Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, Illinois 60611, USA.
Am J Physiol Heart Circ Physiol. 2011 Oct;301(4):H1519-30. doi: 10.1152/ajpheart.01080.2010. Epub 2011 Aug 12.
microRNA-210 (miR-210) is upregulated in hypoxia, but its function in cardiomyocytes and its regulation in response to hypoxia are not well characterized. The purpose of this study was to identify upstream regulators of miR-210, as well as to characterize miR-210's function in cardiomyocytes. We first showed miR-210 is upregulated through both hypoxia-inducible factor (HIF)-dependent and -independent pathways, since aryl hydrocarbon nuclear translocator (ARNT) knockout mouse embryonic fibroblasts (MEF), lacking intact HIF signaling, still displayed increased miR-210 levels in hypoxia. To determine the mechanism for HIF-independent regulation of miR-210, we focused on p53 and protein kinase B (Akt). Overexpression of p53 in wild-type MEFs induced miR-210, whereas p53 overexpression in ARNT knockout MEFs did not, suggesting p53 regulates miR-210 in a HIF-dependent mechanism. Akt inhibition reduced miR-210 induction by hypoxia, whereas Akt overexpression increased miR-210 levels in both wild-type and ARNT knockout MEFs, indicating Akt regulation of miR-210 is HIF-independent. We then studied the effects of miR-210 in cardiomyocytes. Overexpression of miR-210 reduced cell death in response to oxidative stress and reduced reactive oxygen species (ROS) production both at baseline and after treatment with antimycin A. Furthermore, downregulation of miR-210 increased ROS after hypoxia-reoxygenation. To determine a mechanism for the cytoprotective effects of miR-210, we focused on the predicted target, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3), known to induce cell death. Although miR-210 reduced AIFM3 levels, overexpression of AIFM3 in the presence of miR-210 overexpression did not reduce cellular viability either at baseline or after hydrogen peroxide treatment, suggesting AIFM3 does not mediate miR-210's cytoprotective effects. Furthermore, HIF-3α, a negative regulator of HIF signaling, is targeted by miR-210, but miR-210 does not modulate HIF activity. In conclusion, we demonstrate a novel role for p53 and Akt in regulating miR-210 and demonstrate that, in cardiomyocytes, miR-210 exerts cytoprotective effects, potentially by reducing mitochondrial ROS production.
miR-210（miR-210）在低氧条件下上调，但它在心肌细胞中的功能及其对低氧的反应尚未得到很好的描述。本研究的目的是鉴定 miR-210 的上游调控因子，并研究 miR-210 在心肌细胞中的功能。我们首先表明，miR-210 通过缺氧诱导因子（HIF）依赖性和非依赖性途径上调，因为芳香烃核转位蛋白（ARNT）敲除的小鼠胚胎成纤维细胞（MEF）缺乏完整的 HIF 信号通路，但其在低氧条件下仍显示出 miR-210 水平的增加。为了确定 miR-210 非 HIF 依赖性调节的机制，我们集中研究了 p53 和蛋白激酶 B（Akt）。在野生型 MEF 中转染 p53 可诱导 miR-210，而在 ARNT 敲除 MEF 中转染 p53 则不能，表明 p53 通过 HIF 依赖性机制调节 miR-210。Akt 抑制可降低低氧诱导的 miR-210 诱导，而 Akt 过表达可增加野生型和 ARNT 敲除 MEF 中的 miR-210 水平，表明 Akt 对 miR-210 的调节是不依赖 HIF 的。然后，我们研究了 miR-210 在心肌细胞中的作用。miR-210 的过表达可降低氧化应激引起的细胞死亡，并降低基础水平和抗霉素 A 处理后的活性氧（ROS）产生。此外，低氧复氧后 miR-210 的下调会增加 ROS。为了确定 miR-210 细胞保护作用的机制，我们集中研究了预测的靶标，凋亡诱导因子，线粒体相关 3（AIFM3），已知其可诱导细胞死亡。尽管 miR-210 降低了 AIFM3 的水平，但在 miR-210 过表达的情况下过表达 AIFM3，无论是在基础水平还是在过氧化氢处理后，细胞活力均未降低，这表明 AIFM3 并不介导 miR-210 的细胞保护作用。此外，HIF-3α，HIF 信号的负调节剂，是 miR-210 的靶标，但 miR-210 不调节 HIF 活性。总之，我们证明了 p53 和 Akt 在调节 miR-210 方面具有新的作用，并证明在心肌细胞中，miR-210 发挥细胞保护作用，可能通过减少线粒体 ROS 的产生。
