环状RNA _005647通过结合miR-99b-5p抑制心肌细胞中肥厚相关基因的表达

目的研究环状RNA circRNA_005647抑制心肌肥厚相关基因表达的作用机制。方法建立血管紧张素Ⅱ(AngⅡ)灌注诱导的心肌肥厚小鼠模型。原代分离获得C57BL/6乳小鼠心肌细胞(NMVC),AngⅡ处理NMVC建立心肌细胞肥大模型。利用NMVC,分别感染circRNA_005647重组腺病毒(rAd-circRNA_005647)和转染miR-99b-5p模拟物来研究对NMVC肥大的影响。通过双荧光素酶报告基因实验和RNA Pull-down实验验证circRNA_005647与miR-99b-5p的结合作用。实时荧光定量PCR和Western blot检测NMVC中心肌肥厚相关基因的mRNA和蛋白表达水平。结果在AngⅡ诱导的小鼠心肌肥厚模型和心肌细胞肥大模型中,circRNA_005647及其宿主基因肌球蛋白IXA(Myo9a)表达升高。过表达circRNA_005647可抑制AngⅡ诱导的NMVC中肥厚相关基因心房钠尿肽(Anp)和肌球蛋白重链7(Myh7)基因表达升高。circRNA_005647与miR-99b-5p间存在结合作用。过表达circRNA_005647可抑制miR-99b-5p促心肌细胞肥大的作用。结论circRNA_005647通过结合miR-99b-5p发挥抑制心肌细胞肥大的作用。

CircRNA_005647 inhibits the expression of hypertrophy-related genes in cardiomyocytes by sponging miR-99b-5p

Aim To investigate the effect of circRNA_005647 on suppressing the expression of hypertrophy associated genes and the potential mechanism.Methods Mouse model of cardiac hypertrophy induced by angiotensinⅡ(AngⅡ)perfusion was constructed.Primary isolation and culture of C57BL/6 neonatal mouse ventricular cardiomyocytes(NMVC)and AngⅡinduced cell model of cardiac hypertrophy was also established.NMVC were infected with rAd-circRNA_005647 and transfected miR-99b-5p mimic to increase circRNA_005647 and miR-99b-5p levels,respectively.Dual luciferase reporter assay and RNA Pull-down assay were performed to identify the interaction between circRNA_005647 and miR-99b-5p,respectively.mRNA and protein expression of hypertrophy associated genes were determined by quantitative real-time PCR(RT-qPCR)and Western blot assay,respectively.Results circRNA_005647 and its host gene of myosin IXA(Myo9a)were increased in the hypertrophic mouse myocardium and in AngⅡ-treated NMVC.Over-expression of circRNA_005647 could significantly suppress AngⅡ-induced increases of hypertrophy-related genes,including atrial natriuretic peptide(Anp)and myosin heavy chain 7(Myh7),in NMVC.The interaction between circRNA_005647 and miR-99b-5p was identified.Over-expression of circRNA_005647 blocked the expression of hypertrophy associated genes induced by miR-99b-5p in NMVC.Conclusion circRNA_005647 exerts an inhibitory effect on cardiac hypertrophy by sponging miR-99b-5p.