Long T Y, Jing R, Kuang F, Huang L, Qian Z X, Yang T L
Cardiovascular Department, The Xiangya Hospital of Central South University, Changsha City, Hunan Province, China.
Department of Cardiac Surgery, The First Affiliated Hospital of Xiamen University, Xiamen City, Fujian Province, China.
Braz J Med Biol Res. 2017 Mar 23;50(4):e5861. doi: 10.1590/1414-431X20175861.
Myocardial ischemia is a major cause of death and remains a disease with extremely deficient clinical therapies and a major problem worldwide. Cold inducible RNA-binding protein (CIRBP) is reported to be involved in multiple pathological processes, including myocardial ischemia. However, the molecular mechanisms of myocardial ischemia remain elusive. Here, we first overexpressed CIRBP by transfection of pc-CIRBP (pcDNA3.1 containing coding sequenced for CIRBP) and silenced CIRBP by transfection of small interfering RNA targeting CIRBP (siCIRBP). pcDNA3.1 and the negative control of siCIRBP (siNC) were transfected into H9C2 cells to act as controls. We then constructed a cell model of myocardial ischemia through culturing cells in serum-free medium with hypoxia in H9C2 cells. Subsequently, AlamarBlue assay, flow cytometry and western blot analysis were used, respectively, to assess cell viability, reactive oxygen species (ROS) level and apoptosis, and expression levels of IκBα, p65 and Bcl-3. We demonstrated that CIRBP overexpression promoted cell proliferation (P<0.001), inhibited cell apoptosis (P<0.05), reduced ROS level (P<0.001), down-regulated phosphorylated levels of IκBα and p65 (P<0.01 or P<0.001), and up-regulated expression of Bcl-3 (P<0.001) in H9C2 cells with myocardial ischemia. The influence of CIRBP knockdown yielded opposite results. Our study revealed that CIRBP could protect H9C2 cells against myocardial ischemia through inhibition of NF-κB pathway.
心肌缺血是主要的死亡原因,并且仍然是一种临床治疗极为匮乏的疾病,是全球范围内的一个重大问题。据报道,冷诱导RNA结合蛋白(CIRBP)参与包括心肌缺血在内的多种病理过程。然而,心肌缺血的分子机制仍然不清楚。在这里,我们首先通过转染pc-CIRBP(包含CIRBP编码序列的pcDNA3.1)来过表达CIRBP,并通过转染靶向CIRBP的小干扰RNA(siCIRBP)来沉默CIRBP。将pcDNA3.1和siCIRBP的阴性对照(siNC)转染到H9C2细胞中作为对照。然后,我们通过在无血清培养基中对H9C2细胞进行缺氧培养来构建心肌缺血细胞模型。随后,分别使用AlamarBlue检测、流式细胞术和蛋白质印迹分析来评估细胞活力、活性氧(ROS)水平和细胞凋亡,以及IκBα、p65和Bcl-3的表达水平。我们证明,在心肌缺血的H9C2细胞中,CIRBP过表达促进细胞增殖(P<0.001),抑制细胞凋亡(P<0.05),降低ROS水平(P<0.001),下调IκBα和p65的磷酸化水平(P<0.01或P<0.001),并上调Bcl-3的表达(P<0.001)。CIRBP敲低的影响产生了相反的结果。我们的研究表明,CIRBP可通过抑制NF-κB途径保护H9C2细胞免受心肌缺血的影响。