Department of Cardiology, Central Hospital of Zhengzhou University, Zhengzhou, Henan, China (mainland).
Department of Cardiology, No. 1 Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China (mainland).
Med Sci Monit. 2017 Aug 17;23:3981-3988. doi: 10.12659/msm.897542.
BACKGROUND The objective of this study was to identify the pathway responsible for ventricular remodeling. MATERIAL AND METHODS We collected remodeling myocardium tissue (n=18) and control myocardium tissue (n=22), and detected the expression of 4 miRNAs in these 2 groups using real-time PCR. We then searched the miRNA database online to find the candidate genes of miR-93. Real-time PCR and Western blot analysis were used to confirm the regulatory relationship. RESULTS We found that only miR-93 was decreased in remodeling myocardium tissue, and validated CCND1 to be the direct target gene of miR-93, with the "seed sequence" located within the 3'-UTR of the target gene via luciferase reporter assay system. Furthermore, we established the negative regulatory relationship between miR-93 and CCND1 by determining the relative luciferase activity of cells transfected with wild-type or mutant 3'-UTR of CCND1. We also found that The CCND1 protein and mRNA expression level of HL-1 cells treated with 50 nM miR-93 mimics were apparently lower than the scramble control, and those of the cells treated with 100 nM miR-93 mimics and CCND1 siRNA (100 nM) were even lower than those in the 50 nM treatment group. Meanwhile, cells transfected with miR-93 mimics (50 nM) showed evidently downregulated viability when compared with the scramble controls, while cells transfected with (100 nM) and CCND1 siRNA (100nM) showed even lower viability. CONCLUSIONS We showed that CCND1 is a direct target of miR-93, and the dysregulation of the miR-93/CCND1 signaling pathway is responsible for the development of ventricular remodeling.
本研究旨在确定导致心室重构的途径。
我们收集了重构心肌组织（n=18）和对照心肌组织（n=22），并使用实时 PCR 检测了这两组中 4 种 miRNA 的表达。然后，我们在在线 miRNA 数据库中搜索 miR-93 的候选基因。使用实时 PCR 和 Western blot 分析来确认调控关系。
我们发现只有 miR-93 在重构心肌组织中表达降低，并通过荧光素酶报告基因检测系统验证了 CCND1 是 miR-93 的直接靶基因，“种子序列”位于靶基因的 3'-UTR 内。此外，我们通过确定转染野生型或突变型 3'-UTR 的细胞的相对荧光素酶活性，确定了 miR-93 和 CCND1 之间的负调控关系。我们还发现，与 scramble 对照相比，用 50 nM miR-93 模拟物处理的 HL-1 细胞的 CCND1 蛋白和 mRNA 表达水平明显降低，而用 100 nM miR-93 模拟物和 CCND1 siRNA（100 nM）处理的细胞的表达水平甚至更低。同时，与 scramble 对照相比，转染 miR-93 模拟物（50 nM）的细胞活力明显降低，而转染 miR-93 模拟物（100 nM）和 CCND1 siRNA（100 nM）的细胞活力则更低。
我们表明 CCND1 是 miR-93 的直接靶标，miR-93/CCND1 信号通路的失调是导致心室重构发生的原因。
