miR-290在外源性硫化氢保护大鼠脑缺血再灌注损伤中的表达及功能研究

Oxidative stress plays an important role in the cerebral ischemia reperfusion injury(CRI).MicroRNAs could promote and maintain the activation of oxidative stress via ERK or p38MAPK pathway.MiR-290 is the most abundant miRNA after IS. Our preliminary studies have found that the levels of miR-290 significantly increased in MACO rat model treated with exogenous hydrogen sulfide(H2S); in addition, the phosphorylation of ERK and p38MAPK were also increased in this model. Therefore, we propose that H2S may regulate miR-290 and promote phosphorylation of ERK and p38MAPK and motivate dissociation and shifting of Nrf2 and Keap1.In this study, we will assess the levels of miR-290, ERK and p38MAPK by real-time PCR and Western blot .Screen out and verify the expression of miR-290, and make full attempt to figure out whether ERK3 is the target gene of miR-290 by Dual-Luciferase reporter gene assay. Furthermore, we will figure out whether miRNA overexpression or expression inhibition or treatment with exogenous H2S have any influence on cerebral protection by anti-oxidative stress. It would open a new way for developing therapeutic strategy for IS.

氧化应激在缺血性脑卒中(IS)后再灌注损伤中起重要作用。ERK、p38MAPK通路活化是氧化应激的重要环节。MiRNA在氧化应激启动和维持中发挥关键作用。MiR-290在IS后高表达，我们前期研究发现外源性硫化氢（H2S）干预缺血再灌注损伤大鼠模型的脑组织中miR-290显著升高，ERK和p38磷酸化增强，提出外源性H2S进入细胞后通过调控miR-290表达，促进ERK和p38磷酸化，刺激Nrf2和Keap1解离并从胞浆移位到胞核发挥抗氧化作用。本研究拟用Real-time PCR、Western blot等技术，验证miR-290在外源性H2S脑保护作用中的表达，通过报告基因分析确定ERK3是否为miR-290靶基因，然后通过miRNA过表达、表达抑制和外源性H2S干预SH-SY5Y神经细胞，明确H2S是否通过调控miR-290，启动抗氧化发挥脑保护作用，为IS防治药物研发提供新思路。

本研究通过体内外实验中构建的缺血再灌注模型，筛选、验证在外源性硫化氢对脑缺血再灌注损伤保护过程中差异表达的miRNA，预测差异表达miRNA的靶基因；观察外源性硫化氢对该靶基因在缺血再灌注中的影响。通过上、下游信号分子对miR-290表达的影响探讨其在脑缺血再灌注中高表达的分子机制；深入研究外源性硫化氢对氧化应激所致神经细胞损伤保护过程中miR-290对其下游靶基因表达的调控作用及机制。结果发现缺血再灌注后的损伤的神经元中miR-290表达上调，ERK3是miR-290的靶基因。外源性硫化氢可以通过调节miR-290来抑制ERK3的表达和磷酸化，从而达到抗氧化、保护神经细胞的作用，为缺血性脑卒中提供新的治疗靶点。项目资助发表SCI论文1篇，中文论文2篇，待发表1篇，培养博士1名，硕士1名，均已经获得学位。项目投入经费22.48万元，支出20.74万元，各项支出与预算基本相符。