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-2 3'UTR-WT与 mimic-NC共转染组,Bcl-2 3'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 hy-poxia 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 in-duced 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 experi-ments.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 mid-dle 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-2 3′UTR-WT and mimic-NC co-transfection group,the fluorescence activity of Bcl-2 3′-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 ex-pression significantly decreased the apoptosis of SH-SY5Y cells(P<0.001).Conclusion miR-181a can target Bcl-2 and down-reg-ulation of miR-181a can inhibit the OGD/R injury induced apoptosis of SH-SY5Y nerve cells by up-regulating the expression of Bcl-2.