miR-199a-5p通过靶向JunB介导心力衰竭大鼠心肌细胞凋亡的作用机制

Mechanism of miR-199a-5p Mediated Cardiomyocyte Apoptosis through Targeting JunB in Rat with Heart Failure

目的:观察miR-199a-5p对心力衰竭大鼠心肌细胞凋亡的影响,并探讨其可能的作用机制。方法:将120只雄性Wistar大鼠随机分为假手术组、模型组、Antagomir对照组、Antagomir组、Agomir对照组及Agomir组。除假手术组(18只)外,其余组大鼠采用持续14 d结扎左冠状动脉前降支诱导构建心肌梗死后心力衰竭大鼠模型,成功诱导94只心力衰竭大鼠,剔除4只,随机分组,每组大鼠18只。分别给予Antagomir对照组、Antagomir组、Agomir对照组及Agomir组大鼠心肌内注射2×1010PFU/mL miR-199a-5p Antagomir对照液、Antagomir液、Agomir对照液及Agomir稀释液各100μL,假手术组与模型组大鼠均等量注射生理盐水。2,3,5-三苯基氯化四氮唑(TTC)染色法检测心肌梗死面积;苏木精-伊红(HE)染色法检测心肌组织病理损伤情况;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)染色法检测心脏缺血部位细胞凋亡水平;实时荧光定量聚合酶链式反应(RT-qPCR)检测各组大鼠心肌组织中miR-199a-5p、转录因子活化蛋白激酶B(JunB)基因相对表达水平;双荧光素酶试验验证miR-199a-5p与JunB的靶向关系;免疫印迹法(Western Blot)检测各组大鼠心肌组织内活化的天冬氨酸特异性半胱氨酸蛋白酶3(Cleaved-Caspase 3)、JunB、淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达情况。结果:与假手术组比较,模型组大鼠心肌梗死面积增加,组织结构不完整,心肌细胞排列错乱不规则,炎性细胞浸润,细胞间有明显凋亡,miR-199a-5p基因及Cleaved-Caspase 3、Bax蛋白表达水平升高,JunB mRNA及JunB、Bcl-2蛋白表达水平降低(P<0.05);与模型组比较,Antagomir对照组及Agomir对照组大鼠心肌病理组织学变化、相关基因和蛋白相对表达水平比较,差异无统计学意义(P>0.05)。Antagomir组大鼠心肌组织病理情况显著改善,miR-199a-5p基因及Cleaved-Caspase 3、Bax蛋白表达水平降低,JunB mRNA及JunB、Bcl-2蛋白表达水平升高(P<0.05);Agomir组大鼠心肌组织病理损伤加重,miR-199a-5p基因及Cleaved-Caspase 3、Bax蛋白表达水平升高,JunB mRNA及JunB、Bcl-2蛋白表达水平降低(P<0.05)。JunB为miR-199a-5p的靶基因。结论:miR-199a-5p可诱导心力衰竭大鼠心肌细胞凋亡,可能与靶向抑制JunB表达有关。

Objective: To analyze the effect of miR-199a-5p on cardiomyocyte apoptosis in rats with heart failure and its possible mechanism.Methods: One hundred and twenty male Wistar rats were randomly divided into sham group, model group, Antagomir control group, Antagomir group, Agomir control group and Agomir group.Except for 18 rats in the sham-operated group, the rats in the other groups were induced by ligating the anterior descending branch of the left coronary artery for 14 d to construct the heart failure model after myocardial infarction.Ninety-four rats with heart failure were successfully induced, and 4 rats were excluded, with 18 rats in each group.The rats in the Antagomir control group, Antagomir group, Agomir control group and Agomir group were injected with 2×1010PFU/mL miR-199a-5p Antagomir control, Antagomir, Agomir control and Agomir dilutions of 100 μL into myocardium respectively, and the rats in the sham operation group and the model group were injected with the same amount of normal saline.2,3,5-triphenyltetrazolium chloride(TTC) staining was used to detect myocardial infarct area, hematoxylin-eosin(HE) staining was used to to detect myocardial histopathological damage;and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay(TUNEL) staining used to detect the level of apoptosis at the site of cardiac ischemia.The relative expression levels of miR-199a-5p and transcription factor-activated protein kinase B(JunB) in myocardial tissues of rats were measured by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The targeting relationship between miR-199a-5p and JunB was verified by dual luciferase assay.The activated aspartate-specific cysteine protease 3(Cleaved-Caspase 3),JunB,lymphocytoma-2(Bcl-2),and Bcl-2-associated X protein(Bax) protein expression in myocardial tissues of rats were measured by Western Blot.Results: Compared with the sham operation group, the myocardial infarction area of the model group was increased significantly, the tissue structure was incomplete, the arrangement of cardiomyocytes was disordered and irregular, with inflammatory cell infiltration.Significant apoptosis was observed, the expressions of miR-199a-5p gene, Cleaved-Caspase 3 and Bax protein were increased significantly, and the expressions of JunB mRNA,and JunB,Bcl-2 protein were decreased significantly(P<0.05).The myocardial histopathology in the Antagomir group was significantly improved, the expressions of miR-199a-5p gene and Cleaved-Caspase 3,Bax protein were decreased, while the expressions of JunB mRNA and JunB,Bcl-2 protein were increased(P<0.05).The myocardial histopathological injury in the Agomir group was aggravated, the expressions of miR-199a-5p gene and Cleaved-Caspase 3,Bax protein were increased, while the expressions of JunB mRNA and JunB,Bcl-2 protein were decreased(P<0.05).JunB was the target gene of miR-199a-5p.Conclusion: miR-199a-5p could induce cardiomyocyte apoptosis in rat with heart failure, which might be related to targeting inhibition of JunB expression.