脑缺血再灌注损伤后miR-29a及AQP4的表达及关系
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摘要
目的:探讨脑缺血再灌注损伤(CIRI)后miR-29a和水通道蛋白4(AQP4)的表达及相互关系。方法:通过Targetscan软件预测及双萤光素酶报告实验验证AQP4为miR-29的靶基因;将20只雄性成年C57BL/6J小鼠随机分为4组:假手术组、脑缺血后再灌注(I/R)6 h、12 h、24 h组,每组5只,建立CIRI模型。通过采用实时定量PCR法(real time PCR)比较随再灌注时间的逐渐延长各组缺血区域(主要是缺血半暗带)miR-29a的表达情况,同时免疫荧光法检测AQP4表达;另外,将新生小鼠原代培养的星型胶质细胞分为正常组、脂质体转染miR-29a mimics组和miR-29a inhibitor共3组,建立体外糖氧剥夺/复氧损伤(OGD/RX)模型后,采用real time PCR和免疫印迹法(Western Blot)分别比较3组miR-29a及AQP4的表达。结果:Targetscan软件预测miR-29与AQP4 mRNA的3'UTR区存在可能的结合位点,双萤光素酶报告实验验证miR-29是与AQP4序列结合的。体内实验中,随再灌注时间的逐渐延长,与对照组相比,I/R组缺血半暗带区域内miR-29的表达明显下降(P<0. 01);与miR-29a表达趋势相对应,AQP4在半暗带区的表达增强。体外实验中,miR-29a mimics和miR-29a inhibitor转染星型胶质细胞后,可分别增强或抑制miR-29a,而同时抑制或增强AQP4的表达;OGD/RX后,miR-29a表达下降而AQP4的表达增高(P<0. 05)。结论:CIRI后miR-29a显著下调,伴随AQP4的表达升高,提示miR-29a可能通过对星型胶质细胞上AQP4的负调控改善CIRI,从而对CIRI后脑水肿发挥保护作用。
Objective: To explore the expression of microRNA29a(miR-29a) and aquaporin 4(AQP4) after cerebral ischemic/reperfusion(I/R) injury and their relationship.Methods: AQP4 as the target gene of miR-29 was predicted and identified by Targetscans oftware and biluciferase experiment. Transient focal ischemia was induced in C57BL/6J mice by middle cerebral artery occlusion(MCAO) for 1 h.Twenty male C57BL/6J mice were randomly divided into four groups: sham group and cerebral ischemia-reperfusion groups(I/R for 6 h, 12 h and 24 h). The levels of miR-29 were detected by real time PCR and the expression of AQP4 was evaluated by immunofluorescence. Astrocytes derived from newborn mice were cultured and divided into control, miR-29 a mimic and miR-29 a inhibitor groups.The expression of miR-29 a and AQP4 were detected by real time PCR and Western Blot.Results:Targetscan predicted that AQP4 mRNA was the target gene of miR-29 and the 3'UTR region of AQP4 mRNA binding to miR-29. Then biluciferase experiment confirmed the combination of miR-29 with AQP4 sequence. With the gradual extension of reperfusion time, the expression of miR-29 was significantly decreased in the ischemic penumbra(P<0. 05). Corresponding to the trend of miR-29,AQP4 was upregulating expressed in the cerebral penumbra. In vitro experiments, transfection of miR-29 a mimics or inhibitor strengthened or suppressed the level of miR-29 a, respectively. Meanwhile,after performing OGD/RX on astrocytes, we discovered that APQ4 and miR-29 a showed contrary expressing trend(P<0. 05).Conclusion: The expression of miR-29 a is significantly down-regulated in the ischemic penumbra after CIRI accompanied by up-regulation of AQP4 on astrocytes, which implies that miR-29 a possibly targets on AQP4, and negatively regulates AQP4 to exert protective effect on brain edema after CIRI.
