A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p
A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p
复制标题
DOI：
10.7150/thno.27285
复制
发表时间：
2018-01-01
期刊：
影响因子：
12.4
通讯作者：
Wang, Jianxun
中科院分区：
文献类型：
--
作者：
Li, Mengyang;Ding, Wei;Wang, Jianxun
非编码RNA（Non-coding RNA，ncRNA）在包括心血管疾病在内的多种疾病中发挥着重要的调控作用，被认为是生理和病理过程中的重要参与者。环状RNA（circular RNA，circRNA）是近年来发现的一类RNA，在哺乳动物的转录组中占有相当大的比例，并在心血管系统中大量表达。然而，这些circRNA在缺血性心脏病中的调节功能在很大程度上仍然未知。在此，我们研究了钠/钙交换器1（ncx 1）基因转录的circRNA，命名为circNCX 1，在氧化应激诱导的心肌细胞凋亡在缺血性心肌injury.Methods的作用：采用发散聚合酶链反应（PCR）扩增circRNA。通过桑格测序和RNase R消化验证circNCX 1的环状结构。荧光原位杂交（FISH）检测circNCX 1的亚细胞定位。为了检测circNCX 1在氧化应激过程中的表达模式和功能，H9 c2细胞和新生大鼠心肌细胞用H2 O2或缺氧-复氧（H/R）处理。从机制上讲，通过AGO 2-IP和RNA下拉测定来检查circNCX 1与miRNA的相互作用。通过蛋白质印迹和荧光素酶报告基因检测circNCX 1在靶基因表达中的调节作用。结果：circNCX 1可作为内源性miR-133 a-3 p海绵体，促进心肌细胞凋亡，并对活性氧自由基（reactive oxygen species，ROS）产生应答。由于circNCX 1与miR-133 a-3 p的竞争性结合，miR-133 a-3 p对促凋亡基因细胞死亡诱导蛋白（CDIP 1）的抑制活性降低。在小鼠心肌细胞和心脏组织中敲低circNCX 1可降低CDIP 1的水平并减轻凋亡和I/R损伤。结论：我们的研究结果揭示了一种新的调控途径，包括circNCX 1，miR-133 a-3 p和CDIP 1，参与心肌细胞凋亡。该通路可能作为缺血性心脏病的潜在治疗途径。
Non-coding RNAs (ncRNAs) are considered major players in physiological and pathological processes based on their versatile regulatory roles in different diseases including cardiovascular disease. Circular RNAs (circRNAs), a newly discovered class of RNAs, constitute a substantial fraction of the mammalian transcriptome and are abundantly expressed in the cardiovascular system. However, the regulatory functions of these circRNAs in ischemic cardiac disease remain largely unknown. Here, we investigated the role of a circRNA transcribed from the sodium/calcium exchanger 1 (ncx1) gene, named circNCX1, in oxidative stress-induced cardiomyocyte apoptosis during ischemic myocardial injury.Methods: Divergent polymerase chain reaction (PCR) was conducted to amplify the circRNA. The circular structure of circNCX1 was verified by Sanger sequencing and RNase R digestion. The subcellular localization of circNCX1 was detected by fluorescence in situ hybridization ( FISH). To test the expression pattern and function of circNCX1 during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with H2O2 or hypoxia-reoxygenation (H/R). Mechanistically, the interaction of circNCX1 with miRNA was examined by AGO2-IP and RNA pull-down assays. The regulatory role of circNCX1 in target gene expression was tested by western blot and luciferase reporter assays. At the animal level, we constructed a myocardial ischemia-reperfusion (I/R) mouse model to analyze the effect of circNCX1 on heart function, cardiomyocyte apoptosis and cardiac remodeling.Results: circNCX1 was increased in response to reactive oxygen species (ROS) and promotes cardiomyocyte apoptosis by acting as an endogenous miR-133a-3p sponge. Due to competitive binding of circNCX1 to miR-133a-3p, the suppressive activity of pro-apoptotic gene cell death-inducing protein (CDIP1) by miR-133a-3p was reduced. Knockdown of circNCX1 in murine cardiomyocytes and heart tissues reduced the levels of CDIP1 and attenuated the apoptosis and I/R injury.Conclusions: Our findings reveal a novel regulatory pathway that comprises circNCX1, miR-133a-3p and CDIP1, that is involved in cardiomyocyte apoptosis. This pathway may serve as a potential therapeutic avenue for ischemic heart diseases.