Department of Laboratory, The People's Hospital of Tongchuan, Tongchuan, Shaanxi 727000, P.R. China.
Department of Laboratory, Second Affiliated Hospital of Shaanxi Chinese Traditional Medicine, Xianyang, Shaanxi 712000, P.R. China.
Int J Mol Med. 2018 Jul;42(1):290-298. doi: 10.3892/ijmm.2018.3597. Epub 2018 Mar 29.
The present study aimed to identify the association between microRNA (miRNA/miR)‑31a‑5p and the development of hypertension, and its potential molecular mechanism. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses were performed to validate the candidate miRNA and genes involved in hypertension, following which an online miRNA database search, luciferase assay, and RT‑qPCR and western blot analyses were performed to confirm the interaction between miR‑31a‑5p and TP53. A MTT assay and flow cytometric analysis were utilized to determine the effect of miR‑31a‑5p on cell growth and apoptosis. The results revealed that miR‑31a‑5p and TP53 were the candidate miRNA and gene regulating hypertension, and that TP53 was the virtual target gene of miR‑31a‑5p with a binding site located in the TP53 3' untranslated region (3'UTR). It was confirmed by luciferase activity that miR‑31a‑5p markedly reduced the luciferase activity of the Luc‑wild‑type‑TP53‑3'UTR, whereas the mutated putative miR‑31a‑5p binding located on the TP53‑3'UTR was found to eliminate such an inhibitory effect. miR‑31a‑5p had no effect on specificity protein 1, E2F transcription factor 2 or forkhead box P3 luciferase activity. Smooth muscle cells collected from spontaneously hypertensive rats treated with gold nano‑particles containing anti‑rno‑miR‑31a‑5p exhibited a lower growth rate and a higher apoptotic rate. The results of the RT‑qPCR and western blot analyses showed that miR‑31a‑5p negatively regulated the expression of TP53, and transfection with the hsa‑miR‑31a‑5p mimic significantly promoted cell growth and inhibited cell apoptosis, whereas transfection with the anti‑hsa‑miR‑31a‑5p mimic significantly suppressed cell growth and induced cell apoptosis. Taken together, these findings indicated that miR‑31a‑5p is involved in hypertension via the accelerated proliferation of arterial smooth muscle cells and inhibition of apoptosis through targeting TP53.
本研究旨在探讨 microRNA（miRNA/miR）-31a-5p 与高血压发展之间的关联及其潜在的分子机制。通过逆转录-定量聚合酶链反应（RT-qPCR）和 Western blot 分析验证候选 miRNA 和与高血压相关的基因，然后通过在线 miRNA 数据库搜索、荧光素酶测定以及 RT-qPCR 和 Western blot 分析证实 miR-31a-5p 与 TP53 之间的相互作用。通过 MTT assay 和流式细胞术分析来确定 miR-31a-5p 对细胞生长和凋亡的影响。结果表明，miR-31a-5p 和 TP53 是调节高血压的候选 miRNA 和基因，而 TP53 是 miR-31a-5p 的虚拟靶基因，其结合位点位于 TP53 3'UTR。荧光素酶活性证实，miR-31a-5p 显著降低了 Luc-野生型-TP53-3'UTR 的荧光素酶活性，而位于 TP53-3'UTR 上的突变假定的 miR-31a-5p 结合位点则消除了这种抑制作用。miR-31a-5p 对特异性蛋白 1、E2F 转录因子 2 或 forkhead box P3 荧光素酶活性没有影响。用含抗 rno-miR-31a-5p 的金纳米颗粒处理的自发性高血压大鼠的平滑肌细胞表现出较低的生长速度和较高的凋亡率。RT-qPCR 和 Western blot 分析结果表明，miR-31a-5p 负调控 TP53 的表达，转染 hsa-miR-31a-5p 模拟物显著促进细胞生长并抑制细胞凋亡，而转染抗 hsa-miR-31a-5p 模拟物则显著抑制细胞生长并诱导细胞凋亡。综上所述，这些发现表明，miR-31a-5p 通过靶向 TP53 加速动脉平滑肌细胞增殖并抑制细胞凋亡参与高血压的发生。
