| Background:Heart failure is the common result of a variety of diseases progressing to the end stage,and it has now become a major health care burden due to its high morbidity and mortality.Under the influence of hemodynamic stress such as hypertension,cardiomyocytes have compensatory hypertrophy,and progressive loss of cardiomyocytes leads to heart failure.In recent years,with the progress of omics research,new understanding of the pathogenesis of heart failure has been obtained.Clarifying the molecular mechanism of heart failure not only helps to find new serum markers,but also can discover new therapeutic targets and serve clinical medicine.With the development of high-throughput sequencing technology,many studies have found that non-coding RNAs play an important role in the occurrence and development of heart failure.Circular RNAs(circRNAs)are a special class of non-coding RNAs whose circular structure and lack of a 5’-end cap and a special structure of a 3’-end poly A tail make them more stable than linear RNAs.Studies have found that circRNAs play an important regulatory role in tumor and important organs’ ischemia-reperfusion injury.In addition,the expression of circRNAs is also temporally and spatially specific during heart formation.Therefore,the study of the expression patterns of circRNAs in the process of heart failure may provide new targets for the treatment of heart failure.Among the many factors that lead to myocardial contractile dysfunction,the reduction of the number of myocardial cells is one of the important reasons.The decrease in the number of cardiomyocytes in heart failure is mainly seen in cardiomyocyte death and apoptosis.Ferroptosis is a newly discovered way of cell death in recent years,which is mainly characterized by iron-dependent accumulation of cellular lipid peroxides.Ferroptosis is a gene-regulated mode of cell death,but the role of cardiomyocyte ferroptosis regulation in heart failure is rarely studied.This study aims to screen the differential genes that may be involved in ferroptosis in the process of heart failure by bioinformatics means,predict the non-coding RNAs that may interact with these genes through transcriptome data and noncoding RNA database,and reduce cardiomyocyte ferroptosis by regulating circRNAs,thus achieving the purpose of improving cardiac function and providing a new target for the treatment of heart failure.Methods:1.In this study,heart failure was induced in C57BL/6 mice by transverse aortic constriction(TAC).Echocardiography was used to evaluate the cardiac function of the mice through indicators such as left ventricular ejection fraction,left ventricular fractional shortening,left ventricular diameter and volume.The body weight,heart weight and tibia length of the mice were measured to evaluate their heart failure status.HE,Masson and WGA staining and serological indicator tests(ANP,NT-pro BNP)were performed on mouse hearts to evaluate the degree of heart damage and fibrosis in mice after modeling.The content of malondialdehyde,glutathione and ferrous ion in heart tissue of heart failure mice was detected by colorimetric method,the expression levels of marker proteins(GPX4,ACSL4,NOX1 and FTH1)related to ferroptosis were detected by immunohistochemistry and Western Blot,and the degree of cardiomyocyte ferroptosis in mice was evaluated.With the help of online databases(GEO,Proteome Xchange,PUBMED),the transcriptome and protein mass spectrometry data were reanalyzed to screen out differentially expressed proteins and non-coding RNAs related to ferroptosis.By using Target Scan,Star Base and Reg RNA 2.0 databases,non-coding RNAs that might interact with the differential gene were screened and the ceRNA regulatory network was mapped.The predicted genes and proteins were verified by qRT-PCR,immunohistochemistry and Western Blot to determine the correlation between their expression levels and heart failure.2.In this study,circSnx12 overexpressing and silencing adenovirus vectors were constructed to infect HL-1 cells and mouse myocardial tissue to evaluate the regulatory effect of circSnx12 on cardiomyocyte ferroptosis.The concentration of Erastin-induced ferroptosis in HL-1 cells was determined by CCK8 assay.The content of malondialdehyde and total glutathione in HL-1 cells was detected by colorimetric method to evaluate the sensitivity of HL-1 cells induced by Erastin to ferroptosis.In the in vivo experiment,the adenovirus was injected into the mouse heart tissue by way of the origin of the myocardium,and then TAC surgery was performed.In vivo small animal imaging and QRT-PCR were used to evaluate the effect of adenovirus infection on mouse myocardium.Cardiac ultrasound was used to evaluate the cardiac function of the mice through indicators such as ejection fraction,fractional shortening,left ventricular diameter and volume.HE,Masson staining and serological indicator tests(ANP,NT-pro BNP)were performed to detect the effect of circSnx12 on myocardial injury in mice.The