MicroRNA-181b 及Toll 样受体4 在新生大鼠 神经元缺氧缺血损伤中的作用机制研究

MicroRNA-181b regulates hypoxic-ischemic brain damage through TLR4 signaling pathway

摘要:

目的 观察MicroRNA-181b（miRNA-181b）及Toll 样受体4（TLR4）对新生大鼠缺氧缺血脑损 伤（HIBD）的影响并探讨其潜在机制。方法 选取3 只1 ～ 2 日龄新生大鼠脑皮质神经元细胞原代培养7 d， 分为对照组和HIBD 组。CKK-8 法测定原代培养的脑皮质神经元细胞活性，流式细胞术检测细胞凋亡情况， 瞬时转染调节细胞内miRNA-181b 及TLR4 表达。双荧光素酶实验检测miRNA-181b 对TLR 4 基因的靶向 调控，SYBR Green 实时荧光定量PCR 观察miRNA-181b 及TLR4 mRNA 表达变化。Western blot 观察TLR4 蛋白在不同组别神经元细胞中的表达。结果 与对照组比较，miRNA-181b 在新生大鼠缺氧缺血损伤神经元 细胞中表达降低（P <0.05），miRNA-181b mimic 转染后过表达miRNA-181b 能抑制缺血缺氧对新生大鼠神 经元细胞的损伤（P <0.05）。双荧光素酶实验证实miRNA-181b 可与TLR4 mRNA 3'-UTR 直接结合，发挥 对TLR4 转录后翻译的抑制作用。进一步机制研究发现，si-TLR4 转染后抑制TLR4 表达能增加细胞活性，而 miRNA-181b 抑制剂anti-miRNA-181b 转染后降低细胞活性，anti-miRNA-181b 与si-TLR4 共转染能部分 逆转神经元细胞缺血缺氧下的损伤（P <0.05）。结论 新生大鼠HIBD 神经元细胞中miRNA-181b 能够负性 调控TLR4 表达发挥一定的神经保护作用。

Abstract:

Objective To investigate the effects of MicroRNA-181b (miR-181b) and toll like receptor 4 (TLR4) in the progress of Neonatal hypoxic-ischemic brain damage (HIBD) and the underlying mechanism. Methods Brain tissues of 1 or 2 days old SD newborn rats were isolated and cultured as primary neurons. Hypoxia ischemia cell model was established with standardized procedure. Cellular proliferation and cell apoptosis rate of primary cultured cortical neurons were determined by CKK-8 assay and flow cytometry assay, respectively. Expression of miR-181b and TLR4 was modulated by transient transfection, and the activity of TLR4 regulated by miR-181b was evaluated by a dual luciferase reporter assay. Levels of MiR-181b and TLR4 were determined by Real-time quantitative PCR and Western blot. Results Expression of miR-181b was significantly increased in hypoxic-ischemic damaged rat cortical neurons when compared with control group (P < 0.05), while overexpression of miR-181b apparently reversed hypoxia-induced cell damage (P < 0.05). Dual luciferase reporter assay demonstrated that miR-181b directly targeted the 3’-UTR of the TLR4, resulting in inhibition of the post-transcriptional translation of TLR4. Further mechanism researches revealed that knockdown of TLR4 expression by si-TLR4 transfection significantly upregulated cell viability in rat cortical neurons subjected to hypoxic-ischemic damage, which was abolished by anti-miR-181b transfection treatment. Co-transfection of anti-miR-181b and si-TLR4 in rat cortical neurons were partially attenuated hypoxia induced damage (P < 0.05). Conclusions MiR-181b protects rat cortical neurons under hypoxic-ischemic condition through regulating of TLR4.