Effects And Mechanism Of LncRNA LOC102552184/miR-146a-5p/TRAF6 Pathway In Coronary Microembolization-Induced Myocardial Injury

Coronary microembolization(CME)is commonly seen in the spontaneous erosion and rupture of coronary artery atherosclerotic plaques,as well as in the cases of plaque fragmentation during Percutaneous Coronary Intervention(PCI).PCI is the preferred reperfusion strategy for Acute Coronary Syndrome(ACS).However,even after successful PCI,the symptoms of angina may still exist,and CME may be an important potential cause.The prevention and treatments in CME are mainly drug treatment and device protection,with the purpose of reducing the myocardial microinfarct area and reducing the ischemia reperfusion injury.However,whether it can prevent the formation of microembolus,reduce the occurrence of microinfarcts and reduce the microvascular dysfunction still needs further research.Our previous study found that the expression levels of myocardial apoptosis and inflammation-related proteins were significantly increased in the myocardial microinfarction area of CME rats,suggesting that apoptosis and inflammation were involved in the myocardial injury caused by CME.The autophagy level of myocardium decreased significantly after CME.After using the autophagy inhibitor 3-MA,the autophagy level decreased,but the apoptosis of myocardial cells increased significantly,indicating that autophagy plays a protective role in the environment of CME.Since the beginning of the 21 st century,with the emergence of the new generation sequencing and chip technology,the research on the molecular mechanism of non-coding RNA(nc RNA)involved in the pathophysiological conditions of cardiovascular diseases has become a hot spot.Micro RNA(miRNA)is one of the most widely studied non-coding RNA.Our previous study also discussed the role of miRNA in the regulation of myocardial injury caused by CME.Long non-coding RNA(lncRNA)is located in the nucleus or cytoplasm of cells and studies have shown that they can regulate m RNA expression levels in various ways.However,the specific role of lncRNA in myocardial injury caused by CME has not been clear.In particular,whether lncRNA plays a regulatory role in cardiac autophagy after CME has not been reported.In this study,lncRNA LOC102552184 with significant expression changes was selected through high-throughput gene arrays technology,and then through bioinformatics database comparison,miR-146a-5p/TRAF6 was predicted as the potential target of LOC102552184.Our preliminary experiments of RT-qPCR results showed that LOC102552184 and TRAF6 expression was significantly increased after CME,while miR-146a-5p decreased,so we hypothesized that inhibition of LOC102552184 expression could promote cardiomyocytes autophagy,inhibit apoptosis and inflammation of cardiomyocytes,and thereby improve cardiac function.Can lncRNA LOC102552184 targeting inhibit miR-146a-5p/TRAF6,thereby promote autophagy,reduce apoptosis and inflammation,and improve myocardial injury caused by CME? In order to further confirm this hypothesis,cardiomyocyte culture and CME SD rat model were performed for providing a new therapeutic target of precision medicine in CME.Part 1: Effects of LOC102552184 on Myocardial Injury Induced by Coronary MicroembolizationObjective: The aim of this study is to establish the role of lncRNA LOC102552184 in myocardial injury induced by CME in rats and the effects of LOC102552184 on apoptosis,inflammation and autophagy in myocardium after CME.Methods: Random allocation was taken to divide 30 SD rats into 5 groups averagely:Sham group,CME group,CME+AAV-control group,CME+AAVLOC102552184 group and CME+AAV-sh RNA-LOC102552184 group.The CME+AAV-control group,CME+AAV-LOC102552184 group and CME+AAV-sh RNA-LOC102552184 group were administered with adeno-associated virus(AAV)vector,lncRNA LOC102552184 overexpression plasmid and sh RNA-LOC102552184 AAV sequences by tail vein respectively.After 3weeks the CME animal models were set up by clipping aorta and injecting 45?m polyethylene microspheres into the left ventricle of the SD rats,while the Sham group was injected with the same dose of normal saline.After 9 hours the cardiac function was measured by Echocardiography,and the left ventricular myocardium specimens were taken later.TUNEL staining technique was used to detect the myocardial apoptotic index.Autophagy was evaluated using Transmission electron microscopy(TEM).RT-qPCR and western blot were used to evaluate the relative expression levels of LOC102552184,apoptosisrelated proteins(Bax,Cleaved caspase-3,Cleaved caspase-8,Cleaved caspase-9and BCL-2),autophagy-related proteins(LC3?,Beclin-1,ATG5 and p62),and inflammation-related proteins(IL-1?,TNF-? and NF-k B).Results:1.Echocardiography results showed that the cardiac function of the CME group was significantly decreased compared with the Sham group.When lncRNA LOC102552184 was overexpressed after CME,the cardiac function was decreased while the cardiac function was improved when inhibiting the expression of LOC102552184.2.The TUNEL staining of myocardium section results showed that compared with the CME group,the cardiomyocyte apoptosis index was increased significantly after overexpressing LOC102552184,while the cardiomyocyte apoptosis index was significantly reduced when inhibiting the expression of LOC102552184,.3.Transmission electron microscope(TEM)demonstrated that the presence of autophagosome was decreased when overexpression of LOC102552184 and the presence of autophagosome was increased when inhibiting the expression of LOC102552184.4.The results of Western blot showed that after overexpression of LOC102552184,the expression level of apoptosis-related proteins Bax,Cleaved caspase-3,Cleaved caspase-8 and Cleaved caspase-9 increased significantly,while the level of Bcl-2 protein decreased significantly.In