Mechanism Study Of Circular RNA Circ＿0000994-translated Novel Protein SLC8A1-605aa Inhibiting Cardiac Hypertrophy

Cardiac hypertrophy is a compensatory adjustment of the heart in response to stress or volume overload to maintain the original pumping function.Prolonged pathological myocardial hypertrophy can eventually lead to heart failure.Further study on the molecular regulatory mechanisms of myocardial hypertrophy can provide scientific data for the study of intervention targets of myocardial hypertrophy.In recent years,the regulatory role of non-coding RNA in cardiovascular diseases has attracted extensive attention.More and more studies have shown that circular RNA can play a biological role in a variety of ways,including by encoding proteins.Our previous work has shown that the expression of circ₀000994 was significantly upregulated in the myocardium of patients with heart failure.The online database circBank predicted that circ₀000994 might contain two internal ribosome entry sites（IRES）and an open reading frame（ORF）.It is suggested that circ₀000994 may have proteincoding potential,but its regulatory effect and mechanism on myocardial hypertrophy remain unclear.Objective:To investigate the effect of circ₀000994 on cardiomyocyte hypertrophy and the molecular mechanism involved.Methods:1 The expression of circ 0000994 in hypertrophic myocardium1-1 The expression of circ₀000994 and its host gene SLC8A1 was detected in the myocardium of healthy organ donors（n=17）,and patients with heart failure（n=27）by RT-qPCR assay.1-2 Distribution and identification of RNA stability of circ₀000994 in human cardiomyocytes.2 The regulating role of circ 0000994 in the hypertrophy phenotype of cardiomyocytes.2-1 The expression of cardiac hypertrophy-related genes was investigated in NMVCs infected with rAd-circ₀000994.2-2 Phalloidin staining assay was performed to detect the effect of overexpression of circ₀000994 on Ang II-induced hypertrophy of NMVCs.3 Molecular mechanism of circ₀000994 inhibiting cardiomyocyte hypertrophy.3-1 The luciferase activity of the IRES in circ₀000994 was verified by dual-luciferase reporter assay.3-2 The characteristic peptide sequences of SLC8A1-605aa translated by circ₀000994 were identified by mass spectrum shot-gun analysis.3-3 The influence of SLC8A1-605aa on the expression of cardiac hypertrophy-related genes and the hypertrophy phenotype of cardiomyocytes were detected in NMVCs with over-expression of circ₀000994,circ₀000994-ORF,and circ₀000994-ATGmut.34 NMVCs with over-expression of circ₀000994 were transfected by SLC8A1-605aa siRNA to explore the effect of SLC8Al-605aa on suppression of cardiac hypertrophyrelated genes and cardiomyocyte hypertrophy phenotype by circ₀000994.3-5 Distribution of SLC8A1-605aa in cardiomyocytes and analysis of RNA-seq expression profile;3-6 The influence of SLC8A1-605aa and SOD2 on the expression of cardiac hypertrophy-related genes and NF-κB signal were detected in NMVCs with over-expression of circ₀000994,circ₀000994-ORF,and SOD2.Results:1 The expression of circ₀000994 and its host gene SLC8A1 was significantly increased in the myocardium of patients with heart failure.Circ₀000994 has high stability;circ₀000994 is mainly located in the cytoplasm of cardiomyocytes.2 Overexpression of circ₀000994 inhibited the expression of cardiac hypertrophy-related genes,including Myh7,Actal,and Nppa in NMVCs.Circ₀000994 suppressed Ang Ⅱ-induced NMVCs hypertrophy.3 The results of the dual-luciferase reporter gene experiment suggested that the IRES contained in circ₀000994 could mediate protein translation.The results of mass spectrum analysis revealed that circ₀000994 could translate SLC8A1-605aa protein.Over-expression of circ₀000994 and SLC8A1-605aa could consistently inhibit the expression of cardiac hypertrophy-related genes and hypertrophy phenotype in NMVCs.Knock-down of the expression of SLC8A1-605aa could attenuate the inhibitory effect of circ₀000994 on the expression of cardiac hypertrophy-related genes and hypertrophy phenotype in NMVCs.SLC8A1-605aa is mainly located in the nucleus of cardiomyocytes;SLC8A1-605aa could inhibit the expression of cardiac hypertrophyrelated genes by up-regulating superoxide dismutase 2（SOD2）and inhibiting the activation of NF-kB signal in cardiomyocytes.Conclusion:Circular RNA circ₀000994 inhibits cardiomyocyte hypertrophy by encoding SLC8A1-605aa protein.