Expression And Function Of LncRNA H19 In Myocardial Ischemia Preconditioning

Background and purpose:Myocardial ischemic preconditioning（IPC）refers to the myocardial ischemia/reperfusion injury by activating the endogenous protective mechanism of the myocardium after one or more repeated transient ischemia/reperfusion.The mechanism of action of IPC is still not fully understood.Most researchers believe that a brief ischemia can induce the production of trigger factors such as adenosine,free radicals,calcium,etc.,which are regulated by mediators such as protein kinase C（PKC）,nuclear transcription factor KB（NF-KB）,heat shock protein（HSP）and nucleolin,etc.With the in-depth research on the occurrence mechanism and clinical application of IPC,it is found that IPC is the most effective endogenous protective mechanism,which has a significant protective effect on myocardial cells,and is expected to provide a new way for the prevention and treatment of myocardial ischemia/reperfusion injury..Therefore,it is very important to study the molecular mechanism of IPC furtherly.Long noncoding RNA（Lnc RNA）is a type of noncoding RNA with a length of more than 200 nucleotides,accounting for about 98%of the total RNA,most of which are located in the nucleus or cytoplasm,and are commonly found in eukaryotic cells.Transcribe.Lnc RNAs were originally considered to be the"noise"of transcription,a byproduct of RNA polymerase II transcription,with no biological function.However,in recent years,a large number of studies have shown that Lnc RNA is a key regulator in cells.With the wide application of high-throughput screening technology and second-generation sequencing technologies,a series of studies on the role of Lnc RNAs have been carried out.More and more experiments have shown that Lnc RNAs are widely involved in cell growth,differentiation,metabolism and so on.In the cardiovascular system,Lnc RNA is closely related to the development and differentiation of the heart,the occurrence and development of cardiovascular diseases such as cardiac hypertrophy and heart failure.Lnc RNA H19 was the first to be discovered,which is located on human chromosome 11p15.5 and is a maternally imprinted Lnc RNA.The expression level of Lnc RNA H19 in cardiac tissue is about ten times higher than that in other mouse tissues.Existing studies have shown that Lnc RNA H19 is an important regulator of cardiac development and myocardial injury.However,whether Lnc RNA H19 plays a role in myocardial ischemia preconditioning and what is its specific mechanism are still unclear.Nucleolin（also known as C23）is the most abundant RNA-binding protein in the nucleolus,accounting for about 10%of the total nucleolar protein.Its main function is to participate in r RNA splicing,maturation,transport and ribosome assembly through its RNA binding domain.Nucleolin is a multifunctional shuttle protein.In addition to participating in the regulation of ribosome biosynthesis and maturation,as well as the regulation of cell proliferation,growth and other classical biological functions,nucleolin also plays an important role in viral infection,tumor development and other processes.Nucleolin is abundantly expressed in cardiomyocytes.Our previous studies have found that myocardial ischemia preconditioning can significantly up-regulate the expression of nucleolin in myocardium,and myocardial-specific overexpression of nucleolin can significantly reduce myocardial ischemia-reperfusion injury,suggesting that nucleolin is an important cardioprotective protein involved in myocardial ischemia preconditioning,but the mechanism of up-regulation of nucleolin expression in myocardial ischemic preconditioning is still unknown.Through the analysis of bioinformatics software,the possibility of interaction between nucleolin and Lnc RNA H19 is high,and further by protein-RNA co-immunoprecipitation experiment,it is found that Lnc RNA H19 can be combined with nucleolin protein.Combined with the previous research basis of this experiment and the recent experimental results,we propose that Lnc RNA H19 may combine with nucleolin protein to increase the stability of nucleolin protein,thereby upregulating the expression of nucleolin,and exerting the protective effect of myocardial ischemia preconditioning.In this experiment,an animal model of myocardial ischemia preconditioning was established by ligation of the left anterior descending coronary artery of the mouse heart,and the rat primary cardiomyocytes/rat H9C2 cell line were treated with low concentration of H₂O₂or hypoxia/reoxygenation to build the cell model of ischemic preconditioning.The expression and role of Lnc RNA H19 in myocardial ischemic preconditioning,and the effect of Lnc RNA H19 on nucleolin expression were observed.Methods:（1）Using C57BL/6 mice,the left anterior descending coronary artery of the heart was ligated for 5 min,then released to restore reperfusion for 5 min,repeated for 3-5 cycles,to prepare a mouse model of myocardial ischemia preconditioning,and q RT-PCR technology was used to detect the expression of Lnc RNA H19 in myocardial tissue.