MiR-30e调控心肌肥厚的作用机制研究

Mechanism of Cardiac Hypertrophy Regulates by miR-30e

目的:预测并鉴定miR-30e的靶基因,阐明miR-30e调控心肌肥厚的分子机制。方法:分离新生大鼠心肌细胞,用苯肾上腺素(PE)处理心肌细胞构建心肌细胞肥大模型,48小时后通过定量PCR方法检测miR-30e的表达水平变化。利用生物信息学方法预测miR-30e的靶基因,并通过荧光素酶报告基因实验和蛋白免疫印迹方法验证miR-30e的靶基因。结果:与对照组相比,PE处理48hr后,心肌肥厚标志基因nppa表达明显升高,肥大心肌细胞中miR-30e明显下调。生物信息学预测细胞骨架调控蛋白Twinfilin-1(Twf1)3'UTR有两个miR-30e的结合位点。过表达miR-30e能抑制含有Twf1 3'UTR的荧光素酶报告基因的表达,降低Twf1的蛋白表达水平。结论:Twf1为miR-30e的靶基因,miR-30e通过抑制Twf1的表达调控心肌肥厚。

Objective： To investigate the molecular mechanism by which miR-30e regulates cardiac hypertrophy. Methods： Primarily cultured neonatal rat cardiomyocytes were stimulated by phenylephrine （PE） to induce hypertrophy. The expression level of miR-30e was detected by real-time PCR analysis after treated by PE for 48hr. To investigate the mechanism of miR-30e regulating cardiac hypertrophy, bioinformatics analysis was used to predict the potential targets. Then the target was validated by luciferase reporter analysis and Western Blot. Result： The expression level of the cardiac hypertrophic marker gene nppa was obviously increased by PE stimulation compared to that of the control cells, and the level of miR-30e in hypertrophic cardiomyocytes was downregulated. Bioinformatics analysis showed that there were two miR-30e binding sites in the 3＇UTR of Twinfilin- 1 （Twfl）, a cytoskeleton regulatory protein. Overexpression of miR-30e significantly reduced the luciferase activity of the reporter with Twfl 3＇UTR. Furthermore, upregulation of miR-30e decreased the protein level of Twfl. Conclusion： Twfl is a bona fide target of miR-30e, and miR-30e regulated cardiac hypertrophy by inhibiting the expression Of Twfl.