Down-regulating miR-217-5p Protects Cardiomyocytes against Ischemia/Reperfusion Injury by Restoring Mitochondrial Function via Targeting SIRT1
Down-regulating miR-217-5p Protects Cardiomyocytes against Ischemia/Reperfusion Injury by Restoring Mitochondrial Function via Targeting SIRT1
复制标题
下调miR-217-5p通过靶向SIRT1恢复线粒体功能保护心肌细胞免受缺血/再灌注损伤
DOI：
10.1007/s10753-020-01343-5
复制
发表时间：
2020-10-16
期刊：
影响因子：
5.1
通讯作者：
Wu, Zhenhua
中科院分区：
文献类型：
--
作者：
Qi, Yujuan;Zhang, Kai;Wu, Zhenhua
下调miR-217-5p可以保护心肌细胞免受缺血/再灌注(I/R)损伤，但其在恢复I/R损伤心肌细胞线粒体功能中的作用尚不清楚。以缺氧再灌注(H/R)模型为基础，建立H9C2心肌细胞来源的缺血再灌注损伤细胞系。用四甲基偶氮唑盐比色法和流式细胞仪分别检测细胞存活率和细胞凋亡率。测定乳酸脱氢酶(LDH)和三磷酸腺苷(ATP)含量。用流式细胞仪检测细胞内活性氧(ROS)和线粒体膜电位(MMP)的变化。用TargetScan预测了miR-217-5p与Sirtuin1(SIRT1)之间的目的基因和潜在结合位点，并用双荧光素酶报告基因分析进行了验证。采用实时定量聚合酶链式反应(qRT-PCR)和Western印迹法检测SIRT1的相对表达及自噬相关基因和细胞凋亡相关基因的表达。H9C2心肌细胞经I/R处理后，细胞存活率和ATP含量降低，LDH和ROS含量增加，细胞凋亡率增加，miR-217-5p、p62和裂解caspase-3表达增加，SIRT1、LC3(轻链3)、PINK1(PTEN诱导的激酶1)、Parkin、Bcl2和c-IAP(凋亡抑制蛋白)表达减少。然而，下调miR-217-5p的表达逆转了I/R的作用，SIRT1被预测并被证实是miR-217-5p的靶点，而沉默SIRT1则逆转了下调miR-217-5p对I/R损伤细胞的影响。下调miR-217-5p可通过靶向SIRT1恢复线粒体功能，从而保护心肌细胞免受I/R损伤。
Downregulating miR-217-5p could protect cardiomyocytes against ischemia/reperfusion (I/R) injury, but its role in restoring mitochondrial function of I/R-injured cardiomyocytes remained unclear. H9C2 cardiomyocyte-derived cell line with I/R injury was establishedin vitroon the basis of hypoxia/reperfusion (H/R) model. Cell viability and apoptosis were respectively detected by MTT assay and flow cytometry. Contents of lactate dehydrogenase (LDH) and adenosine triphosphate (ATP) were determined. Flow cytometry was performed to measure the production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Target gene and potential binding sites between miR-217-5p and Sirtuin1 (SIRT1) were predicted by TargetScan and confirmed by dual-luciferase reporter assay. Relative SIRT1 and expressions of autophagy-related and apoptosis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. After I/R treatment, the viability of H9C2 cardiomyocyte-derived cell line and ATP contents were reduced, but LDH and ROS contents were increased, at the same time, cell apoptosis and the expressions of miR-217-5p, p62 and cleaved caspase-3 were increased, whereas the expressions of SIRT1, LC3 (light chain 3), PINK1 (PTEN-induced kinase 1), Parkin, Bcl-2, and c-IAP (inhibitor of apoptosis protein) were reduced. However, downregulating miR-217-5p expression reversed the effects of I/R. SIRT1 was predicted and verified to be the target of miR-217-5p, and silencing SIRT1 reversed the effects of downregulating miR-217-5p on I/R-injured cells. Downregulating miR-217-5p could help restore mitochondrial functionviatargeting SIRT1, so as to protect cardiomyocytes against I/R-induced injury.