摘要
目的探讨miR-378在氧-糖剥夺(OGD)致小鼠成神经瘤N2A细胞缺血损伤中的作用及其可能分子机制研究。方法离体培养N2A细胞,采用3 h OGD/24 h复糖复氧模拟缺血/再灌细胞模型,3-(4,5-二甲基噻唑)-2,5-二苯基四氮唑溴盐(MTT)比色法检测N2A细胞生存率,蛋白印迹(Western blot)检测caspase-3蛋白表达,实时定量RT-PCR检测miR-378和caspase-3 mRNA表达,荧光素酶报告基因验证miR-378对caspase-3 mRNA 3'非翻译区(UTR)的直接调控作用。结果 miR-378在N2A细胞内表达水平,随着3 h OGD复糖复氧时间的增加,而显著降低(P<0.05,n=5);在3 h OGD/24 h复糖复氧致N2A细胞缺血损伤中,上调或下调miR-378的表达水平可显著提高或降低N2A细胞生存率(P<0.05,n=6);而在非OGD条件下,miR-378表达水平改变对N2A细胞生存率则无明显影响;同样,在3 h OGD/24 h复糖复氧条件下,miR-378表达水平的改变可显著影响caspase-3蛋白表达(P<0.05,n=3)而非caspase-3 mRNA表达水平;共转染pri-miR-378可显著抑制含caspase-3 mRNA 3'-UTR的荧光素酶报告基因的表达(P<0.05,n=6)。结论 miR-378可通过负性调节caspase-3蛋白表达水平,来减轻OGD致N2A细胞缺血损伤,所获实验结果有助于从miRNAs水平为缺血性脑卒中提供潜在的治疗靶点。
Objective To explore the role of miR-378 in oxygen-glucose deprivation （OGD）-induced N2A cell is- chemic injury and its mechanism. Methods 3 h OGD/24 h reoxygenation of N2A cells was established to mimic isehemia/reperfusion in vitro, and the N2A cell survival rate was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide （MTT） assay. The caspase-3 protein level and the expression level of miR-378 and caspase-3 mRNA were determined by Western blot and real time RT-PCR respectively. Luciferase reporter assay was performed to identify the direct binding of miR-378 with 3＇-UTR of caspase-3 mRNA. Results The miR-378expression level decreased significantly （ P 〈 0. 05, n = 5 group） in response to reoxygenation time following 3 h OGD treatment in N2A cells. Under the condition of 3 h OGD/24 h reoxygenation non-OGD, up-and down-regula- tion of miR-378 expression level by transfection with pri-miR-378 or anti-miR-378 failed to enhance nor reduce N2A cell survival rate （P 〈 0. 05, n = 6）. Similarly, miR-378 negatively regulated caspase-3 protein expression levels, but no significant affection on caspase-3 mRNA expression levels was observed in N2A cells after 3 h OGD/24 h reoxygenation treatment. In addition, overexpression of miR-378 by co-transfection with pri-miR-378 signifi- cantly decreased the luciferase activities which were expressed by plasmid containing 3 ＇-UTR of caspase-3 mRNA in N2A cells （P 〈0. 05, n =6）. Conclusions miR-378 attenuates OGD-induced N2A cell ischemic injuries by neg-atively regulating caspase-3 protein expression, which may provide a potential therapeutic target for ischemic stroke at miRNAs level.
出处
《基础医学与临床》
CSCD
北大核心
2013年第11期1446-1451,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(31071048
31171147
31200895)
科技部973计划前期研究专项(2011CB512109)
北京市自然科学基金(7132025
7132070)
