Circular RNA CircRNA₀00203 Aggravates Cardiac Hypertrophy Via Suppressing MiR-26b-5p And MiR-140-3p Binding To Gata4

Heart failure is the one leading cause of mortality in cardiovascular diseases.Cardiac hypertrophy in response to chronic hypertension is associated with significantly increased risk for heart failure and sudden death.Over the few years,growing evidence has confirmed that some circular RNAs are crucial for the cardiac hypertrophy.The molecular mechanisms that mediate these effects,however,remain largely obscure.Therefore,it is urgently needed to explore the molecular mechanisms of a potential therapeutic target circRNA underlying pathological cardiac hypertrophy and that might be useful in preventing and treating heart failure.Our previous study revealed that circRNA₀00203 was up-regulated in the diabetic mouse myocardium with pro-fibrotic effect.However,the potential molecular mechanism of circRNA₀00203 on cardiac hypertrophy is still unknown.Objective:The aim of this study was to investigate the effect of circRNA₀00203 on cardiac hypertrophy in vitro and in vivo,and the molecular mechanism by which circRNA₀00203 modulates cardiac hypertrophic phenotype and to explore the potential molecule target.Methods:1.The expression levels of circRNA₀00203 in the Ang-? infusion-induced hypertrophy animal and cell model and cardiac tissue of 22 patients with heart failure,20 healthy controls were assessed by real-time quantitative polymerase chain reaction?RT-qPCR?.2.We verified circRNA₀00203 function in cardiac hypertrophic phenotype in vitro by using phalloidin staining to detect cell surface and by RT-qPCR and Western Blot analysis to detect the mRNA and protein level of the hypertrophy related gene Myh7 and Anp in NMVCs infected with rAd-circ203.3.To further verify circRNA₀00203 function on cardiac hypertrophic phenotype in vivo,we established the Tg-circ203 transgenic mice with cardiac-specific overexpression of circRNA₀00203 and assessed heart function of Tg-circ203 mice by echocardiography,detected the size of cardiomyocytes by wheat germ agglutinin staining and the expression levels of hypertrophy related gene Myh7 and Anp by RT-qPCR and Western Blot assay in the myocardium of Tg-circ203 mice.4.Based on the bioinformatics analysis,combining with the experimental techniques on RNA antisense purification,dual-luciferase assays,RNA pull-down and RT-qPCR,we explored whether predicted microRNAs?miR-26b-5p and miR-140-3p?could directly bind to circRNA₀00203,and confirmed the correlation between circRNA₀00203 levels and miR-26b-5p or miR-140-3p levels in NMVCs.5.RT-qPCR,Western Blot and dual luciferase reporter assay were performed to identify the interaction between miR-26b-5p or miR-140-3p and target gene Gata4 and the regulation relationship between them in NMVCs.6.RT-qPCR and Western Blot assay were performed to verify the regulatory relationship between these three and their effect on cardiac hypertrophic phenotype.7.The dual luciferase reporter and RT-qPCR assay were used to explore whether the transcription factor NF-?B p65 could bind to the gene Myo9a promoter and regulate the transcription activity of circRNA₀00203.Results:1.Up-regulation of circRNA₀00203 in the hypertrophic myocardium of Ang-?-infused mice and cardiac tissue of patients with heart failure.2.The cell surface of cardiomyocytes and the mRNA and protein level of the hypertrophy related gene Myh7 and Anp were significantly increased in NMVCs infected rAd-circ203.3.The cardiac function was significantly decrease,and cell size of myocardium and the mRNA and protein levels of Myh7 and Anp were significantly increased in Ang-II-infused Tg-circ203 mice.4.RAP,dual luciferase reporter assay and RNA pull-down assay confirmed that circRNA₀00203 could directly interact with miR-26b-5p and miR-140-3p.Further,overexpression of circRNA₀00203 showed down-regulated expressions of miR-26b-5p and miR-140-3p in vitro and in vivo.5.Overexpression of miR-26b-5p or miR-140-3p showed down-regulated expression of Gata4 in NMVCs.Overexpression of circRNA 000203 showed an up-regulated expression of Gata4 in vitro and in vivo.Furthermore,dual luciferase reporter assay confirmed that Gata4 could directly bind to miR-26b-5p or miR-140-3p,and circRNA₀00203 abolished the interactions of miR-26b-5p,miR-140-3p with the common target gene of Gata4.6.Phalloidin staining assay and Western Blot results demonstrated that cell size of cardiomyocytes and protein levels of ?-MHC,ANP and GATA4 were markedly increased in Ang-II-treated or circRNA₀00203-modified NMVCs but could be ameliorated after transfection with miR-26b-5p,miR-140-3p and Gata4 si-RNA,respectively.7.Dual luciferase reporter assay result confirmed that transcription factor NF-?B p65 could bind to the gene Myo9a promoter and promote the transcription activity of circRNA₀00203.Conclusion:1.CircRNA₀00203 is elevated in the hypertrophic myocardium of Ang-?-infused mice and cardiac tissue of patients with heart failure.2.CircRNA₀00203 enhances hypertrophy in vitro and in vivo.3.CircRNA₀00203 exacerbates cardiac hypertrophy via suppressing miR-26b-5p and miR-140-3p binding to Gata4.4.Up-regulation of circRNA₀00203 in Ang-II-treated NMVCs through the NF-?B pathway.In summary,our study demonstrated that circRNA₀00203 could sponge miR-26b-5p,miR-140-3p to increase GATA4 expression,contributing to enhance the cardiac hypertrophy in vivo and in vitro.We also conclude that the transcription factor NF-?B p65 participates in the up-regulation of circRNA₀00203 in cardiac hypertrophy,and identify circRNA₀00203 might be as a potential therapeutic target in heart failure.