Long Noncoding RNA AK12348 Is Involved In The Regulation Of Myocardial Ischemia-reperfusion Injury By Targeting PARP And Caspase-3

Background:Although intravenous thrombolysis and precutaneous coronnary artery lumen keratoplasty coronary artery stent implantation,such as heart reperfusion therapy,can significantly improve the survival rate of patients,but ischemic myocardial blood perfusion in the recovery process,appears reperfusion injury(ischemia reperfusion assiciated,IRI).The mechanism of myocardial ischemia reperfusion injury is not yet fully understood,but how to prevent and control the reperfusion injury has become the hotspot in research of cardiovascular.Small RNA(microRNA,miRNA)is a kind of endogenous non coding small molecule RNA with gene regulatory function.It can be combined with the target messenger RNA(message RNA,mRNA)by identifying the non coding region of the 3'terminal(3'-uncoding region,3'UTR),thus degrading the target mRNA or repressing the target mRNA translation and reducing the protein expression level.In the non coding RNA,which occupies 98.5%of the whole human genome,most of the RNA molecules that are transcribed more than 200nt,which are called long non-coding RNA(LncRNA).LncRNA is involved in the regulation of important life processes such as cell differentiation and ontogeny at various levels,and is closely related to major human diseases.The results of GSE50378 chip data analysis were found that the expression of LncRNA-AK1234833 gene was significantly up-regulated in A/R cardiomyocytes.Therefore,it is believed that there is a close relationship between LncRNA-AK1234833 and the process of myocardial ischemia reperfusion injury,but how to participate in the mechanism of myocardial ischemia reperfusion injury is the concern of this study.We aim to construct a model of hypoxia/reoxygenation by using rat cardiomyocytes(H9c2)to study the interaction of LncRNA-AK1234833 and myocardial ischemia reperfusion related genes(PARP and caspase-3),and to clarify the regulation mechanism of LncRNA-AK1234833 in myocardial ischemia-reperfusion injury.Methods:EdgeR bioconductor package was used to screen differentially expressed IncRNAs in myocardial IRI,and IncRNAAK123483 was selected.The mRNA levels of lncRNA AK123483 in normal and anoxia/reoxygenation(A/R)cardiomyocytes were determined by qRT-PCR.After transfection with siRNA-lncRNAAK123 4 83,LDH release and cell apoptotic rates in normal and A/R cardiomyocytes were determined.The protein expression values of PARP(Poly ADP-ribose Polymerase)and Caspase-3 were also determined by western blotting.Results:In this study,we screened the differentially expressed lncRNAs in GSE50378 and GSE22282 which were downloaded from GEO,and the top 26 differentially expressed genes were obtained.LncRNA AK123483 was the most expressed gene in GSE50378 and selected for the further study.The relative level of lncRNAAK123483,LDH release and cell apoptotic rate in A/R cardiomyocytes was significantly higher than that in normal cardiomyocyte.After transfection with siRNA-LncRNA-AK 123483,LDH release and cell apoptotic rates in A/R cardiomyocyteswere reduced,while the values in normal cardiomyocytes had almost no change.Lactate dehydrogenase(LDH)has long been considered a useful clinical marker of intravascular haemolysis.It exists in sera of cancer patients and is taken as a marker for prognosis for patients with pancreatic carcinoma,osteosarcoma,renal or testicular carcinoma.LDH-5 could catalyse the reversible transformation of pyruvate to lactate,which plays a key role in the anaerobic cellular metabolism.In non-small-cell lung cancer,it is associated with tumour hypoxia,angiogenic factor production and poor prognosis.Our study showed AR enhanced LDH release and knockdown of IncRNAAK123483 reduced LDH release.Then,The protein expression values of PARP(Poly ADP-ribose Polymerase)and Caspase-3 were also determined by western blotting.The results of western blotting showed AR increased the protein expression levels of PARP and Caspase-3.The protein expression values of PARP and Caspase-3 in A/R cardiomyocytes were much higher than the Control.After transfection with siRNA-lncRNA AK123483,the level decreased.PARP(Poly ADP-ribose Polymerase)is responsible for post-translational modification of proteins in response to numerous endogenous and environmental genotoxic agents.It is a conserved nuclear protein that binds rapidly and directly to both single-and double-strand breaks.It constitutes a family of enzymes involved in the regulation of many cellular processes such as DNA repair,gene transcription,cell cycle progression,cell death,chromatin functions and genomic stability.PARP-1 is involved in DNA repair and activated by DNA damage.Inhibition of PARP-1 could increase mitochondrial metabolism through activating SIRT1.Apoptosis has gained central importance in the study of many biological processes,including neoplasia,neurodegenerative diseases,and development.Caspase-3 has been identified as a key protease in the execution of apoptosis.During programmed cell death,activation of Caspase-3 leads to proteolysis of DNA repair proteins,cytoskeletal proteins,and the inhibitor of Caspase-activated deoxyribonuclease,culminating in morphologic changes and DNA damage defining apoptosis.Caspase-3 could activate ROCK-1 which plays an essential role in cardiac myocyte apoptosis.Conclusion:In conclusion,the current results suggest that the upregulation of lncRNA AK123483 expression contributes to myocardial ischaemia-reperfusion injury.Knockout of LncRNA-AK1234833affects the expression of PARP and caspase-3 genes,thereby restoring and protecting damaged myocardial cells induced by ischemia reperfusion.This may provide a promising approach to treating patients suffering from heart disease.In this study,the knockout of LncRNA-AK123483 gene in the A/R treatment group significantly decreased the apoptosis rate of cardiomyocytes,but the knockout of LncRNA-AK123483 in the control group had no significant effect on the apoptosis of myocardial cells.Therefore,the LncRNA-AK123483 gene may be involved in the induction of apoptosis pathway by external stress factors such as hypoxia/reoxygenation,and not participate in the process of programmed cell death.However,the present study has limitations.In this study,we only explored the role of lncRNA AK123483 on cardiomyocyte death and apoptosis,the effects of lncRNA AK123483 on cardiomyocyte migration also need be explored.A number of important molecular markers were also found in this study,such as Ki-67,Caspase 6,Bax,Bcl-2,Bax-X1.In our future study,we will conduct functional verification on the above molecular markers and analyze their role in the occurrence of ischemia reperfusion injury.The experimental results show that long non-coding RNAs AK123483 could be potential therapeutic targets for the treatment of myocardial IRI.