The Functional Roles Of CircRNA CHRC Through MiR-431-5p/KLF15 Signaling Axis In The Progression Of Heart Failure

Heart failure is the ultimate manifestation of decompensatory response in cardiac function,in which the heart is no longer able to pump enough blood to meet the body’s needs,and this complex clinical syndrome is a serious threat to human life.As an endogenous regulatory mechanism,the heart converts mechanotransduction into biochemical reactions and molecular signals in response to stress,forming a complex biomolecular regulatory network and causing structural and functional changes in the myocardium under spatio-temporal regulation.In particular,key molecules in the network(e.g.transcription factors,non-coding RNAs)can directly lead to changes in gene expression and signaling pathways that affect the progression of heart failure.Hence,one of the important directions in heart failure research is to identify molecules associated with the occurrence of heart failure from the cardiac molecular signaling regulatory network and to stimulate endogenous molecular protective mechanisms early in the cardiac remodeling response.Circular RNA(circ RNA)is a class of non-coding RNAs with a closed-loop structure,which can be involved in the occurrence and progression of human cardiovascular disease as competing endogenous RNAs.Previous studies have shown that pathological cardiac hypertrophy,an early clinical manifestation of heart failure,is present with a large number of differentially expressed circ RNAs,suggesting its association with the progression of cardiac hypertrophy.However,the functional mechanism of circ RNA affecting the occurrence of heart failure is unclear.If we can use the signaling molecular regulatory network to discover circ RNAs associated with the occurrence of heart failure and explore their regulatory mechanisms,it will provide important clues for us to understand the dynamic process of heart failure development.In view of this,this study constructed a competitive endogenous regulatory network based on heart failure expression profile data,screened key molecules and signaling pathways,as well as experimentally validated their functional mechanisms in heart failure.The contents are as follows:First,this study collected RNA-seq datasets from the GEO database and related articles,and screened 120 circ RNAs,80 miRNAs and 2916 m RNAs that were significantly differentially expressed in heart failure,suggesting that circ RNAs and their downstream molecules are correlated with the occurrence of heart failure.Targetscan,miRanda,RNAhybrid and miRWalk databases were used to predict the target molecules of differential miRNAs,and 418 circ RNA-miRNA and 9401 miRNAm RNA pairs were obtained.For the screening of miRNA-m RNA pairs,we used gene functional enrichment analysis to further screen out genes associated with the pathological process of cardiac hypertrophy and heart failure,and combined with the result of negative correlation between miRNA and m RNA expression,we obtained 131 pairs consistent with the mechanism of miRNA-mediated gene silencing.According to the molecular competition relationship in ce RNA theory,the circ RNA-miRNA-m RNA competitive interaction network was constructed,which contained 150 nodes(56circ RNAs,38 miRNAs and 56 m RNAs)and 405 edges.Secondly,we detected m RNA expression levels of candidate genes in the transverse aortic constriction(TAC)model,and obtained 16 genes down-regulated during the occurrence of heart failure.According to the relevant functions in physiological and pathological processes,we focused on the gene Klf15 in both cellular and animal experiments showed an inhibitory effect on pathological cardiac hypertrophy and fibrosis.Therefore,we used the gene Klf15 as a core functional molecule in the signaling axis and detected the expression levels of target molecules linked to it in the network.Based on the ce RNA mechanism,miR-431-5p was found to have an opposite expression trend to its downstream gene Klf15,and the upstream molecule circ RNA CHRC(Cardiac hypertrophy related circ RNA)is significantly down-regulated in heart failure.Thus,we identified that the heart failure-related circ RNA CHRC targets the miR-431-5p/Klf15 signaling axis.Then,for the above molecules in the signaling axis,we explored the role of circ RNA CHRC in the process of early cardiac hypertrophy in heart failure.Silencing of CHRC expression in primary mouse cardiomyoocytes(pm CMs)revealed a significant hypertrophic phenotype of the cells after 48 h.Overexpression of CHRC in a model of hypertrophy induced by Ang II in pm CMs enhanced its role as a miR-431-5p sponge and significantly improved the hypertrophic phenotype of pm CMs.By constructing an adeno-associated virus(AAV)-based CHRC overexpression vector and injecting TAC mice via tail vein,miR-431-5p was affected by the sponge effect to reduce the inhibition of target gene Klf15 and effectively alleviated cardiac hypertrophy.Therefore,we suggest that CHRC has the function of alleviating the symptoms of pathological hypertrophy and may have specific competitive interactions with miR-431-5p.Finally,we explored the mechanism of CHRC in the heart failure process,and confirmed the specific interaction among miR-431-5p,CHRC and Klf15 by dualluciferase reporter system,Ago2 RIP and q PCR.To further reveal that the posttranscriptional regulation of CHRC on the target gene Klf15 is affected by the competitive inhibition of miR-431-5p,the CHRC overexpression plasmid and miR-431-5p mimic were simultaneously transfected into pm CMs and found that the upregulation of Klf15 caused by overexpression of CHRC is blocked.These demonstrate that CHRC plays a cardioprotective function by competitively binding miR-431-5p,activating Klf15 to protect the heart during cardiac hypertrophy.In summary,we screened for the functional molecule circ RNA CHRC by constructing a circ RNA competitive endogenous regulatory network and demonstrated in vivo and in vitro experiments that circ RNA CHRC functions in cardiac hypertrophy by targeting the miR-431-5p/Klf15 core signaling axis.The study showed that during the pathological process,CHRC activates the expression of downstream transcription factor KLF15 through competitive binding of miR-431-5p,thereby reducing the dysfunction caused by cardiac hypertrophy and slowing the progression of heart failure.The results of this project will help to improve the knowledge of epigenetic molecules in cardiovascular diseases and provide a new reference for the pathogenesis of heart failure.