CircFoxo3调节miR-130a-5p/TEAD1轴对心力衰竭大鼠心肌细胞凋亡影响的实验研究

An experimental study on the effect of CircFoxo3 regulating miR-130a-5p/TEAD1 axis on myocardial cell apoptosis in heart failure rats

目的探讨环状RNA叉头框蛋白O3(CircFoxo3)调节miR-130a-5p/TEA域转录因子1(TEAD1)轴对心力衰竭(HF)大鼠心肌细胞凋亡的影响。方法将SD大鼠分为对照组、HF组、si-NC组、si-CircFoxo3组、agomir NC组、miR-130a-5p agomir组、si-CircFoxo3+antagomir NC组、si-CircFoxo3+miR-130a-5p antagomir组,每组18只。除对照组外,其他组大鼠均通过腹腔注射盐酸阿霉素的方法构建HF大鼠模型。建模成功后进行药物处理,每3 d给药一次,持续3周。应用反转录实时荧光定量聚合酶链式反应(qRT-PCR)法检测心肌组织中CircFoxo3、miR-130a-5p表达水平。应用彩色多普勒超声仪检测大鼠左室收缩末期内径(LVESD)、左室舒张末期内径(LVEDD)、左室射血分数(LVEF)水平。采用酶联免疫吸附试验(ELISA)法检测大鼠血清人基质裂解素2(ST_(2))、N端脑钠肽前体(NT-pro BNP)水平。应用HE染色、Masson染色检测大鼠心肌组织病理变化和心肌纤维化情况。通过TUNEL染色检测心肌细胞凋亡水平。应用Western blot法检测大鼠心肌组织TEA域转录因子1(TEAD1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解的天冬氨酸特异性半胱氨酸蛋白酶-3(cleaved caspase-3)蛋白表达水平。通过双荧光素酶报告基因实验验证CircFoxo3与miR-130a-5p、miR-130a-5p与TEAD1的关系。结果与对照组比较,HF组大鼠心肌组织病理损伤、心肌纤维化严重,LVESD、LVEDD水平上升,心肌组织CircFoxo3及TEAD1、Bax、cleaved caspase-3蛋白表达水平升高,血清ST_(2)、NT-pro BNP水平升高,心肌细胞凋亡率升高,LVEF、miR-130a-5p和Bcl-2蛋白水平降低,差异有统计学意义(P<0.05)。与HF组、si-NC组比较,si-CircFoxo3组大鼠心肌组织病理损伤减轻,LVESD、LVEDD水平降低,心肌组织CircFoxo3及TEAD1、Bax、cleaved caspase-3蛋白表达水平降低,血清ST_(2)、NT-pro BNP水平降低,心肌细胞凋亡率降低,LVEF、miR-130a-5p及Bcl-2蛋白水平升高,差异有统计学意义(P<0.05)。与HF组、agomir NC组比较,miR-130a-5p agomir组大鼠心肌组织病理损伤、心肌纤维化有所改善,LVESD、LVEDD水平降低,心肌组织TEAD1、Bax、cleaved caspase-3蛋白表达水平降低,血清ST_(2)、NT-pro BNP水平降低,心肌细胞凋亡率降低,LVEF、miR-130a-5p及Bcl-2蛋白水平升高,差异有统计学意义(P<0.05)。与si-CircFoxo3组、si-CircFoxo3+antagomir NC组比较,si-CircFoxo3+miR-130a-5p antagomir组大鼠心肌组织病理损伤、心肌纤维化加剧,LVESD、LVEDD水平升高,心肌组织TEAD1、Bax、cleaved caspase-3蛋白表达水平升高,血清ST_(2)、NT-pro BNP水平升高,心肌细胞凋亡率升高,LVEF、miR-130a-5p、Bcl-2蛋白水平降低,差异有统计学意义(P<0.05)。CircFoxo3与miR-130a-5p、miR-130a-5p与TEAD1存在靶向关系。结论沉默CircFoxo3可能通过海绵化上调miR-130a-5p来抑制TEAD1表达,进而抑制HF大鼠心肌细胞凋亡。

