脑梗死过程中微RNA-181d和肿瘤坏死因子α的表达及机制研究

The expression of microRNA-181d and tumor necrosis factor-αin cerebral infarction and its mechanism

目的检测临床脑梗死患者和小鼠脑缺血再灌注模型血清中miR-181d和肿瘤坏死因子-α(TNF-α的表达变化,并探讨其相关性。方法选取发病48h内入住承德医学院附属医院的脑梗死患者32例(脑梗死损伤组),另选取同期13例健康体检者(对照组)。取受试者外周血血清,采用实时定量PCR(qRT-PCR)和酶联免疫吸附试验(ELISA)分别检测血清中miR-181d和TNF-α的表达。同时采用线栓法建立小鼠大脑中动脉阻塞(MCAO)模型,分别在0、1、3、7、14和28d检测血清miR-181d和TNF-α的表达水平。分析miR-181d和TNF-α表达的关系。在人胚肾HEK-293T细胞通过荧光素酶报告基因方法,并在人外周血单核细胞THP-1中利用蛋白质印迹法(Western blot)检测miR-181d对TNF-α的抑制作用。结果脑梗死损伤组患者血清miR-181d表达水平低于对照组(P<0.05)。在小鼠MCAO模型中,随着脑梗死病程延长,血清miR-181d表达水平逐渐下调。TNF-α表达水平在脑梗死患者和小鼠MCAO模型中均上调,并与miR-181d的表达呈负相关(r=-0.703 8,P=0.000 4;r=0.965 3,P=0.001 8)。miR-181d可以结合到TNF-α的3′非翻译区(3′-UTR)并抑制后者的表达。在人外周血单核细胞THP-1中,miR-181d可以抑制TNF-α蛋白的产生。结论 miR-181d的表达下调可能导致TNF-α升高,从而使炎性反应加重。

Objective To investigate the expression of microRNA-181d(miR-181d)and tumor necrosis factor-α(TNF-α)in serum samples derived from patients with cerebral infarction and cerebral ischemia-reperfusion mice,and explore the relationship between circulating miR-181d and TNF-α.Methods The serum samples of 32 cases of patients with cerebral infarction admitted to the Affiliated Hospital of Chengde Medical University within 48 h after onset(the brain infarction injury group)and 13 healthy volunteers(the control group)were collected.The real-time quantitative PCR(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of miR-181d and TNF-αin the serum samples.At the same time,the middle cerebral artery occlusion(MCAO)mice models were established by using the suture method.The expression levels of serum miR-181d and TNF-αwere detected at 0,1,3,7,14 and 28 d,respectively.The relationship between miR-181d and TNF-αwas analysed.Furthermore,the inhibitory effect of miR-181d on TNF-αwas determined by dual-luciferase reporter gene assay and western blot analysis in human embryonic kidney HEK-293T cells and peripheral blood mononuclear THP-1 cells,respectively.Results The serum expression of miR-181d in the brain infarction injury group was significantly lower than that in the control group(P<0.05).In the MCAO mice models,the serum expression of miR-181d was gradually down-regulated as the course of cerebral infarction prolonged.The serum expression levels of TNF-αwere up-regulated in both patients and MCAO mice models,and were negatively correlated with the expression levels of miR-181d,respectively(r=-0.703 8,P=0.000 4;r=-0.965 3,P=0.001 8).Moreover,miR-181d negatively regulate the expression of TNF-αby directly binding to its 3′-UTR.In human peripheral blood mononuclear cell THP-1,miR-181d inhibited the expression of TNF-αprotein.Conclusion Down-regulation of miR-181d leads to an increase of TNF-αexpression,which may aggravate the inflammatory response.