The Effects Of MiR-221-3p On Coronary Microembolization In Rat Associated With Involvement Of C-Fos/beclin-1 Signaling Pathway

ObjectiveTo investigate the effects of miR-221-3p-mediated c-Fos/beclin-1 signaling pathway on myocardial autophagy after coronary microembolization（CME）in rats.MethodsTwenty-four Sprague-Dawley（SD）rats were randomly divided into sham operation group（Sham group）,CME group,CME+miR-221-3p inhibitor group（CME+shRNA group）and CME+miR-221-3p negative control group（CME+NC group）（n=6,per group）.The CME model was induced by injecting polyethylene microspheres（42μm）into the left ventricle except the sham group,and the sham operation group was injected with normal saline instead.The CME+shRNA and CME+NC groups were injected with 1.1x10¹¹vg of adeno-associated virus（AAV）containing shRNA-miR-221-3p and miR-221-3p negative control respectively 4 weeks before CME.Cardiac function was performed 9 hours after operation in each group;The plasma cardiac troponin I（cTn-I）was examined by ELISA;HE staining was used to assess the myocardial morphological changes;HBFP staining was used to detect and evaluate the myocardial micro-infarction area.Transmission electron microscopy（TEM）and immunofluorescence were used to observe myocardial autophagy;The relative expression of miR-221-3p and c-Fos mRNA and Beclin-1 mRNA were detected by Reverse transcription quantitative real-time polymerase chain reaction（RT-qPCR）;Western blot was used to detect the relative expression levels of autophagy-related proteins such as c-Fos,beclin-1,LC3B and P62;The SPSS17.0 statistical software was used for data analysis.Measurement data were expressed as mean±standard deviation（X±SD）.Comparison among multiple groups was analyzed by One-Way ANOVA.ResultsThe results of echocardiography showed that compared with the sham operation group,the left ventricular ejection fraction（LVEF）in the CME group was significantly reduced[（90.06±2.03）%and（59.17±5.91）%,P<0.05].In comparison,the left ventricular ejection fraction（LVEF）in the CME+shRNA group was significantly improved,compared with the CME group[（59.17±5.91）%and（78.00±3.98）%,P<0.05].Nine hours after coronary microembolization（CME）in rats,the plasma cTn-I level of rats was increased significantly in the CME group[（213.58±21.07）pg/ml vs（30.04±5.47）pg/ml,P<0.05]and CME+NC group[（210.78±19.77）pg/ml vs（30.04±5.47）pg/ml,P<0.05],compared with the Sham group.In addition,compared with the CME group,the plasma cTn-I level in the CME+shRNA group was significantly reduced[（98.62±17.67）pg/ml vs（213.58±21.07）pg/ml,P<0.05].It is no obvious microinfarction that we found in the myocardium of the Sham group by HBFP staining.However,obvious red myocardial microinfarction of different areas were seen in the CME group,CME+shRNA group and CME+NC group.In addition,the area of myocardial microinfarction in the CME+shRNA group was significantly smaller than CME group[（7.59±1.79）%and（15.54±2.75）%,P<0.05].Transmission electron microscope（TEM）observation results showed that mitochondrial membrane was intact in the Sham group,and the myofibril structure of the myocardial cells was normal,while the myocardial myofibrils in the CME group were fragmented,the mitochondria were swollen,and the myofilaments were disordered.Compared with the CME group,the typical bilayer membrane structure of autophagic vesicles was observed in the CME+shRNA group,and the degree of myofibril fragmentation and mitochondrial swelling were all alleviated.The CME model was established 4 weeks after transfection with AAV containing shRNA-miR-221-3p and miR-221-3p negative control.Total RNA was extracted from myocardial tissues in rats 9 hours after CME.RT-qPCR results showed that AAV vector in the CME+shRNA group inhibited miR-221-3p successfully in myocardial tissues.Compared with the Sham group,the expression level of miR-221-3p was significantly up-regulated;and compared to the CME group,the expression level of miR-221-3p in the CME+shRNA group was significantly down-regulated,while the CME+NC group was not significantly changed.The expression levels of c-Fos mRNA and Beclin-1 mRNA in the CME group were significantly reduced compared to the Sham group;and the expression levels of c-Fos mRNA and Beclin-1 mRNA in the CME+shRNA group were significantly increased compared with the CME group.All the differences were statistically significant（P<0.05）.The results of Western blot suggested the expression of autophagy-related proteins such as LC3B,p62,Beclin-1,and c-Fos/beclin-1 pathway proteins in rats myocardial tissue after the establishment of miR-221-3p-shRNA transfection and CME.Compared with the Sham group,the expression levels of LC3â…¡,c-Fos,and Beclin-1 proteins in the CME group were significantly decreased,while the expression levels of p62 protein were significantly increased.What is more,the expression levels of LC3â…¡,c-Fos,Beclin-1 protein in the CME+shRNA group were significantly higher than those in the CME group or CME+NC group.Meanwhile,p62 protein expression level in the CME+shRNA group were significantly lower than those in the CME group or CME+NC group.All the differences were statistically significant（P<0.05）.This suggest that after down-regulating miR-221-3p,myocardial autophagy levels are partially restored,and the myocardial injury induced by CME may be regulated by the c-Fos/beclin-1signaling pathway.ConclusionThis study shows that the expression level of miR-221-3p in rats myocardial tissue increased after CME,myocardial autophagy was suppressed,and cardiac function was significantly reduced at the same time.Myocardial autophagy level after CME is closely related to miR-221-3p expression.Inhibition of miR-221-3p can reduce myocardial injury and improve cardiac function in rats after CME,which may partially upregulate myocardial autophagy through the c-Fos/beclin-1signaling pathway,providing a new idea for the prevention and treatment of CME-induced myocardial injury.