process of cell death.As a newly discovered type of cell death,pyroptosis is a programmed cell death process,often accompanied by immune inflammatory response.CircRNA is a circular RNA with a stable structure.Recent studies have found that circRNA is involved in the regulation of many diseases,including the occurrence of some tumors and the regulation of heartrelated diseases.Based on the main research background of injury and pyroptosis after cardiac ischemia-reperfusion,this paper explores the regulatory process and related molecular mechanism of circRNA involved in myocardial pyroptosis,and provides new treatment methods and research ideas for inflammatory response after myocardial ischemia-reperfusion.Method In this study,the primary cardiomyocytes of neonatal mice were used as the research object.The pyroptosis process was induced by hypoxia / reoxygenation(H/R)in vitro.The hypoxia time gradient of 0 h,6 h,12 h,24 h and 48 h and the condition of reoxygenation for 6 h were set up to screen out the optimal time point for inducing pyroptosis in primary cardiomyocytes of neonatal mice.High-throughput sequencing was used to screen differentially expressed circRNAs under H/R conditions as the research object.Real-time fluorescence quantitative PCR was used to detect significantly differentially expressed genes.It was found that circRNA45524 was expressed stably and the difference was obvious.Therefore,circRNA45524 was used as the main research object.The distribution of circRNA45524 in primary cardiomyocytes of neonatal mice was detected by nuclear separation and fluorescence in situ hybridization.The in vitro cell knockdown / overexpression model was constructed by transfecting si RNA and infected adenovirus into mouse primary cardiomyocytes.The expression of pyroptosis-related proteins in cells was detected by Western Blotting,and the content of lactate dehydrogenase in cell supernatant was detected by LDH method to measure cell death.PI staining was used to detect the level of cell death.the target molecules that may bind to circRNA45524 were detected by co-immunoprecipitation.Bioinformatics analysis was used to analyze the targets that interact with circRNA45524 and may be involved in the regulation of pyroptosis.Finally,the relationship between circRNA45524 and downstream targets was detected by molecular protein interaction verification.Results1.The H/R model of neonatal mouse cardiomyocytes was constructed to simulate the pyroptosis process in vitro.The results showed that the degree of pyroptosis was the most obvious under the condition of hypoxia for 6 h and reoxygenation for 6 h.2.After H/R,high-throughput sequencing and real-time fluorescence quantitative PCR verified that circRNA45524 expression was stable and significantly different,so circRNA45524 was identified as the main research object.3.The circRNA looping verification showed that circRNA45524 had a circular structure.4.The results of nuclear separation and fluorescence in situ hybridization showed that circRNA45524 was distributed in both cytoplasm and nucleus.5.After knocking down circRNA45524 in neonatal mouse primary cardiomyocytes,the expression of pyroptosis marker protein GSDMD and GSDMD-C was significantly increased,the expression of pyroptosis pathway-related proteins Caspase-1 and IL-1β was also significantly increased,the LDH content in the cell supernatant was increased,and the number of PI staining positive cells was increased.6.After overexpression of circRNA45524 in neonatal mouse primary cardiomyocytes and H/R treatment,the expression of pyroptosis marker protein GSDMD and GSDMD-C was significantly reduced,and the expression of pyroptosis pathway-related proteins Caspase-1 and IL-1β was also significantly reduced,the LDH content in the cell supernatant was reduced,and the number of PI staining positive cells was reduced.7.Co-immunoprecipitation assay showed that there were different bands in the pull down group compared with the control group,indicating that there may be molecules that interact with circRNA45524.8.Bioinformatics analysis and molecular protein interaction experiments showed that there was an interaction between circRNA45524 and Drg2,so Drg2 was the main object of our downstream mechanism research.Conclusion In this study,the H/R model of primary cardiomyocytes of neonatal mice was constructed in vitro to explore the regulatory effect of circRNA45524 on pyroptosis.The results showed that the pyroptosis process and the expression of related proteins in primary cardiomyocytes of neonatal mice were the most obvious under the condition of hypoxia for6 h and reoxygenation for 6 h.Knockdown of circRNA45524 in primary cardiomyocytes of neonatal mice will aggravate the pyroptosis process,while overexpression will effectively reverse the pyroptosis process,and circRNA45524 regulates the death process of cardiomyocytes by Caspase-1—GSDMD pyroptosis classical signaling pathway.The results of this study clarified the regulatory mechanism of circRNA45524 in regulating pyroptosis of cardiomyocytes after H/R and provided theoretical support,and also provided ideas and references for the injury of cardiomyocytes after cardiac ischemia-reperfusion.