Objective To explore the effect of circular RNA forkhead box protein O3(CircFoxo3)regulating micro ribonucleic acid-130a-5p(miR-130a-5p)/TEA domain transcription factor 1(TEAD1)axis on myocardial cell apoptosis in heart failure(HF)rats.Methods Sprague-Dawley(SD)rats were divided into control group,HF group,si-NC group,si-CircFoxo3 group,agomir NC group,miR-130a-5p agomir group,si-CircFoxo3+antagomir NC group and si-CircFoxo3+miR-130a-5p antagomir group,with 18 rats in each group.Except for the rats in the control group,the rats in the other groups were injected with doxorubicin hydrochloride intraperitoneally to construct HF rat models.After successful modeling,medication was administered once every 3 days for 3 weeks.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was applied to detect the expression levels of CircFoxo3 and miR-130a-5p in the myocardial tissues.Color Doppler ultrasound was applied to detect the levels of left ventricular end systolic diameter(LVESD),left ventricular end diastolic diameter(LVEDD),and left ventricular ejection fraction(LVEF)in the rats.Enzyme-linked immunosorbent assay(ELISA)method was applied to detect the levels of human stromelysin-2(ST_(2))and N-terminal pro-brain natriuretic peptide(NT-pro BNP)in the serum of the rats.Haematoxylin-eosin(HE)staining and Masson staining were used to detect the pathological changes and the myocardial fibrosis in the myocardial tissues of the rats.TUNEL staining was applied to detect the level of cardiomyocyte apoptosis.Western blot method was applied to detect the expression levels of TEAD1,B cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cleaved aspartate-specific cysteine protease-3(cleaved caspase-3)in the myocardial tissues of the rats.Double luciferase reporter gene experiment was applied to verify the relationship between CircFoxo3 and miR-130a-5p,and between miR-130a-5p and TEAD1.Results Compared with those in the control group,the rats in the HF group had pathological injury of myocardial tissues and severe myocardial fibrosis,elevated levels of LVESD and LVEDD,elevated levels of CircFoxo3,TEAD1,Bax and cleaved caspase-3 protein expressions in the myocardial tissues,elevated levels of ST_(2) and NT-pro BNP in the serum,elevated cardiomyocyte apoptosis rate,and decreased levels of LVEF,miR-130a-5p and Bcl-2 protein,and the differences were significant(P<0.05).Compared with those in the HF group and the si-NC group,the rats in the si-CircFoxo3 group had mitigated pathological injury of myocardial tissues,decreased levels of LVESD and LVEDD,decreased expression levels of CircFoxo3,TEAD1,Bax and cleaved caspase-3 protein expression in the myocardial tissues,decreased levels of ST_(2) and NT-pro BNP in the serum,decreased cardiomyocyte apoptosis rate,and elevated levels of LVEF,miR-130a-5p and Bcl-2 protein,and the differences were significant(P<0.05).Compared with those in the HF group and the agomir NC group,the rats in the miR-130a-5p agomir group had improved pathological injury and myocardial fibrosis in the myocardial tissues,decreased levels of LVESD and LVEDD,decreased levels of TEAD1,Bax,cleaved caspase-3 protein expressions in the myocardial tissues,decreased levels of ST_(2) and NT-pro BNP in the serum,decreased cardiomyocyte apoptosis rate,and elevated levels of LVEF,miR-130a-5p and Bcl-2 protein,and the differences were significant(P<0.05).Compared with those in the si-CircFoxo3 group and the si-CircFoxo3+antagomir NC group,the rats in the si-CircFoxo3+miR-130a-5p antagomir group had aggravated pathological injury and myocardial fibrosis in the myocardial tissues,elevated levels of LVESD and LVEDD,elevated levels of TEAD1,Bax,cleaved caspase-3 protein expressions in the myocardial tissues,elevated levels of ST_(2) and NT-pro BNP in the serum,elevated cardiomyocyte apoptosis rate,and decreased levels of LVEF,miR-130a-5p and Bcl-2 protein,and the differences were significant(P<0.05).CircFoxo3 had a targeted relationship with miR-130a-5p,and miR-130a-5p had a targeted relationship with TEAD1.Conclusion Silencing CircFoxo3 may inhibit the expression of TEAD1 via sponging miR-130a-5p upwards,thereby inhibiting cardiomyocyte apoptosis in HF rats.