content of malondialdehyde,glutathione and ferrous ion in mouse heart tissue was detected by colorimetric method,and the related ferroptosis indicators(GPX4,ACSL4,NOX1,FTH1)were detected by immunohistochemistry and Western Blot to evaluate the effect of circSnx12 on mouse cardiomyocyte ferroptosis regulation.3.Based on the previous re-analysis and experimental verification of protein mass spectrometry and transcriptome data,immunofluorescence in situ hybridization was used to localize circSnx12,and RNA immunoprecipitation experiment was used to verify the interaction between circSnx12 and AGO2 protein,and miR-224-5p and AGO2 protein.The interaction between circSnx12 and miR-224-5p and miR-224-5p and FTH1 was detected by dual-luciferase reporter gene assay.In the in vivo experiment,the model was established after infecting mouse myocardium with miR-224-5p-silencing adenovirus vector in vivo.Cardiac injury in mice was evaluated by cardiac ultrasound,pathological staining and serological detection,and the ferroptosis of mouse cardiomyocytes was evaluated using malondialdehyde,glutathione and ferrous ion content and protein indexes.In vitro,HL-1 cells were co-infected with miR-224-5p and circSnx12,and the contents of malondialdehyde and total glutathione were detected to evaluate whether miR-224-5p could restore the effect of circSnx12 on Erastin-induced ferroptosis in HL-1 cells.The mechanism of circSnx12 regulating cardiomyocyte ferroptosis was verified.Results:1.After TAC surgery,the cardiac function of mice was reduced,and there was obvious myocardial injury or fibrosis.Compared with the control group,the serum ANP and NTpro BNP contents were significantly increased.Biochemical detection showed that after TAC surgery,the contents of malondialdehyde and ferrous ions in the heart tissue of mice were significantly increased,and GSH was significantly decreased.At the same time,Western Blot and immunohistochemistry showed that the expressions of ACSL4 and NOX1 were significantly increased,and the expression of GPX4 was down-regulated,suggesting that TAC can induce ferroptosis in mouse cardiomyocytes.The differential expression of ATP6V1 A and FTH1 in heart failure was screened based on bioinformatics.Through database prediction and transcriptome data re-analysis,5 miRNAs and 7circRNAs that may interact with them were obtained,and the ceRNA regulatory network was drawn.Using qRT-PCR,Western Blot and immunohistochemical detection,it was found that the expression of FTH1 was significantly down-regulated in heart failure,which was consistent with the results of mass spectrometry and correlated with the expression of circSnx12 and miR-224-5p,which may interact with it.2.In vitro,after overexpression of circSnx12,compared with Erastin group,the content of malondialdehyde in HL-1 cells was significantly decreased,and the content of total glutathione was increased.Compared with the control group,circSnx12 silencing had no significant effect on malondialdehyde and total glutathione contents in HL-1 cells.In vivo experiment found that overexpression of circSnx12 had a significant protective effect on cardiac function in mice.After TAC surgery,left ventricular ejection fraction and fractional shortening were significantly higher than those in the TAC group,and myocardial tissue damage and fibrosis were alleviated.3.Immunofluorescence in situ hybridization found that circSnx12 was localized in the HL-1 cytoplasm.RNA immunoprecipitation experiments confirmed that circSnx12 and miR-224-5p could bind to AGO2 protein,which determined the basis of its ceRNA action.The dual-luciferase reporter gene showed that miR-224-5p could significantly reduce the fluorescence intensity of wild-type circSnx12 vector and wild-type FTH-1-UTR vector,but had no effect on the fluorescence intensity of mutant vector.Furthermore,miR-224-5p overexpression could counteract the protective effect of circSnx12 overexpression on Erastin-induced ferroptosis in HL-1 cells.In vivo experiments found that TAC surgery after silencing miR-224-5p had a similar protective effect on cardiac function in mice with circSnx12 overexpression group,and reduced the sensitivity of cardiomyocytes to ferroptosis.Conclusion:1.TAC surgery can lead to ferroptosis in mouse cardiomyocytes and induce heart failure in mice.circSnx12 and FTH1 were down-regulated in myocardial tissue of heart failure mice,while miR-224-5p was up-regulated.It indicated that the expressions of circSnx12,miR-224-5p and FTH1 were correlated with heart failure.2.Overexpression of circSnx12 can protect cardiomyocytes and reduce the sensitivity of cardiomyocytes to ferroptosis.3.Overexpression of miR-224-5p can counteract the protective effect of circSnx12 overexpression on cardiomyocyte ferroptosis.circSnx12 maintains the homeostasis of intracellular iron metabolism by competing with FTH1 for binding to miR-224-5p,and regulates ferroptosis in cardiomyocytes of heart failure mice. |
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