大鼠原代星形胶质细胞牵张损伤后差异表达microRNA的芯片筛选及生物信息学分析

目的探究大鼠原代星形胶质细胞体外牵张损伤后微小核糖核酸（microRNA）表达谱差异，并通过生物信息学分析预测差异表达microRNA在星形胶质细胞被损伤诱导活化后的作用。方法使用细胞损伤控制仪Ⅱ型构建大鼠原代星形胶质细胞体外牵张损伤模型，阀门压力设置为40 PSI，模拟重型颅脑损伤。损伤后24 h收集对照组及损伤组星形胶质细胞总RNA，使用Agilent microRNA芯片检测差异表达microRNA，通过R语言软件构建火山图及聚类分析热图；利用DIANA TOOLS和miRDB数据库预测microRNA的靶基因，并对靶基因集进行基因本体论（GO）和京都基因和基因组百科全书（KEGG）富集分析；使用 STRING数据库构建蛋白互作（PPI）网络，应用Cytoscape软件筛选关键基因。结果大鼠原代星形胶质细胞体外牵张损伤24 h后共有9个microRNA差异表达［|Log2 Fold Change(FC)|≥0.26，P＜0.05］，其中5个表达上调，分别是rno-miR-34a-5p、rno-miR-34b-5p、rno-miR-129-5p、rno-miR-140-3p和rno-miR-7a-5p；4个表达下调，分别是rno-miR-199a-3p、rno-miR-199a-5p、rno-miR-221-3p和rno-miR-1306-3p。rno-miR-34b-5p差异表达倍数最为显著，对其进行靶基因预测共获得154个基因，进一步的GO和KEGG富集分析发现，rno-miR-34b-5p靶基因参与蛋白磷酸酶2A结合及磷脂酶C活性正向调节，并调控磷脂酶D信号通路、MAPK信号通路及PI3K-AKT信号通路，与颅脑创伤（TBI）后神经修复及再生密切相关。同时使用靶基因集构建PPI网络，筛选5个关键基因，分别为Tp53、Notch1、Kitlg、Pdgfrb和Pdgfra。结论大鼠原代星形胶质细胞体外牵张损伤后rno-miR-34b-5p显著升高，生物信息学预测rno-miR-34b-5p靶向抑制Notch1及 PDGFRA基因的表达，降低PI3K-AKT信号通路活性，可能使损伤星形胶质细胞向A1型转化，阻碍TBI后神经修复及再生。本研究为TBI后康复治疗提供了潜在的作用新靶点及新的研究方向。

Objective To explore the difference of microRNA expression profile in primary astrocytes of rats after stretch injury in vitro, and predict the role of differentially expressed microRNA in astrocytes after injury and activation by bioinformatics analysis. Methods The primary astrocyte stretch injury model of rats was established by using the cell damage controller type Ⅱ, and the valve pressure was set at 40 PSI to simulate severe brain injury. The total RNA of astrocytes in the control group and the injured group was collected 24 hours after injury. The Agilent microRNA chip was used to detect the differential expression of microRNAs. Volcanic maps and cluster analysis heat maps were constructed using R language software. DIANA TOOLS and miRDB databases were used to predict the target genes of microRNA, and enrich and analyze the target gene set in gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG). The protein interaction(PPI) network was constructed using STRING database, and key genes were screened using Cytoscape software. Results There were 9 differentially expressed microRNA in rat primary astrocytes 24 h after stretch-induced injury in vitro(|Log2FC|≥0.26,P<0.05). Among them, 5 microRNA were upregulated, including rno-mir-34a-5p, rno-mir-34b-5p, rno-mir-129-5p, rno-mir-140-3p, and rno-mir-7a-5p. 4 microRNA were downregulated, including rno-mir-199a-3p, rno-mir-199a-5p, rno-mir-221-3p, and rno-mir-1306-3p. Rno-mir-34b-5p was the most significantly changed microRNA in rat primary astrocyte after stretch-induced injury. A total of 154 predicted genes of rno-mir-34b-5p were obtained, and these genes were involved in the processes of protein phosphatase 2A binding and positive regulation of phospholipase C activities. Moreover, they regulated the phospholipase D signaling pathway, MAPK signaling pathway and PI3K-AKT signaling pathway, which were highly related to neurological protection and regeneration after traumatic brain injury(TBI). Following PPI network analysis identified 5 hub genes, including Tp53, Notch1, Kitlg, Pdgfrb, and Pdgfra. Conclusion Rno-mir-34b-5p might inhibit the PI3K-AKT signaling pathway by suppressing Notch1 and PDGFR expression, which may induce type A1 astrocyte and impede neurological protection and regeneration after TBI. Our findings may provide a potential therapeutic target for TBI treatment.