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目的:观察神经细胞氧化损伤时microRNA-199a(miRNA-199a)和Sirt1表达的变化,探讨miRNA-199a对Sirt1的调控作用。方法:不同浓度双氧水(600μmol/L,1 200μmol/L)刺激体外培养的SH-SY5Y细胞12h造成损伤。MTT检测细胞活力,DCFA-DA探针检测细胞内活性氧水平,Hochest染色分析细胞凋亡,RT-PCR检测miRNA-199a的表达,细胞免疫荧光和Western blot检测Sirt1蛋白水平。结果:600μmol/L和1 200μmol/L双氧水刺激SH-SY5Y后细胞活力显著下降,分别降低27.0%和53.6%;ROS荧光强度细胞凋亡显著上升且具有浓度依赖性;miRNA-199a的表达明显上升,分别上升23.0%和51.0%;Sirt1蛋白表达量下降具有浓度依赖性。结论:双氧水刺激SH-SY5Y神经细胞时miRNA-199a表达升高,Sirt1的表达降低。Sirt1水平降低与miRNA-199a通过调控Sirt1表达参与氧化损伤有关[1]。
OBJECTIVE: To observe the changes of microRNA-199a (miRNA-199a) and Sirt1 in oxidative damage of neurons and to explore the role of miRNA-199a in regulating Sirt1. Methods: SH-SY5Y cells cultured in vitro were exposed to different concentrations of H2O2 (600μmol / L, 1 200μmol / L) for 12h. Cell viability was measured by MTT assay, reactive oxygen species (ROS) levels were detected by DCFA-DA probe, apoptosis was detected by Hochest staining, miRNA-199a expression was detected by RT-PCR, Sirt1 protein was detected by immunofluorescence and Western blot. Results: The viability of SH-SY5Y cells was significantly decreased after treated with 600μmol / L H2O2 and 1 200μmol / L H2O2, respectively, with a decrease of 27.0% and 53.6%, respectively. ROS fluorescence intensity significantly increased and concentration-dependently, and the expression of miRNA- , Up 23.0% and 51.0% respectively; the expression of Sirt1 protein decreased in a concentration-dependent manner. Conclusion: When hydrogen peroxide stimulates SH-SY5Y neurons, the expression of miRNA-199a is up-regulated and the expression of Sirt1 is down-regulated. Decreased Sirt1 level is associated with the involvement of miRNA-199a in oxidative damage through the regulation of Sirt1 expression [1].