Department of Cardiac Center, The Third Central Clinical College of Tianjin Medical University, Tianjin, China.
Department of the Ministry of Health Subsidiary Engineering Research Center for Artificial Cells, Tianjin Third Central Hospital, Tianjin, China.
J Cell Biochem. 2018 Feb;119(2):2427-2439. doi: 10.1002/jcb.26405. Epub 2017 Nov 16.
Mitochondrial dysfunction has emerged as a critical pathophysiological factor of myocardial ischemia/reperfusion (I/R) injury. A thorough understanding of mitochondrial dysfunction during I/R at the molecular level is urgently needed. One prominent microRNA, miR-410, was previously reported to be dynamically regulated in diverse cardiomyopathies, but its mechanism is unclear. In the present study, in a cardiac I/R injury mice model, the expression of miR-410 was significantly upregulated, accompanied with decreased mitochondrial function and mitophagy deficit. After an unbiased search for downstream messenger RNA targets of miR-410, effects of the target gene in mitochondrial dysfunction during I/R injury and the underlying mechanism were further explored in cultured human adult cardiac myocytes (HACMs). The results showed that MitoTracker Red-labeled HACMs mitochondria overlapped with GFP-LC3-labeled autophagosomes, suggesting the presence of mitophagy. MiR-410 expression was significantly increased in hypoxia/reoxygenation (H/R)-stimulated HACMs. MiR-410 overexpression further inhibited cell viability, ATP production, mitochondrial membrane potential and mitophagy level, and increased caspase-3 activity, Bax expression and cytochrome c release. Conversely, inhibition of miR-410 attenuated these effects. We found that miR-410 directly interacted with the 3'-untranslated region of the suppressor of high-mobility group box 1 protein (HMGB1) by Dual-Luciferase assay. Moreover, pcDNA3.1-HMGB1 pretreatment effectively reduced the inhibition effects of cell viability and mitophagy brought by H/R, while all those effects can be attenuated by pretreatment with HSPB1 siRNA transfection. Taken together, our results suggest that miR-410 may inhibit mitophagy after cardiac I/R injury by modulating HSPB1 activity via directly targeting HMGB1.
线粒体功能障碍已成为心肌缺血/再灌注（I/R）损伤的关键病理生理因素。迫切需要从分子水平深入了解 I/R 期间的线粒体功能障碍。先前有研究报道，一种突出的 microRNA（miR-410）在多种心肌病中呈动态调节，但具体机制尚不清楚。在本研究中，在心肌 I/R 损伤小鼠模型中，miR-410 的表达显著上调，伴随着线粒体功能下降和噬线粒体缺陷。miR-410 的下游信使 RNA 靶基因进行了无偏搜索，进一步在培养的人成年心肌细胞（HACMs）中探讨了靶基因在 I/R 损伤期间的线粒体功能障碍中的作用及其潜在机制。结果显示，MitoTracker Red 标记的 HACMs 线粒体与 GFP-LC3 标记的自噬体重叠，表明存在噬线粒体现象。miR-410 在缺氧/复氧（H/R）刺激的 HACMs 中表达显著增加。miR-410 过表达进一步抑制细胞活力、ATP 生成、线粒体膜电位和噬线粒体水平，并增加 caspase-3 活性、Bax 表达和细胞色素 c 释放。相反，抑制 miR-410 可减轻这些作用。我们发现，通过双荧光素酶测定，miR-410 直接与高迁移率族蛋白 1 抑制物（HMGB1）的 3'-非翻译区相互作用。此外，pcDNA3.1-HMGB1 预处理可有效减轻 H/R 引起的细胞活力和噬线粒体抑制作用，而 HSPB1 siRNA 转染预处理可减轻所有这些作用。综上所述，我们的研究结果表明，miR-410 可能通过直接靶向 HMGB1 调节 HSPB1 活性来抑制心肌 I/R 损伤后的噬线粒体作用。
