Mechanism Of LncRNA SOX2-OT Regulating METTL3 In Pathological Cardiac Hypotrophy

Objective: It has been reported that the expression of lncRNA SOX2-OT is increased in heart failure,but it is not clear whether SOX2-OT is involved in pathological cardiac hypertrophy.METTL3 has the function of promoting pathological cardiac hypertrophy,but the specific mechanism still to be further tapped.The purpose of this study is to identify whether lncRNA SOX2-OT is involved in the regulation of pathological cardiac hypertrophy through the neonatal rat model of ventricular hypertrophy,and to explore whether METTL3 is involved in the process of lncRNA SOX2-OT regulating pathological cardiac hypertrophy and its possible mechanism.Methods: 1.The pathological cardiac hypertrophy models of neonatal rat ventricular myocytes(NRVMs)and H9c2 cardiomyocytes were induced by isoproterenol(ISO)stimulation,and RT-q PCR and Western blot were applied to detect the expression level of related genes.2.SOX2-OT overexpression vector plasmid,Let-7g-3p mimic and si RNA of SOX2-OT and METTL3 were used to change the gene expression of cardiomyocytes,and RT-q PCR and Western blot were applied to detect the expression level of related genes.3.The relative surface area of cardiomyocytes in each group was observed by phalloidine immunofluorescence experiment.4.Bioinformatics methods were used to predict the interaction between Let-7g-3p and SOX2-OT,Let-7g-3p and METTL3 and the subcellular localization of SOX2-OT.5.The interactions between Let-7g-3p and METTL3,Let-7g-3p and SOX2-OT were verified by dual-luciferase reporter assay.Fluorescence in situ hybridization and immunofluorescence assays(FISH)were applied to verify the subcellular localization of SOX2-OT.Results: 1.The analysis of GSE77399 datasets in GEO database showed that SOX2-OT was overexpressed in heart failure myocardium,and the results of pathological cardiac hypertrophy in neonatal rat ventricular myocytes(NRVMs)showed that the expression of SOX2-OT was up-regulated in pathological cardiac hypertrophy.2.Pathological cardiac hypertrophy experiments of SOX2-OT overexpression showed that up-regulation of SOX2-OT could induce and promote pathological cardiac hypertrophy,while pathological cardiac hypertrophy experiments of SOX2-OT knock-down showed that SOX2-OT knock-down could inhibit pathological cardiac hypertrophy.3.Dual-luciferase reporter assays showed that 3’UTR of METTL3 m RNA was the specific binding and action target of Let-7g-3p,and there was a specific binding target of Let-7g-3p on lncRNA SOX2-OT.4.Let-7g-3p phenotypic rescue assays showed that Let-7g-3p mimics could partially reverse the pathological cardiac hypertrophy phenotype caused by overexpression of SOX2-OT,which confirmed that Let-7g-3p was a downstream of SOX2-OT in the function of promoting pathological cardiac hypertrophy.5.METTL3 phenotypic rescue assays showed that knocking down the expression level of METTL3 could partially reverse the pathological cardiac hypertrophy phenotype caused by SOX2-OT overexpression,which confirmed that METTL3 was a downstream of SOX2-OT in the function of promoting pathological cardiac hypertrophy.Conclusion: Overexpression of SOX2-OT can induce pathological myocardial hypertrophy.The specific mechanism is that SOX2-OT promotes the expression of METTL3 by sponging Let-7g-3p,while METTL3,as a downstream molecule of SOX2-OT,can rescue the function of SOX2-OT in promoting pathological myocardial hypertrophy.