The HPC-BMSC Derived Exosomal LncRNA LOC100360491 Inhibits Autophagy And Apoptosis In Myocardial Ischemia-reperfusion Injury By Targeting MiR107-5p/PAK3 Axis

Background: Morbidity and mortality of the ischemic cardiomyopathy at home and abroad also significantly increased,under the control of Acute Myocardial Infarction(AMI)by Percutaneous Transluminal Coronary Intervention(PCI).The process of PCI can itself induce cardiomyocyte injury,even cell death.We call it myocardial ischemic-reperfusion injury,for which there is still no effective therapy.Following PCI,researchers have detected cell injury and death in reperfused heart of dog.The research about Myocardial ischemic-reperfusion injury focus on inflammation,Oxidative stress,calcium overload,autophagy and apoptosis.Autophagy and apoptosis take main position in Myocardial ischemic-reperfusion injury,as knowing that inflammation,Oxidative stress and calcium overload can damage cell morphology and function via regulating autophagy and apoptosis.Autophagy is a cellular recycling process by degrading damaged or senescent cytoplasmic organelles,proteins.The active and gene-regulated cell death,apoptosis aimed at adapting to the microenvironment.Accumulating evidence suggests that autophagy and apoptosis are important in the regulation of Myocardial ischemic-reperfusion injury.As the ideal stem cell therapy strategy,Bone Marrow Mesenchyml Stem Cell(MSC)which can significantly improve the AMI has already entered the clinical research stage.With the deepening of research,we know that MSC mediated its cardioprotective effect by paracrine mechanism.Compared to MSC,exosomes without being affected by the ischemic microenvironment have more stable performance and provided cytoprotective effects,such as antiapoptosis,antifibrosis and promote angiogenesis.In 2019,Qin discoveried that AMI active monocyte,secrete and migrate exosomes to injury area.The exosomes could repair cardiac function.In previous study,we found that MSC exogenous miR-19 b via exosomes to protect H9c2 cardiomyocytes against hypoxia/reoxygenation-induced autophagy and apoptosis.However,as the upstream molecule,the roles of LncRNA in Myocardial ischemic-reperfusion injury is unknown.This study investigate the role and molecular mechanism of novel lncRNA screened by transcriptome microarray in ischemia-reperfusion injury.Part ? Screening and Functional studies of LOC100360491 which is a New regulatory molecule of ischemia-Reperfusion Objective: H9c2 myocardial cell were subjected to hypoxia/reoxygenation(HR).Using gene microarray Screen and Investigate LncRNA which is associated with apoptosis and autophagy out of the H9c2.Methods:(1)We establish the apoptosis model of H9c2 after hypoxia 24 h and reoxygenation 6h(HR).We compared HR group with Normal group using LncRNA microarray to find LncRNA,and then detected the result using RT-PCR.(2)Co-culturring HPC-BMSC derived exosomes(HPC-Exo)with H9c2 for12 h estabilish the HPC-Exo group.We compared HPC-Exo group with HR group using LncRNA microarray to find LncRNA,and then detected the result using RT-PCR.(3)We analyzed microarray data using GO/pathway,coexpression net work,and KEGG.In vitro,we overexpression/knock down LncRNA by transfecting lentivirus into H9c2 to observe the role of LncRNA about apoptosis and autophagy.Results:(1)According significance analysis(fold changed>2,p<0.05),we found that375 LncRNAs,1398 mRNA have differential expression.Among them,308 LncRNAs and 1154 mRNA were up-regulation.Others were down-regulation.GO analysis found that up-regulated mRNA enriched in regulation of multicellular organismal process,cell-cell signaling,ion transport.Down-regulated mRNA enriched in cellular metabolic process,RNA binding,protein binding.KEGG analysis found that up-regulated mRNA enriched in Complement and coagulation cascades,Glutamatergic synaps,Calcium signaling pathway,Inflammatory mediator regulation of TRP channels.Down-regulated mRNA enriched in Protein processing in endoplasmic reticulum,Cell cycle,RNA transport and Oxidative phosphorylation.Compared with HR group,HPC-Exo group have 840 LncRNAs and 1937 mRNAs up-regulated,340 LncRNAs and 801 mRNAs down-regulated.About biological processes up-regualted mRNA enriched in Cellular metabolic process,Cellular nitrogen compound metabolic process,and Gene expression and so on.About cell components,up-regulated mRNA enriched in Intracellular,Intracellular part,Organelle and so on.About molecular function,up-regulated mRNA enriched in Binding,Protein binding,Heterocyclic compound binding and so on.On the contrtrary,down-regulated mRNAenriched in Response to organic substance,Endoplasmic reticulum and Protein domain specific binding.Pathway analysis founf that up-regulated mRNA enriched in Ribosome,Oxidative phosphorylation,down-regulated mRNA enriched inPI3K-Akit signal pathway.Using coding-non-coding gene co-experssion network(CNC network)we found that mRNASmndc1,Mef2 d,Bnip1,Bad,Aqp2,Xbp1,Rack1,Plekhf1,Hif3 a may have relation withapoptosis.LncRNA XR-351145,XR-343601,XR-599858,XR-344466,XR-594628,XR-594627 and so on have direct relation with them.