The Effect And Mechanism Of LncRNA H19 In Hypoxia Postconditioning To Senescent Cardiomyocytes

Background:Ischemic postconditioning(I/Post)is an endogenous protection mechanism that reduces injury induced by ischemia-reperfusion(I/R).It remains controversial whether I/Post protects against I/R injury to the aging heart.The long non-coding RNA,H19 protects H9c2 cells against hypoxia-induced injury.In clinical and basic studies,dexmedetomidine plays an important part in myocardial protective effect,whose function and mechanism in aged cardiomyocytes ischemia-reperfusion injury still need to be further studied.RNA N6-methyladenosine(m6A)is the most abundant endogenous RNA modification.The aim of this study was to elucidate the role of H19 in the hypoxic postconditioning(H/Post)and dexmedetomidine postconditioning of aged cardiomyocytes,detect the level of RNA m6 A modification on Ipost or dexmedetomidine postconditioning and the expression of RNA m6A modified regulatory genes.To explore the mechanism of RNA m6A modified regulatory gene regulating the expression of ncRNA H19 and its role in the H/post.Methods:Senescence induced by D-galactose in primary cardiomyocytes from neonatal Sprague-Dawley rats was measured by senescence-associated ?-galactosidase staining.Hypoxic injury was evaluated by cell viability and apoptosis assays.H19 expression before and after hypoxia-reoxygenation(H/R)and H/Post was evaluated by real-time polymerase chain reactions.MicroRNA binding sites in H19 and target gene were predicted by bio informatics analysis,and interaction was verified by co transfection of microRNA mimics and inhibitor with luciferase reporter constructs.The effects of altered H19,micro RNA and target gene expression on the viability and apoptosis of senescent cardiomyocytes following H/Post were determined.Hydrogen peroxide(H2O2)was used to induce the senescence of myocardial cell line H9c2.After 6h of hypoxia,H9c2 was treated by different final concentration of dexmedetomidine(0,500nM,1?M and 2?M)for 6h.Then detect the damage of myocardial cells.After overexpression or knockdown of H19 and its downstream genes,dexmedetomidine postconditioning experiments were performed and we detect myocardial cell injury.The global m6A levels in the H/R,H/post and dexmedetomidine postconditioning were measured by colorimetric m6ARNA Methylation Quantification Kit,and the RNA m6A methylation regulation genes mediating H19 expression were screened.The mechanism of its regulation of H19 expression and its function in the H/Post and dexmedetomidine postconditioning of aged myocardial cells were further verified by m6A RNA immunoprecipition(MeRIP).Results:H/Post prevented H/R injury in normal but not senescent cardiomyocytes.H19 expression was remarkably down-regulated after H/Post in senescent compared with normal cardiomyocytes.Small interfering RNA-mediated knockdown of H19 in senescent cardiomyocytes increased H/Post-induced injury.miR-29b-3p was regulated by H19 and led to a greater injury.miR-29b-3p directly targeted the 3' untranslated region(3'-UTR)of cIAP1 and suppressed its expression.Furthermore,knockdown cIAPI damaged senescent cardiomyocytes following H/post.Dexmedetomidine had the beneficial effects on anti-hypoxic reoxygenation injury of aged myocardial cells through H19/miR-29b-3p/cIAPl.H/Post and dexmedetomidine postconditioning reduced the increased methylation of RNA m6A caused by H/R.RNA demethylase gene AKLBH5 regulate the expression of H19,and played an important role in the H/R injury of aged cardiomyocytes.Conclusions:The H/R injury in aged cardiomyocytes prevented by dexmedetomidine postconditioning,but not by H/post.H19 mediated the antiapoptotic effect of H/R-induced injury following H/Post by inhibiting miR-29b-3p/cIAPl and was regulated by the RNA m6A demethylase,ALKBH5.Dexmedetomidine postconditioning plays a protective effect by regulating ALKBH5 in senescence cardiomyocytes.