Effect Of MiR-760-3p Inhibition On H9c2 Cell Apoptosis Induced By H₂O₂ And Its Mechanism

Background:Acute myocardial infarction is the most serious and fatal disease in the world.Reperfusion can restore the coronary blood flow of ischemic tissue,which is beneficial to reverse myocardial ischemia,and is the most effective treatment strategy.However,reperfusion therapy such as coronary intervention or drug thrombolysis itself can also cause damage to cardiomyocytes,resulting in mycardial cell apoptosis or even cardiac arrest and other serious consequences.Studies have shown that the extent of myocardial damage depends not only on the ischemia period,but also on the second wave of damage caused by reperfusion,which accounts for 50%of all damage.Although many studies have explored strategies for the treatment of myocardial ischemia-reperfusion injury and have been demonstrated in animal and cell experiments,the translation of these beneficial strategies into clinical applications has often disappointing.Studies have shown that apoptosis is the main mechanism of myocardial ischemia-reperfusion injury and inhibition of apoptosis can reduce the extent of myocardial infarction.miRNA is a small non-coding RNA molecule,which has a variety of biological functions,and through complete or incomplete complementary pairing with the 3’-untranslated region of the target mRNA,negatively regulates the expreession of the target mRNA or inhibits its translation at the post-transcriptional level,and down-regulates the expression of related proteins.The abnormal expression level of miRNA is related to the pathogenesis of a variety of heart diseases.More and more evidences show that miRNA is abnormally expressed in the process of myocardial ischemia-reperfusion injury,suggesting that miRNA is involved in the development of myocardial ischemia-reperfusion injury.In addition,PI3K/AKT is one of the strongest survival promoting signaling pathways in cells.Studies have shown that activation of PI3K/AKT signaling pathway can inhibit myocardial cell apoptosis and reduce myocardial infarction area in the process of myocardial ischemia reperfusion injury.Studies have shown that miR-26a-5p activates PI3K/AKT signaling pathway by inhibiting the expression of target gene PTEN,promoting PI3K/AKT phosphorylation,thereby inhibiting the apoptosis of cardiomyocytees,improving the activity of cardiomyocytes and playing a protective role.It was found that miR-506 was abnormally expressed in the myocardial injury rats,compared with the normal control group,the expression leve of miR-506 was decreased in the myocardial I/R injury group,and the overexpression of miR-506 could alleviate the myocardial injury through the PI3K/AKT signaling pathway.However,the role of miR-760-3p in myocardial ischemia-reperfusion injury remains unclear.H2O2 is widely used in vitro cell models to induce apoptosis during I/R injury,So,H2O2 was used to induce apoptosis of H9c2 cardiomyocytes in our study.Objective:1.To investigate the expression of miR-760-3p in H9c2 cardiomyocyte apoptosis model induced by H2O2;2.To investigate the role and mechanism of miR-760-3p in the apoptosis model of H9c2myocardial cells induced by H2O2,and to provide some experimental basis for expaoring the treatment methods of myocardial ischemia repefusion injury.Methods:H2O2 induce apoptosis of H9c2 cardiomyocytes to establish an vitro injury model.CCK8 kit was used to detect cardiomyocyte viability to determine the appropriate intervention conditions.Then Western Blot was used to detect apoptosis-related proteins and TUNEL was used to detet apoptosis,which confirmed the success of vitro modeling.The expression of miR-760-3p after H9c2 cardiomyocyte apoptosis induced by H2O2 was detected by RT-PCR.The cells were transfected with miR-760-3p inhibitor;and apoptosis was detected by Western Blot and flow cytometry.Target genes of miR-760-3p were predicted using the TargetScan7.2 online database,and the possible biological functions of miR-760-3p were speculated by GO and KEGG enrichment analysis of these target genes through the DAVID6.8 database.LY294002 was used to block the PI3K/AKT signaling pathway by inhibiting PI3K and its downstream AKT phosphorylation.Results:Compared with the normal control group,the expression of miR-760-3p was up-regulated in H2O2-induced apoptosis(P<0.05);while the expression of miR-760-3p was inhibitored,and Cleaved caspase-3 and Bax protein was decreased by Western Blot,Bcl-2 was increased,and cell apoptosis was decreased by flow cytometry(P<0.05),Compared with H2O2 group alone,p-AKT protein in H2O2+miR-760-3p inhibitor group was increased by Western Blot,and cell apoptosis in H2O2+miR-760-3p inhibitor group was increased by flow cytometry detection(P<0.05).TargetScan7.2 online database to predict miR-760-3p 347 target genes,the target genes biological process was mainly involed in cell metabolism,adhesion,phosphatidylinositol 3-kinase signal conditioning process,molecular function mainly invole cell adhesion,phosphatidylinositol-4,5-diphosphate-3-kinase activity,etc.cell localzation is mainly in nucleosome,plasma membrane,exosome,etc.KEGG signaling pathway mainly involves systemic lupus erythematosus,cancer transcriptional dysregulation,ErbB signaling pathway,phosphatidylinositol signaling pathway,etc(P<0.05).p-AKT protein detection and flow cytometry showed that compared with H2O2+miR-760-3p inhibitor intervention group,p-AKT protein decreased and cell apoptosis increased in H2O2+miR-760-3pinhibitor+LY294002 combined intervention group(P<0.05),suggesting that LY294002 inhibitor may block the anti-apoptosis effect of miR-760-3p inhibitor.Conclusions:miR-760-3p expression is up-regulated in H2O2-induced apoptosis,which is harmful to cells,while inhibition of miR-760-3p expression is beneficial to cells and may reduce cell apoptosis through activation of the PI3K/AKT pathway.