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目的 探讨氧化应激对小鼠海马神经元microRNAs(miRNAs)表达的影响及其意义。方法 原代分离培养快速老化小鼠(SAMR1)的胎鼠(孕18~19d)海马神经元,用不同浓度的H2O2刺激神经元制备氧化应激细胞模型,分别采用MTT法和TUNEL染色检测细胞活力和凋亡情况;应用miRNA芯片检测miRNAs表达谱的改变,对差异性表达的miRNAs进行生物信息学分析,筛选目标miRNAs并采用Real-timePCR方法进行验证。结果 采用不同浓度H2O2刺激后海马神经元细胞活力明显降低,细胞凋亡增多(除100μmol/L外,P均<0.01)。芯片检测结果提示氧化应激可导致小鼠海马神经元中101个miRNAs的表达发生改变(P<0.05),其中17个miRNAs表达变化在1.5倍以上。生物信息学分析示这些差异性表达的miRNAs可能参与细胞增殖、分化、凋亡、神经营养因子转运等多个生物学过程。Real-timePCR结果表明miR-135b和miR-708的表达量在氧化应激状态下升高(P<0.05)。结论 氧化应激可导致小鼠海马神经元miRNAs表达失调,这些差异性表达的miRNAs可能通过MAPK等信号通路参与神经元的氧化应激损伤过程。
Objective To investigate the effect of oxidative stress on the expression of microRNAs (miRNAs) in hippocampal neurons of mice and its significance. Methods The hippocampal neurons of fetus (pregnant 18 ~ 19d) with rapid aging mice (SAMR1) were primarily isolated and cultured. The neurons were stimulated with different concentrations of H2O2 for oxidative stress. Cell viability was measured by MTT assay and TUNEL staining. MiRNAs were detected by miRNA microarray. Bioinformatic analysis of differentially expressed miRNAs was carried out. The target miRNAs were screened and verified by Real-time PCR. Results The viability of hippocampal neurons was significantly decreased and the apoptosis increased after stimulated with different concentrations of H2O2 (P <0.01, all except 100 μmol / L). Chip test results suggest that oxidative stress can cause the expression of 101 miRNAs in hippocampal neurons of mice changed (P <0.05), of which 17 miRNAs expressed more than 1.5 times. Bioinformatics analysis showed that these differentially expressed miRNAs may be involved in many biological processes such as cell proliferation, differentiation, apoptosis, and neurotrophin transport. Real-time PCR results showed that the expression levels of miR-135b and miR-708 increased under oxidative stress (P & lt; 0.05). Conclusions Oxidative stress can lead to dysregulation of miRNAs in hippocampal neurons. These differentially expressed miRNAs may be involved in neuronal oxidative stress injury through MAPK signaling pathway.