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MicroRNA-223下调心脏特异性新激酶表达对心肌细胞肥大的抑制效应研究
MicroRNA-223 protects cardiomyocyte from hypertrophy by targeting cardiac troponin I-interacting kinase
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DOI:
作者:
王耀晟,周璟,李毅刚
WANG Yaosheng,ZHOU Jing,LI Yigang
作者单位:
1.上海交通大学医学院附属新华医院心内科 2.上海医药高等专科学校
1.Department of Cardiology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 2.Shanghai Institute of Health Sciences
关键词:
微小RNA-223;心脏特异性新激酶;心肌细胞;细胞肥大
摘要:
目的 探讨微小RNA-223(miR-223)通过下调心脏特异性新激酶(TNNI3K)基因对心肌细胞肥大的调控作用。方法 原代培养新生大鼠心肌细胞,以内皮素-1(ET-1)作为心肌细胞肥大人工诱导。miR-223的表达水平通过实时定量聚合酶链式反应(real-time PCR)测定。miR-223的过表达通过化学修饰RNA模拟物转染实现。TNNI3K的过表达通过重组腺病毒转染实现。抗α-辅肌动蛋白(α-actinin)荧光染色法测量细胞大小。以real-time PCR检测心肌细胞肥大标志性基因的表达。Western Blots免疫印迹法测定TNNI3K、cTnI蛋白表达。荧光素酶检测法判定miR-223与TNNI3K的3’端非编码区(3’UTR )接合。结果 与正常心肌细胞相比,肥大心肌细胞中的miR-223表达较低。外源性miR-223能有效抑制ET-1诱导的心肌细胞肥大。具体效应为细胞表面积减小,以及心肌细胞肥大标志性基因ANP、α-辅肌动蛋白、myh6、MYH7的表达下调。荧光素酶检测表明,TNNI3K是miR-223的直接靶基因。miR-223过表达可使正常及肥大的心肌细胞中TNNI3K蛋白表达明显下调。此外,cTnI的磷酸化也受到miR-223的抑制。结论 miR-223通过直接下调TNNI3K基因表达抑制心肌细胞肥大,此调控效应与cTnI的磷酸化受限有关。
Objective This study was designed to investigate the cellular and molecular effects of miR-223 on cardiomyoctye hypertrophy, focusing on the role of TNNI3K. Methods Neonatal rat cardiomyocytes (CMs) were cultured, and CMs hypertrophy was induced by endothelin-1 (ET-1). In vivo cardiac hypertrophy was induced by transverse aorta constriction (TAC) in rats. Expression of miR-223 in CMs and myocardium was detected by real-time PCR (RT-PCR). MiR-223 and TNNI3K were overexpressed in CMs via chemically modified sense RNA (miR-223 mimic) transfection or recombinant adenovirus infection, respectively. Cell size was measured by surface area calculation using fluorescence microscopy after anti-α-actinin staining. Expression of hypertrophy-related genes was detected by RT-PCR. The protein expression of TNNI3K and cTnI was determined by Western blots. Luciferase assay was employed to confirm the direct binding of miR-223 to the 3'UTR of TNNI3K mRNA. Results miR-223 was downregulated in ET-1 induced hypertrophic CMs and in hypertrophic myocardium compared with respective controls. miR-223 overexpression in CMs alleviated ET-1 induced hypertrophy, evidenced by smaller cell surface area and downregulated ANP, α-actinin, Myh6 and Myh7 expression. Luciferase reporter gene assay showed that TNNI3K serves as a direct target gene of miR-223. In miR-223-overexpressed CMs, the protein expression of TNNI3K was significantly downregulated. MiR-223 overexpression also rescued the upregulated TNNI3K expression in hypertrophic CMs. Furthermore, cTnI phosphorylation was downregulated post miR-223 overexpression. Conclusion By direct targeting TNNI3K, miR-223 could suppress CMs hypertrophy via downregulating cTnI phosphorylation. miR-223 / TNNI3K axis may thus be major players of CMs hypertrophy.
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