摘要
目的皮层是响应脑缺血缺氧最为敏感的组织之一,基于前期深度测序技术,我们筛选获得响应脑缺血缺氧应激的皮层区目标基因miR-181a及Bcl-2。本研究旨在SH-SY5Y细胞株氧糖剥夺/复糖复氧模型验证二者靶向调控关系及功能,明确miR-181a—Bcl-2调控网络在OGD/R诱导的神经细胞凋亡中的作用。方法采用线栓法构建大鼠缺血缺氧再灌注损伤模型,脑切片TTC染色及行为学评分法评估模型。应用qRT-PCR及Western Blot验证目标基因的表达。生物信息学分析miR-181a与Bcl-2的靶向结合位点并比对结合位点的保守性,双荧光素酶报告基因实验验证miR-181a与Bcl-2靶向结合的特异性。采用OGD/R细胞模型体外模拟脑缺血再灌注损伤,检测凋亡相关蛋白表达及Hoechst荧光染色评估细胞凋亡。结果大鼠大脑中动脉阻塞后miR-181a、Bcl-2表达变化趋势相反。RNA hybird软件预测miR-181a可结合Bcl-2的3′-UTR区,且结合区域高度保守。双荧光素酶报告基因实验发现,相对于Bcl-23′UTR-WT与mimic-NC共转染组,Bcl-23′UTR-WT与miR-181a mimic共转染后的荧光活性更低(P<0.001),而Bcl-2-Mut与miR-181a mimic共转染组,荧光活性无显著差异(P>0.05)。分别用miR-181a的模拟物及抑制物转染OGD/R诱导的SH-SY5Y细胞,miR-181a可以抑制Bcl-2 mRNA及其蛋白的表达水平(P<0.001)。过表达miR-181a显著增加了SH-SY5Y细胞的凋亡(P<0.001),而抑制miR-181a表达可使SH-SY5Y细胞凋亡显著降低(P<0.001)。结论miR-181a可靶向结合Bcl-2,下调miR-181a可通过促进Bcl-2的表达进而抑制SH-SY5Y神经细胞OGD/R损伤诱导的细胞凋亡。
Objective The cortex is one of the most sensitive tissues in response to cerebral hypoxia-ischemia.Based on the previous deep sequencing technology,we screened and obtained cortical target genes miR-181a and Bcl-2 that closely respond to cerebral hypoxia ischemia stress.The aim of this study is to verify the above targeted relationships and functions in SH-SY5Y cell line induced by the oxygen-glucose deprivation/reperfusion(OGD/R),and to clarify the role of miR-181a-Bcl-2 regulatory network in OGD/R induced nerve cell apoptosis.Methods The model of hypoxic-ischemic and reperfusion injury in rats was established by suture method,and the model was evaluated by TTC staining and behavioral score.Target gene expression was verified by qRT-PCR and Western Blot.Bioinformatics was used to analyze the target binding sites of miR-181a and Bcl-2 and compare the conservation of the binding sites.The specificity of targeted binding between miR-181a and Bcl-2 was confirmed through dual-luciferase reporter gene experiments.The OGD/R model was used to simulate cerebral ischemia-reperfusion injury in vitro.Cell apoptosis was detected by apoptosis-related protein expression and Hoechst fluorescence staining.Results The expression of miR-181a and Bcl-2 was reversed after middle cerebral artery occlusion in rats.RNA hybird software predicted that miR-181a could bind to the 3′-UTR region of Bcl-2 mRNA,and the nucleotides in the binding region were highly conserved.Dual luciferase reporter gene assay showed that compared with the Bcl-23′UTR-WT and mimic-NC co-transfection group,the fluorescence activity of Bcl-23′-UTR and miR-181a mimic co-transfection group was lower(P<0.001).There was no significant difference in the fluorescence activity of Bcl-2-Mut co-transfected with miR-181a mimic group(P>0.05).After the OGD/R model was constructed,SH-SY5Y cells were transfected with miR-181a mimics and inhibitors,respectively.It was found that miR-181a could inhibit the expression levels of Bcl-2 mRNA and protein(P<0.001).Overexpression of miR-181a significantly increased the apoptosis of SH-SY5Y cells(P<0.001),while inhibition of miR-181a expression significantly decreased the apoptosis of SH-SY5Y cells(P<0.001).Conclusion miR-181a can target Bcl-2 and down-regulation of miR-181a can inhibit the OGD/R injury induced apoptosis of SH-SY5Y nerve cells by up-regulating the expression of Bcl-2.
作者
袁珊
杨玉莹
许梅梅
胡广泽
高蕊
YUAN Shan;YANG Yuying;XU Meimei;HU Guangze;GAO Rui(Department of Biochemistry,School of Medicine/The Key Laboratory of Ministry of Education for Xinjiang Endemic&Ethnic Disease,Shihezi University,Shihezi,Xinjiang 832000,China)
出处
《石河子大学学报（自然科学版）》
CAS
北大核心
2024年第1期91-100,共10页
Journal of Shihezi University(Natural Science)
基金
国家自然科学基金项目(32260142)
新疆生产建设兵团区域创新引导计划项目(2021BB002)。
