MiR-874-3p Targeting STAT3 Protects Against Coronary Microembolization-induced Cardiomyocyte Apoptosis In Rats

Background:Coronary microembolization(CME)is a coronary microcirculation embolism and myocardial microinfarction caused by thrombogenic components such as lipids and debris during primary percutaneous coronary intervention(PCI)or spontaneous rupture of unstable atherosclerotic plaque in patients with acute coronary syndrome,which seriously affects the prognosis of patients and is a strong predictor of long-term major adverse cardiovascular events.Multiple apoptotic pathways are activated after CME,and myocardial cells in infarct related areas are apoptotic and induce cardiac dysfunction,which is one of the main mechanisms of myocardial injury after CME.Signal transducer and activator of transcription 3(STAT3)is a common acute reactive factor,involved in regulating cell growth,apoptosis,cancer and other life activities.During myocardial ischemia and hypoxia,the rapid phosphorylation of STAT3 can improve the tolerance of cardiomyocytes to ischemia and hypoxia environment,inhibit cardiomyocyte apoptosis and promote cardiomyocyte survival.However,it is unclear whether STAT3 is involved in regulating CME-induced cardiomyocyte apoptosis.MicroRNAs(miRs,miRNAs)negatively regulate gene expression by complementary binding to the 3’-untranslated region of mRNA,promoting its hydrolysis or inhibiting its translation.MiR-874-3p is involved in regulating cell apoptosis.However,it is not clear whether miR874-3p can affect myocardial cell apoptosis in CME.Therefore,this study aims to observe the expression levels of miR-874-3p and STAT3 and myocardial cell apoptosis in rats after CME,and to investigate the role of miR-874-3p in CME-induced myocardial cell apoptosis and cardiac dysfunction.Object:Detect the expression levels of miR-874-3p and STAT3 in CME model of rats,and verify their relationship by dual luciferase assay.Methods:16 SD rats were randomly divided into CME group and Sham group,with 8 rats in each group.The CME model group was established by injecting microembolic sphere into the left ventricle,and the sham operation group was replaced by injecting the same amount of normal saline.Cardiac function was determined by ultrasonography hours after surgery 6 h.HE staining of myocardial tissue was performed.Relative expression levels of miR-874-3p and STAT3 mRNA were detected by RT-PCR.The dual luciferase assay verified whether miR-874-3p and STAT3 had a targeted regulatory relationship.Results:1.Compared with Sham group,LVEF and FS of rats in CME group decreased significantly,while LVEDs increased significantly(P<0.05);Plasma cTn-I in CME group was increased significantly(P<0.05);Microspheres were found in the myocardial tissue sections in the CME group.The nuclei around the microspheres dissolved or disappeared,and the surrounding cells were edema and degeneration,and there were infiltration of white blood cells and exudation of red blood cells around the microspheres.2.Compared with the Sham group,the relative expression level of miR-8743p in the myocardium of rats in the CME group was increased significantly(P<0.05),and the relative expression level of STAT3 mRNA was decreased significantly(P<0.05).3.Dual luciferase assay:Compared with NC group,rno-miR-874-3p downregulated luciferase expression of R-STAT3-3UTR-WT significantly(P<0.001).After mutation,rno-miR-874-3p failed to down-regulate luciferase expression of R-STAT3-3UTR-MUT compared with NC group(P>0.05).Conclusions:The expression of miR-874-3p increases and the relative expression of STAT3 mRNA decreases in CME model,STAT3 is the target gene of miR-8743p,and miR-874-3p and STAT3 may be involved in the regulation of myocardial injury induced by CME.Objective:To investigate the effect of miR-874-3p on cardiomyocyte apoptosis in CME rats and explain its mechanism.Methods:The expression of miR-874-3p in myocardium of rats was decreased by tail vein injection of adeno-associated virus containing which inhibited the expression of miR-874-3p.Another 16 SD rats were randomly divided into CME+miR-8743p suppressed adeno-associated virus group(CME+shRNA group)and CME+miR-874-3p empty adeno-associated virus group(CME+NC group),with 8 rats in each group.Six