MiR-221-3p调控c-Fos/beclin-1对大鼠冠状动脉微栓塞后心肌细胞自噬的影响

Effects of miR-221-3p regulating c-Fos/beclin-1 signaling pathway on autophagy of myocardial cells in rats after coronary microembolization

目的:探究miR-221-3p介导的c-Fos/beclin-1信号通路在大鼠冠状动脉微栓塞(CME)后对心肌细胞自噬的影响及意义。方法:将24只SD大鼠按随机数字表法分为Sham组(假手术组)、CME组(微栓塞组)、CME+shRNA组(CME+AAV9-miR-221-3p shRNA组)和CME+NC组(CME+AAV9-Control shRNA组),每组6只。经左心室注入微栓塞球建立CME模型,Sham组用等量生理盐水代替。各组术后9 h行心脏超声及心肌肌钙蛋白(IcTn-I)检测;组织取材做苏木精碱性复红苦味酸染色(HBFP)、透射电镜(TEM)、免疫荧光以及实时荧光定量聚合酶链反应(PCR)、蛋白质印迹(Western blot)等评估miR-221-3p介导的c-Fos/beclin-1信号通路在功能、形态和分子方面对CME的影响。结果:CME后心肌细胞中miR-221-3p表达升高,与Sham组相比,心脏功能抑制,cTn-I及心肌微梗死面积明显升高,c-Fos、beclin-1以及自噬指标LC3-Ⅱ蛋白表达降低(P<0.05)。下调miR-221-3p后,与CME组相比,cTn-I及心肌梗死面积显著下降(P<0.05),c-Fos、beclin-1以及LC3-Ⅱ蛋白表达增加(P<0.05),TEM提示自噬泡的双层膜结构数量增加,自噬部分恢复,心肌损伤减轻,心脏功能改善。结论:抑制miR-221-3p可改善大鼠CME后心脏功能,其可能通过c-Fos/beclin-1途径调节心肌自噬参与CME后诱导的心肌损伤。

Objective:To investigate the effect of miR-221-3p mediated c-Fos/beclin-1 signaling pathway on myocardial autophagy in rats after coronary microembolization(CME).Meth ods:A total of 24 SD rats were randomly divided into the Sham group,the CME group,the CME+AAV9-miR-221-3p shRNA group(CME+shRNA group),and the CME+AAV9-Control shRNA group(CME+NC group),with 6 rats in each group.CME model was established by injecting polyethylene microspheres(42μm)into the left ventricle,and an equal amount of saline was used in the Sham group.Cardiac ultrasound and cardiac troponin I(cTn-I)were measured in each group after 9 h operation.Tissue samples were taken for hematoxylin-basic fuchsin-picric acid(HBFP),transmission electron microscope(TEM),immunofluorescence,fluorescent quantitation polymerase chain reaction(RT-qPCR),and Western blot to evaluate the effect of miR-221-3p mediated c-Fos/beclin-1 signaling pathway on CME's function,shape,and molecular aspect.Results:Compared with the Sham group,the expression of miR-221-3p in cardiomyocytes was increased,cardiac function was suppressed,cTn-I and myocardial microinfarction areas were significantly increased,and the protein levels of c-Fos,beclin-1,and LC3-Ⅱwere decreased of the CME group(P<0.05).Compared with the CME group,cTn-I and myocardial infarction areas were significantly reduced after miR-221-3p was down-regulated(P<0.05);protein levels of c-Fos,beclin-1,and LC3-Ⅱwere increased(P<0.05).TEM image showed that double membranes of autophagic vacuoles were increased,autophagy was partially recovered,myocardial damage was reduced,and cardiac function was improved.Conclusion:Inhibition of miR-221-3p can improve cardiac function in rats after CME,which may regulate myocardial autophagy and participate in CME-induced myocardial injury via c-Fos/beclin-1 pathway.