Circular RNA CircRNA₀05647 Inhibits Cardiomyocyte Hypertrophy Via Sponging MiR-99b-5p

Cardiac hypertrophy is the adaptive response of the heart to various physiological and pathological factors such as congenital or acquired environment.When the heart is decompensated,cardiovascular adverse events such as heart failure,arrhythmia,and even sudden death will occur.CircularRNA?circRNA?is a type of single-stranded closed-loop non-codingRNA.It is an important way of epigenetic regulation and its mechanism involved in cardiac hypertrophy has not been elucidated.Objective: To investigate the effect of circRNA₀05647 on the expression of cardiac hypertrophy associated genes in mouse cardiomyocytes and mechanism involved.Methods: 1.Ang-II capsule pump?1.46 mg/kg/day for 2 weeks?was used to establish a mouse model of cardiac hypertrophy in C57BL/6 mice.2.Primary isolation and culture of neonatal mouse ventricular cardiomyocytes?NMVCs?from C57BL/6 mice at 0 to 3 days of age,induced by 50nmol/L Ang-II for cell model.3.Expression of cardiac hypertrophy associated genes,circRNA₀05647 and Myo9 a in the myocardium of Ang-II-infused mice and in Ang-II-treated NMVCs was detected by Western blot andRT-qPCR assay,respectively.4.Actinomycin D treatment andRNaseR exonuclease digestion were performed to test the stability of circRNA₀05647 in NMVCs.5.Detected the distribution of circRNA₀05647 in NMVCs by separation of cytoplasmic and nuclearRNA components.6.Construction the recombinant adenovirus of circRNA₀05647?r Ad-circRNA₀05647?.7.Over-expression of circRNA₀05647 was achieved in NMVCs with infection of r Ad-circRNA₀05647.The expression of cardiac hypertrophy associated genes,circRNA₀05647 and Myo9 a was detected in NMVCs byRT-qPCR and Western blot assay,respectively.8.Dual luciferase reporter assay andRNA pull-down assay were performed to identify the interaction between circRNA₀05647 and miR-99b-5p.9.The expression of cardiac hypertrophy associated genes in NMVCs transfected with miR-99b-5p,byRT-qPCR and Western blot assay,respectively.Results: 1.CircRNA₀05647 was up-regulated in the myocardium of Ang-II-infused mice and in Ang-?-treated NMVCs.2.CircRNA₀05647 was more stable than its host gene of Myosin IXA?Myo9a?when it was subjected to actinomycin D andRNaseR exonuclease treatment,respectively.3.The expression of hypertrophy associated genes was down-regulated in NMVCs with over-expression of circRNA₀05647.4.circRNA₀05647 located in the cytoplasm of NMVCs.5.Dual luciferase reporter assay andRNA pull-down assay revealed the interaction between miR-99b-5p and circRNA₀05647.6.MiR-99b-5p could enhance the hypertrophic phenotype,but reversed the inhibitory effect of circRNA₀05647 on the expression of hypertrophy associated genes in NMVCs.Conclusions: 1.CircRNA₀05647 is up-regulated in cardiac hypertrophy.2.CircRNA₀05647 inhibits the expression of hypertrophy-related genes in NMVCs.3.CircRNA₀05647 plays a role in inhibiting cardiac hypertrophy through sponging miR-99b-5p in NMVCs.