Zhan Renya, Xu Kangli, Pan Jianwei, Xu Qingsheng, Xu Shengjie, Shen Jian
Department of Neurosurgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China.
Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China.
Biochem Biophys Res Commun. 2017 Aug 26;490(3):700-706. doi: 10.1016/j.bbrc.2017.06.104. Epub 2017 Jun 17.
This study aimed to explore the mechanism of lncRNA MEG3 on angiogenesis after cerebral infarction (CI).
The rat brain microvascular endothelial cells (RBMVECs) isolated from rat was used to establish CI model, which were treated with oxygen-glucose deprivation/reoxygenation (OGD/R). The genes mRNA and protein expression levels in RBMVECs were determined by the quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, respectively. The flow cytometry was used to measured cell apoptosis and intracellular reactive oxygen species (ROS) generation. The RBMVECs activities was detected by MTT method. The RNA-immunoprecipitation (RIP) assay was used to detect the interaction between MEG3 and p53, and the relationship between p53 and NOX4 was proved by chromatin co-immunoprecipitation (chip) assay.
The results showed that OGD or OGD/R increased MEG3 and NOX4 expression, and there was positive correlation between MEG3 and NOX4 expression in RBMVECs. Next, knockdown of MEG3 indicated that inhibition of MEG3 was conducive to protect RBMVECs against OGD/R-induced apoptosis, with decreased NOX4 and p53 expression, further enhanced pro-angiogenic factors (HIF-1α and VEGF) expression, and reduced intracellular ROS generation. And then the RIP and CHIP assay demonstrated that MEG3 could interacted with p53 and regulated its expression, and p53 exerted significant binding in the promoters for NOX4, suggesting that MEG3 regulated NOX4 expression via p53. At last, knockdown of NOX4 indicated that inhibition of NOX4 protected RBMVECs against OGD/R-induced apoptosis, with increased cell viability and pro-angiogenic factors expression, and reduced ROS generation.
LncRNA MEG3 was an important regulator in OGD/R induced-RBMVECs apoptosis and the mechanism of MEG3 on angiogenesis after CI was reduced ROS by p53/NOX4 axis.
本研究旨在探讨长链非编码RNA MEG3在脑梗死（CI）后血管生成中的作用机制。
采用从大鼠分离的大鼠脑微血管内皮细胞（RBMVECs）建立CI模型，并用氧糖剥夺/复氧（OGD/R）处理。分别采用定量实时聚合酶链反应（RT-qPCR）和蛋白质印迹法测定RBMVECs中基因的mRNA和蛋白质表达水平。采用流式细胞术检测细胞凋亡和细胞内活性氧（ROS）生成。采用MTT法检测RBMVECs活性。采用RNA免疫沉淀（RIP）试验检测MEG3与p53之间的相互作用，并通过染色质共免疫沉淀（chip）试验证实p53与NOX4之间的关系。
结果显示，OGD或OGD/R可增加MEG3和NOX4表达，且RBMVECs中MEG3与NOX4表达呈正相关。接下来，敲低MEG3表明抑制MEG3有利于保护RBMVECs免受OGD/R诱导的凋亡，NOX4和p53表达降低，促血管生成因子（HIF-1α和VEGF）表达进一步增强，细胞内ROS生成减少。然后，RIP和CHIP试验表明MEG3可与p53相互作用并调节其表达，p53在NOX4启动子上有显著结合，提示MEG3通过p53调节NOX4表达。最后，敲低NOX4表明抑制NOX4可保护RBMVECs免受OGD/R诱导的凋亡，细胞活力和促血管生成因子表达增加，ROS生成减少。
长链非编码RNA MEG3是OGD/R诱导的RBMVECs凋亡的重要调节因子，CI后MEG3促进血管生成的机制是通过p53/NOX4轴降低ROS水平。