Objective: To explore the expression of microRNA29a(miR-29a) and aquaporin 4(AQP4) after cerebral ischemic/reperfusion(I/R) injury and their relationship.Methods: AQP4 as the target gene of miR-29 was predicted and identified by Targetscans oftware and biluciferase experiment. Transient focal ischemia was induced in C57BL/6J mice by middle cerebral artery occlusion(MCAO) for 1 h.Twenty male C57BL/6J mice were randomly divided into four groups: sham group and cerebral ischemia-reperfusion groups(I/R for 6 h, 12 h and 24 h). The levels of miR-29 were detected by real time PCR and the expression of AQP4 was evaluated by immunofluorescence. Astrocytes derived from newborn mice were cultured and divided into control, miR-29 a mimic and miR-29 a inhibitor groups.The expression of miR-29 a and AQP4 were detected by real time PCR and Western Blot.Results:Targetscan predicted that AQP4 mRNA was the target gene of miR-29 and the 3'UTR region of AQP4 mRNA binding to miR-29. Then biluciferase experiment confirmed the combination of miR-29 with AQP4 sequence. With the gradual extension of reperfusion time, the expression of miR-29 was significantly decreased in the ischemic penumbra(P<0. 05). Corresponding to the trend of miR-29,AQP4 was upregulating expressed in the cerebral penumbra. In vitro experiments, transfection of miR-29 a mimics or inhibitor strengthened or suppressed the level of miR-29 a, respectively. Meanwhile,after performing OGD/RX on astrocytes, we discovered that APQ4 and miR-29 a showed contrary expressing trend(P<0. 05).Conclusion: The expression of miR-29 a is significantly down-regulated in the ischemic penumbra after CIRI accompanied by up-regulation of AQP4 on astrocytes, which implies that miR-29 a possibly targets on AQP4, and negatively regulates AQP4 to exert protective effect on brain edema after CIRI.
引文
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[2]Xie X,Peng L,Zhu J,et al.miR-145-5p/Nurr1/TNF-alpha signaling-induced microglia activation regulates neuron injury of acute cerebral ischemic/reperfusion in rats[J].Front Mol Neurosci,2017,10:383.
[3]Ouyang YB,Lu Y,Yue S,et al.miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo[J].Neurobiol Dis,2012,45(1):555-563.
[4]Shi G,Liu Y,Liu T,et al.Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury[J].Exp Brain Res,2012,216(2):225-230.
[5]Wang W,Jiang B,Sun H,et al.Prevalence,incidence,and mortality of stroke in China:Results from a nationwide population-based survey of 480 687 adults[J].Circulation,2017,135(8):759-771.
[6]Zhu SM,Xiong XX,Zheng YY,et al.Propofol inhibits aquaporin 4 expression through a protein kinase C-dependent pathway in an astrocyte model of cerebral ischemia/reoxygenation[J].Anesth Analg,2009,109(5):1 493-1 499.
[7]Schratt GM,Tuebing F,Nigh EA,et al.A brain-specific microRNA regulates dendritic spine development[J].Nature,2006,439(7 074):283-289.
[8]Hollander JA,Im HI,Amelio AL,et al.Striatal microRNA controls cocaine intake through CREB signalling[J].Nature,2010,466(7 303):197-202.
[9]Liu DZ,Tian Y,Ander BP,et al.Brain and blood microRNA expression profiling of ischemic stroke,intracerebral hemorrhage,and kainate seizures[J].J Cereb Blood Flow Metab,2010,30(1):92-101.
[10]Lee ST,Chu K,Jung KH,et al.MicroRNAs induced during ischemic preconditioning[J].Stroke,2010,41(8):1 646-1 651.
[11]Tan KS,Armugam A,Sepramaniam S,et al.Expression profile of MicroRNAs in young stroke patients[J].PLoS One,2009,4(11):e7689.
[12]Jeyaseelan K,Herath WB,Armugam A.MicroRNAs as therapeutic targets in human diseases[J].Expert OpinTher Targets,2007,11(8):1 119-1 129.
[13]Papadopoulos MC,Saadoun S,Binder DK,et al.Molecular mechanisms of brain tumor edema[J].Neuroscience,2004,129(4):1 011-1 020.