Lv Guangwei, Shao Suxia, Dong Hua, Bian Xiaohua, Yang Xingwei, Dong Shimin
Department of ICU, The Third Hospital of Hebei Medical University, Shijiazhuang City, Hebei Province, P.R. China.
J Cell Biochem. 2014 Jan;115(1):93-101. doi: 10.1002/jcb.24636.
Reactive oxygen species (ROS)-induced cardiac myocyte injury resulting from changes in the expression levels of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. The purpose of this study was to determine the potential roles of microRNA-214 (miR-214) in hydrogen peroxide (H2O2)-mediated gene regulation in cardiac myocytes. In this study, we used quantitative real-time RT-PCR (qRT-PCR) to demonstrate that miR-214 was upregulated in cardiac myocytes after treatment with H2O2. We transfected cells with pre-miR-214 to upregulate miR-214 expression and transfected cells with a miR-214 inhibitor (anti-miR-214) to downregulate miR-214 expression. H2O2-induced cardiac cell apoptosis was detected by flow cytometry. The level of apoptosis was increased by the miR-214 inhibitor and decreased by pre-miR-214. Therefore, we believe that miR-214 plays a positive role in H2O2-induced cardiac cell apoptosis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is constitutively active and is considered to be the primary downregulator of the pro-oncogenic PI3K/Akt pathway. Western blot analysis revealed that the expression of the PTEN protein in cardiac myocytes decreased after H2O2 induction. Anti-miR-214 increased PTEN protein expression level, in contrast, pre-miR-214 decreased the PTEN protein expression level in cultured cardiac myocytes. These results indicate that PTEN is regulated by miR-214 and serves as an important target of miR-214 in cardiac myocytes. In conclusion, miR-214 is sensitive to H2O2 stimulation, and miR-214 protects cardiac myocytes against H2O2-induced injury via one of its targets, PTEN.
活性氧（ROS）诱导的心肌细胞损伤是由多个基因表达水平的变化引起的，在多种心脏病的发病机制中起关键作用。本研究的目的是确定微小RNA-214（miR-214）在过氧化氢（H2O2）介导的心肌细胞基因调控中的潜在作用。在本研究中，我们使用定量实时RT-PCR（qRT-PCR）证明，用H2O2处理后心肌细胞中miR-214上调。我们用pre-miR-214转染细胞以上调miR-214表达，并用miR-214抑制剂（抗miR-214）转染细胞以下调miR-214表达。通过流式细胞术检测H2O2诱导的心肌细胞凋亡。miR-214抑制剂增加了凋亡水平，而pre-miR-214降低了凋亡水平。因此，我们认为miR-214在H2O2诱导的心肌细胞凋亡中起积极作用。10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN）具有组成性活性，被认为是促癌PI3K/Akt途径的主要下调因子。蛋白质印迹分析显示，H2O2诱导后心肌细胞中PTEN蛋白的表达降低。相反，抗miR-214增加了PTEN蛋白表达水平，而pre-miR-214降低了培养心肌细胞中PTEN蛋白的表达水平。这些结果表明，PTEN受miR-214调控，并作为miR-214在心肌细胞中的重要靶点。总之，miR-214对H2O2刺激敏感，并且miR-214通过其靶点之一PTEN保护心肌细胞免受H2O2诱导的损伤。
