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该研究旨在运用生物信息学方法预测固有免疫信号分子hsa-mi R-146b与脑卒中关联的分子调控网络。实验运用UCSC基因组浏览器、人类mi RNA疾病数据库、TF-mi RNA调控数据库、mi RNA靶基因预测验证数据库和Genecards数据库研究hsa-mi R-146b的上游转录因子、下游靶基因及信号通路的多个调控途径,绘制hsa-mi R-146b的核心调控网络图。采用脂多糖(lipopolysaccharide,LPS)刺激慢病毒感染的小胶质细胞BV2细胞,采用实时定量PCR检测早期生长反应基因1(early growth response 1,EGR1)、Toll样受体4(Toll-like receptor 4,TLR4)、mi R-146b、脑源性神经营养因子(brain derived neurotrophic factor,BDNF)和基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)的m RNA水平变化,对hsa-mi R-146b调控网络进行验证。UCSC数据库中显示,hsa-mi R-146b在多个物种中具有高度保守性。生物信息学分析显示,hsa-mi R-146b受转录因子EGR1调控的同时,又调控下游TLR4、BDNF和MMP9等39个靶基因。所有基因构成一个以hsa-mi R-146b为核心的调控网络,在脑卒中的发生与发展中起着重要作用。在LPS刺激的BV2细胞中,EGR1、TLR4、mi R-146b、BDNF和MMP9m RNA水平升高,而RNA慢病毒技术干扰小胶质细胞中的EGR1的表达后,TLR4、mi R-146b和MMP9 m RNA水平降低,BDNF升高,表明EGR1是调节mi R-146b表达和下游信号分子TLR4、BDNF和MMP9的关键信号分子。综上所述,生物信息学方法预测并初步验证了hsa-mi R-146b分子在脑卒中疾病中的调控网络,为深入阐明hsa-mi R-146b在脑卒中疾病中的机制奠定了实验基础。
The aim of this study was to predict the molecular regulatory network associated with stroke by using the bioinformatics method, hsa-mi R-146b. The experiment used UCSC genome browser, human miRNA disease database, TF-mi RNA regulation database, miRNA target gene prediction verification database and Genecards database to study the upstream transcription factors, downstream target genes and signal pathways of hsa-mi R-146b A regulatory approach, draw hsa-mi R-146b core regulatory network map. The microglia BV2 cells infected with lentivirus were stimulated by lipopolysaccharide (LPS). The expression of early growth response 1 (EGR1), Toll-like receptor 4 , TLR4, mi R-146b, brain derived neurotrophic factor (BDNF) and matrix metalloproteinase 9 (MMP9) authenticating. The UCSC database shows that hsa-mi R-146b is highly conserved across multiple species. Bioinformatics analysis showed that hsa-mi R-146b regulates 39 TLR4, BDNF and MMP9 target genes under the regulation of transcription factor EGR1. All genes form a regulatory network centered on hsa-mi R-146b, which plays an important role in the occurrence and development of stroke. In LPS-stimulated BV2 cells, the levels of EGR1, TLR4, mi R-146b, BDNF and MMP9mRNA were elevated, whereas TLR4, mi R-146b and MMP9 were up-regulated after RNA lentivirus technology interfered with the expression of EGR1 in microglia The decrease of m RNA and the increase of BDNF indicate that EGR1 is a key signaling molecule that regulates mi R-146b expression and downstream signaling molecules TLR4, BDNF and MMP9. In summary, bioinformatics methods predict and preliminary verify the regulatory network of hsa-mi R-146b in stroke disease, which lays the foundation for further elucidating the mechanism of hsa-mi R-146b in stroke diseases .