(2)Using RT-PCR to detected the microarray datas,RGD1563905,AABR06065797.1,AABR06096630.1 and AABR06033510.1.The results of RT-PCR were the same as microarray data.(3)In conclusion,LOC100360491 and RGD1563905 were candidate LncRNA.Combine with the result of RT-PCR in HR group,Normal group and HPC-Exo group,we found LOC100360491 being target LncRNA.Then,we OE/shLOC100360491 by transfecting lentivirus into H9c2 to observe the early apoptosis,the delivery of reactive oxygen species(ROS)and the Fragmentation of DNA.The result shows,compairing with other groups,in the OELOC100360491 group,the early apoptosis,the delivery of ROS and the Fragmentation of DNA has been significantly decreased.At the protein level,compairing with HR group,In the HPC-Exo Group,actived-caspase3,actived-caspase9,Bcl-2,LC3?/? has been significantly decreased;Bax,p62 significantly been increased.However,shLOC100360491 in HPC-BMSC derived exosomes,actived-caspase9 has been no significant change,actived-caspase3,Bcl-2 have been significant up-regulation compairing with HPC-Exo group.Bax and p62 have been down-regulated in HPC-Exo+shLOC100360491.Conclusion: Under hypoxia reoxygenation,LOC100360491 which comes from HPC-BMSC derived exosomes has the function of anti-apoptosis and anti-autophagy.Part ? The HPC-BMSC derived exosomal LncRNA LOC100360491 inhibits autophagy and apoptosis in myocardial ischemia-reperfusion injury by targeting miR107-5p/PAK3 axisObjective: Investigating the molecular mechanism of LOC100360491 about anti-apoptosis and anti-autophagy in H9c2.Methods: Detected the subcellular localization of LOC100360491 in H9c2 cells by Fluorescence in situ hybridization(FISH);Obtained full-length sequence of LOC100360491 by 3',5'RACE;Analyzed targets of LOC100360491 by Biological Informatics Analysis and Dual Luciferase Reporter Assay(DLRTM);Detected apoptosis by Flow cytometry(FCM),transcription by RT-PCR;Proteins were detected by Western Blot(WB).Results:(1)FISH shows LOC100360491 were located in the cytoplasm of the H9c2 cardiomyocytes.We found 7 target genes including miR103-1-5p,miR107-5p,miR125a-5p,miR125b-5p,miR351-5p,miR423-5p,miR3573-5p of LOC100360491 by bioinformatics.RT-PCR shows that the miRNAs were down-regulated in OELOC100360491 group.MiR103-1-5p,miR107-5p and miR125b-5p were up-regulated in shLOC100360491.All of them,miR107-5p has the most significant difference.We guessed that LOC100360491 maybe connected with miR107-5p.RT-PCR shows PAK3 and CNNM2 have no statistic difference in each groups.WB shows CNNM2 has no statistic difference in each groups.However,PAK3 was down-regulated in miR107-5p mimic group,up-regulated in miR107-5p inhibitor group.DLRTM shows LOC100360491-miR107-5p-PAK3 has direct binding site.(2)To investigate the role of LOC100360491-miR107-5p in H9c2 cardiomyocytes which were subjected to Hypoxia reoxygenation,we transfected miR107-5p mimic plasmid,inhibitor plasmid into H9c2 cardiomyocytes.The results show that apoptosis were significantly decreased in miR107-5p inhibitor group,significantly increased in miR107-5p mimic group.Comparing with HPC-Exo,effects of anti-apoptosis in HPC-Exo+miR107-5p mimic has significantly decreased.WB shows that actived-caspase3,Bax have significantly up-regulated in HR group.In HPC-Exo group,they were down-regulated.Activedl-caspase3,Bax has significantly difference.Bcl-2 was opposite.About autophagy,compairing with HR groups,in HPC-Exo group,LC?/? was down-regulated,in HPC-Exo+miR107-5p mimic groups LC3?/? up-regulated again.P62 was opposite.Conclusion: LOC100360491 anti-apoptosis and anti-autophagy through binding miR107-5p liking Sponge.Part ? The HPC-BMSC derived exosomal LOC100360491 inhibits autophagy and apoptosis in rat myocardial infarction Objective: Verifying roles of LOC100360491 in rat AMI model.Methods: ShLOC100360491 and shRNA NC by intramyocardial injected lentivirus.Ligating left anterior descending ramus of the rat heart to establish AMI model.Detected infarct area by TTC,myocardial fibrosis by HE,expression of proteins associating with apoptosis and autophagy by WB.Results: Compairing with sham group,infarct area was significantly increased in MI group.Compairing with MI group,infarct area was significantly decreased in HPC-Exo group.shRNA NC has no significance.Actived-caspase3,Bax,LC3?/? were up-regulated significantly in MI group,Bcl-2 and p62 were down-regulated significantly in MI group.However,Compairing with HPC-Exo group,cleaved-caspase3,Bax,LC?/? were up-regulated significantly in shLOC100360491 group.Bcl-2 and p62 were down-regulated in shLOC100360491 group.shRNA NC group has no significance.Conclusion: LOC100360491 has the function of anti-apoptosis and anti-autophagy in rat AMI models.