addition,the relative expression level of autophagy-related proteins LC3 II,Beclin-1 and ATG5 protein reduced,while the level of p62 protein increased significantly.At the same time,the expression level of inflammation-related proteins IL-1 ?,TNF-?,NF-k B increased significantly.On the contrary,inhibition of LOC102552184 expression can reduce CME-induced myocardial apoptosis and inflammation,and promote autophagy.Conclusion: LOC102552184 promotes CME-induced myocardial injury via aggravating inflammation,apoptosis and inhibiting autophagy.Part 2: Effects of lncRNA LOC102552184 on Cardiomyocyte Injury Induced by Ischemia and HypoxiaObjective: The aim of this study is to investigate the effects of lncRNA LOC102552184 on cardiomyocyte injury induced by ischemia/hypoxia(IH)via establishing a IH model in neonatal primary cardiomyocytes.Methods: Neonatal primary cardiomyocytes were cultured in vitro and the9 hours after exposed to IH was the time point for investigating.The cardiomyocytes were divided into control(Con)group,ischemia/hypoxia(IH)group,IH+ lenti-control group and IH+ lenti-sh RNA-LOC102552184 group.The serum-free medium was applied to set up the ischemic environment,and the anoxic chamber was used to simulate the hypoxic environment.The IH+lenticontrol group and the IH+lenti-sh RNA-LOC102552184 group were transfected with vector lentivirus and sh RNA-LOC102552184 lentivirus respectively.The viability of the cardiomyocytes was examined by MTS and the LDH leakage was tested to evaluate the injury of cardiomyocytes.Cardiomyocyte apoptosis was inspected by flow cytometry.Confocal microscope was observed to determin the autophagic flux after m RFP-GFP-LC3 lentivirus infection.The presence of autophagosomes or autolysosomes was evaluated by TEM.The expression levels of apoptosis,autophagy and inflammation-related proteins were measured by Western blot.Results:1.Compared with the controls,the viability of the cardiomyocytes was significantly reduced in IH group.Compared with the IH group,the viability of cardiomyocytes was significantly enhanced and the injury of cardiomyocytes was improved when the expression of LOC102552184 was inhibited.2.The results of Flow cytometry demonstrated that the cardiomyocytes apoptosis was obviously reduced when LOC102552184 level was inhibited after IH.3.The TEM results indicated that after exposed to the IH environment there were visible mitochondrial swelling or vacuolation.Compared with the IH group,the presence of autophagosomes significant increased and the mitochondrial swelling was alleviated when the expression of LOC102552184 was inhibited.The confocal microscopy results demonstrated that the autophagy level of hypoxic primary cardiomyocytes was enhanced when the expression of LOC102552184 was inhibited.4.Western blot analysis indicated that when inhibiting the expression of LOC102552184 under IH conditions,the apoptosis-related proteins Bax,Cleaved caspase-3,caspase-8 and caspase-9 significantly reduced,while Bcl-2elevated.The autophagy related proteins LC3 ?,Beclin 1and ATG5 increased obviously,while p62 declined accordingly.The inflammation-related proteins IL-1?,TNF-? and NF-k B reduced significantly.Conclusion: Inhibiting the LOC102552184 expression under the IH condition can increase cardiomyocytes autophagy,reduce cardiomyocytes apoptosis and inflammation.Part 3: LOC102552184 Targeting miR-146a-5p /TRAF6 to Regulate the Myocardial Injury Induced by Coronary MicroembolizationObjective: The purpose of this study is to investigate the intrinsic mechanism of lncRNA LOC102552184 targeting miR-146a-5p/TRAF6 in the regulation of myocardial injury induced by CME.Methods: Thirty SD rats were divided into five groups by random allocation: Sham group,CME group,CME+AAV-control group,CME+AAVLOC102552184 group and CME+AAV-sh RNA-LOC102552184 group.The CME+AAV-control group,CME+AAV-LOC102552184 group and CME+AAV-sh RNA-LOC102552184 group were injected with adeno-associated virus injections loading vector,LOC102552184 and sh RNA-LOC102552184 sequences respectively before CME.After three weeks the CME model was established by administering 45?m microspheres into the left ventricle of rats,and the Sham group was administered with saline.The relationship between LOC102552184 and miR-146a-5p was determined by the dual luciferase assay.The targeting site of lncRNA LOC102552184 was verified by Fluorescence in situ hybridization(FISH).Rt-qPCR was tested to evaluated the expression levels of miR-146a-5p and TRAF6 after CME.Western blot analysis was used to assess the expression level of TRAF6 protein.Results:1.Dual luciferase reporter assay confirmed that miR-146a-5p can bind to LOC102552184 and inhibit its expression.2.FISH result showed that lncRNA LOC102552184 is mainly positive cytoplasm.3.The results of RT-qPCR and Western blot demonstrated that when inhibiting of LOC102552184,the miR-146a-5p expression level markedly enhanced while the expression level of TRAF6 obviously reduced.Overexpression LOC102552184 the miR-146a-5p level declined and the TRAF6 expression level elevated accordingly.4.The correlation analysis results showed that the expression level of LOC102552184 was inverse correlated with miR-146a-5p(r=-0.8440)and positively correlated with TRAF6(r= 0.8929).The miR-146a-5p expression level was inverse correlated with TRAF6(r=-0.8591).5.The correlation analysis showed that apoptosis-related protein Cleaved caspase-3 was positively related with TRAF6(r= 0.974).The autophagy-related protein LC3 II was reverse correlated with TRAF6 expression level(r=-0.8355).Inflammation-related protein NF-k B was positively related with TRAF6(r=0.974).Conclusion: lncRNA LOC102552184 can targeting regulate TRAF6 by sponging miR-146a-5p,which can aggravate apoptosis and inflammatory myocardial injury after CME,and inhibit autophagy,thus leading to myocardial injury.