Long Non-coding RNA HOTAIR Promotes Ischemic Infarct Through Up-regulating The Expression Of NOX2

BackgroundMounting studies have illustrated an important role of HOTAIR in cancer progress,but few studies have reported itsfunction in brain disease,The study of ischemic cerebral infarction has not been reported.The pathophysiological mechanism of cerebral infarction,the oxidative stress caused damage to brain tissue is the key,it can lead to reactive oxygen species(ROS)generated in the brain increased further aggravate cerebral ischemia and hypoxia,leading to cell apoptosis and death,nerve function defect.Many enzymes can produce ROS,NADPH oxidase 2(NOX2)has attracted much attention,the focal cerebral ischemia occurs,the expression of NOX2 increased,resulting in increased production of ROS,the irreversible brain injury.The purpose of this study is to study the effect of HOTAIR on the expression level of NOX2 protein under hypoxia,and further reveal the possible functional significance and influence of HOTAIR in the process of cerebral infarction formation.Methods1.the establishment of MCAO mice induced by hypoxia ischemia brain artery model,and HT22 in hippocampal neurons in vitro hypoxia,respectively using RT-PCR method and Western Blot method to detect the different time of ischemia and hypoxia cerebral infarct and isolated HT22 cells HOTAIR and NOX2 protein expression.2.transfection of pcDNA-HOTAIR,si-HOTAIR to HT22 cells respectively,to detect the expression level of NOX2 protein,and to observe the effect on apoptosis of HT22 cells.3.the possible association between HOTAIR and NOX2 proteins was further investigated by RNA pull-down and RNA binding protein immunoprecipitation(RIP)Assay.Results1.the expression of HOTAIR and mRNA in the solitary brain infarction and HT22 cells in the mouse brain was detected successfully by RT-PCR,and showed a time dependent manner.2.by Western blot,the expression of NOX2 protein in the cerebral solitary infarction foci and HT22 cells in mice was up regulated and showed a time dependent manner.3.with the increase of hypoxia time,the percentage of TUNEL staining and the percentage of apoptotic positive cells in HT22 cells increased significantly.4.in HT22 cells,the percentage of apoptotic positive cells in TUNEL transfected with pcDNA-HOTAIR was significantly higher than that in the control group.Meanwhile,the products of NOX2 protein increased significantly.5.The silencing of HOTAIR gene leads to a marked reduction in NOX2 protein production in HT22 cells,while the percentage of apoptotic cells in TUNEL staining is markedly decreased.6.RNA pull-down experiments confirmed that HOTAIR and NOX2 protein can be highly specific binding,HOTAIR can bind NOX2 protein,so that its expression increased.7.RNA immunoprecipitation experiments confirmed that the specific binding of RNA to NOX2 was HOTAIR,and there was a specific association between HOTAIR and NOX2.ConclusionHOTAIR can promote the hypoxic ischemic brain infarction by regulating the expression of NOX2,which can increase our understanding of the molecular mechanism of ischemic cerebral infarction.