hours after CME,the cardiac function of rats in each group was detected by echocardiography,the level of serum troponin-Ⅰ(cTn-Ⅰ)was detected by ELISA method,the myocardial microinfarction area was observed by HE and HBPF staining,cardiomyocyte apoptosis was detected by Tunel staining,the relative expression levels of miR-874-3p and STAT3 mRNA in myocardial tissue were detected by qRT-PCR,and the relative expression levels of STAT3,Bax,Bcl-2 and cleaved caspase-3 protein in myocardial tissue were detected by Western blot.Results:1.Echocardiography:Compared with Sham group,LVEF and FS decreased significantly,LVEDd and LVEDs increased significantly in CME group and CME+NC group.Compared with CME group and CME+NC group,LVEF and FS increased significantly,LVEDd and LVEDs decreased significantly in CME+shRNA group.There was no significant difference between CME group and CME+NC group.2.Plasma cTn-Ⅰ level:Compared with Sham group,plasma cTn-Ⅰ level in CME group and CME+NC group was significantly higher,and cTn-Ⅰ in CME+shRNA group was significantly lower than that in CME group and CME+NC group(P<0.05).There was no significant difference in cTn-Ⅰ between CME group and CME+NC group.3.HE staining:No microspheres and myocardial infarction lesions were observed in the myocardial tissue of the Sham group.The myocardial fibers were neatly arranged,no myocardial rupture,inflammatory cell infiltration,and no nucleolysis were observed.Microspheres were observed in the myocardial tissue sections of rats in the CME group,the CME+shRNA group and the CME+NC group.The nuclei of the myocardium around the microspheres dissolved or disappeared,and the surrounding cells were edema and degeneration,surrounded by infiltration of white blood cells and exudation of red blood cells.Compared with the CME group and the CME+NC group,the structural changes of cardiomyocytes in the CME+shRNA group were reduced,and the infiltration of inflammatory cells was reduced.4.Fluorescence microscope observation:Green fluorescence was observed in the cytoplasm of cardiomyocytes in CME+shRNA group and CME+NC group.5.HBFP staining:There was no obvious microinfarction in the myocardium of Sham group.Compared with the Sham group,obvious microinfarction was observed in the myocardium of rats in the CME group and the CME+NC group(P<0.05).Compared with the CME group and the CME+NC group,the myocardial microinfarct area in the CME+shRNA group decreased significantly(P<0.05).There was no significant difference in the area of myocardial microinfarction between the CME group and the CME+NC group.6.qRT-PCR results:Compared with Sham group,the relative expression of miR-874-3p increased significantly and the relative expression level of STAT3 mRNA decreased significantly in CME group and CME+NC group.Compared with the CME group and the CME+NC group,the relative expression level of miR-874-3p decreased significantly and the relative expression level of STAT3mRNA increased significantly in the CME+shRNA group(P<0.05).There was no significant difference in the relative expression levels of miR-874-3p and STAT3mRNA between the CME group and the CME+NC group.7.Western blot results:Compared with Sham group,the relative expression levels of STAT3 and Bcl-2 were decreased significantly in CME group and CME+NC group(P<0.05),while the relative expression levels of Bax and cleaved caspase-3 were increased significantly(P<0.05).Compared with CME group and CME+NC group,the protein relative expression levels of STAT3 and Bcl-2 in CME+shRNA group were increased significantly(P<0.05),while the protein relative expression levels of Bax and cleaved caspase-3 in CME+shRNA group were decreased significantly(P<0.05).There was no significant difference in the relative expression levels of STAT3,Bcl-2,Bax and cleaved caspase-3 proteins between the CME group and the CME+NC group.Conclusion:After CME,the expression of miR-874-3p in myocardium increases,cardiomyocyte apoptosis increases,and cardiac contractile function decreases.Inhibiting the expression of miR-874-3p can increase the expression of STAT3,further decrease the expression of pro-apoptotic protein Bax,increase the expression of anti-apoptotic protein Bcl-2,inhibit cardiomyocyte apoptosis and improve cardiac function in CME rats.