论文部分内容阅读
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 predict and identify the target gene of miR-30e and clarify the molecular mechanism by which miR-30e regulates cardiac hypertrophy. Methods: Cardiomyocytes were isolated from neonatal rat hearts. Cardiomyocytes were treated with phenylephrine (PE) to establish cardiomyocyte hypertrophy model. The expression of miR-30e was detected by quantitative PCR 48 hours later. The target gene of miR-30e was predicted by bioinformatics method and the target gene of miR-30e was verified by luciferase reporter assay and western blotting. Results: Compared with the control group, the expression of nppa, a marker of cardiac hypertrophy, significantly increased after PE treatment for 48 hours, and miR-30e was significantly down-regulated in hypertrophic cardiomyocytes. Bioinformatics prediction The cytoskeleton regulatory protein Twinfilin-1 (Twf1) 3’UTR has two miR-30e binding sites. Overexpression of miR-30e inhibited the expression of luciferase reporter gene containing Twf1 3’UTR and decreased the expression of Twf1 protein. Conclusion: Twf1 is the target gene of miR-30e, and miR-30e regulates cardiac hypertrophy by inhibiting the expression of Twf1.