（2）The primary cardiomyocytes/rat H9C2 cell line were treated with low-concentration H₂O₂or hypoxia/reoxygenation to construct a myocardial preconditioning cell model,and q RT-PCR was used to detect Lnc RNA H19 level.（3）Gene transfection and RNA interference technology were used were used to detect the role of Lnc RNA H19 overexpression or low expression in cardiomyocytes oxidative stress or hypoxia preconditioning.（4）The expression levels of nucleolin in different myocardial ischemia preconditioning models were detected by Western-blot assay at the overall and cellular levels.（5）Gene transfection and RNA interference techniques were used to observe the effect of Lnc RNA H19overexpression or knockdown on the expression level of nucleolin in myocardial ischemia preconditioning.（6）The interaction between Lnc RNA H19 and nucleolin was verified by protein-RNA co-immunoprecipitation experiment.（7）The protein translation was blocked by cycloheximide（CHX）treatment,and the stability changes of nucleolin protein were observed.（8）Co-transfection of Lnc RNA H19overexpression vector and nucleolin RNA interference vector was used to explore the effect of nucleolin on the protective effect of Lnc RNA H19 in myocardial ischemia preconditioningResults:（1）In the mouse myocardial ischemia preconditioning model,the expression level of Lnc RNA H19 in mouse myocardial tissue increased significantly at 12h,24h,and 36h,and peaked at 24h;The expression level of Lnc RNA H19 was significantly increased after 3,4,and 5 transient ischemia/reperfusion cycles,and peaked after 4 cycles of transient ischemia/reperfusion.（2）After different cycles of transient hypoxia/reoxygenation preconditioning,the expression level of Lnc RNA H19 peaked at 3 and 4 cycles;The expression level of Lnc RNA H19gradually increased,and the expression level reached a peak at 24h after treatment.（3）After Lnc RNA H19 RNA interfered with H9C2cardiomyocytes for 72 hours,the expression level of Lnc RNA H19 was significantly decreased;CCK-8 detection showed that low concentration of H₂O₂treatment can reduce the oxidative stress-induced cardiomyocytes.However,the knockdown of Lnc RNA H19 significantly reduced the activity of cardiomyocytes and relieved the protective effect of low-concentration H₂O₂treatment;Caspase-3 activity detection kit also found that low-concentration H₂O₂treatment can reduce the caspase-3 activity caused by oxidative stress,while Lnc RNA H19 knockdown abolished the protective effect of low-concentration H₂O₂treatment.These results suggest that knockdown of Lnc RNA H19 can significantly relieve the protective effect of myocardial ischemia preconditioning.（4）After 48h transfection with the Lnc RNA H19 overexpression vector,the expression of Lnc RNA H19 was significantly increased;Low-concentration H₂O₂treatment can alleviate the myocardial cell damage caused by oxidative stress,while Lnc RNA H19 overexpression significantly increased cardiomyocyte survival rate,decreased apoptosis,and enhanced the protective effect of low-concentration H₂O₂treatment.These results suggest that Lnc RNA H19 overexpression can significantly enhance the protective effect of myocardial ischemia preconditioning.（5）After pretreatment with low concentration of H₂O₂,the expression level of nucleolin in cardiomyocytes was significantly increased,and low expression of Lnc RNA H19 could inhibit the expression of nucleolin in cardiomyocytes pretreated with low concentration of H₂O₂;while overexpression of Lnc RNA H19 could promote the expression of nucleolin in cardiomyocytes.These results suggest that the expression of nucleolin is dependent on the regulation of Lnc RNA H19 in a low-concentration H₂O₂preconditioning model of cardiomyocytes.（6）Nucleolin can interact with Lnc RNA H19 under normal conditions and myocardial ischemia preconditioning,and the interaction between them is further enhanced during myocardial ischemic preconditioning.（7）After cardiomyocytes were treated with Cycloheximide（CHX）,the overexpression of Lnc RNA H19 significantly increased the stability of nucleolin protein,but had no significant effect on the stability of the control protein Tubulin,which suggested that Lnc RNA H19 could specifically increase the stability of nucleolin protein.（8）After Lnc RNA H19 overexpression vector and nucleolin RNA interference co-transfected cardiomyocytes,it was found that nucleolin si RNA interference could eliminate the effect of Lnc RNA H19 overexpression on nucleolin expression;Nucleolin ablation could relieve the protective effect of Lnc RNA H19 on myocardial cell injury,manifested as a decrease in cell activity and an increase in the activity of Caspase-3.Conclusion:After myocardial ischemia preconditioning in mice,the expression level of Lnc RNA H19 in myocardial tissue increased significantly,reaching a peak at 24 h;Lnc RNA H19 played an important protective role in myocardial ischemic preconditioning.The enhanced interaction between Lnc RNA H19 and nucleolin protein increases the stability of nucleolin protein,thereby up-regulating the expression of nucleolin and exerting a cardioprotective effect.17 pictures,1 table,106 references...