School of Medicine, Creighton University, 2500 California Plaza, Omaha, NE, 68178-0010, USA.
Biology Department, Creighton University, 2500 California Plaza, Omaha, NE, 68178-0100, USA.
J Neuroinflammation. 2021 Jan 6;18(1):16. doi: 10.1186/s12974-020-02051-5.
Microglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity.
Microglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity.
We report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity.
Our data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.
小胶质细胞是中枢神经系统中固有免疫的、具有吞噬功能的细胞。受到微生物信号（如细菌脂多糖（LPS）、病毒 RNA 或炎性细胞因子）刺激的促炎小胶质细胞具有神经毒性，与多种神经退行性疾病的发病机制有关。长非编码 RNA（lncRNA）作为重要的组织特异性调节分子，指导细胞分化和功能状态，可能有助于指导小胶质细胞的促炎反应。目前已鉴定出促炎小胶质细胞中上调的 lncRNA，如 NR_126553 或 2500002B13Rik，现称为 Nostrill（诱导型一氧化氮合酶转录调控基因间 lncRNA 基因座），这增加了我们对中枢神经系统固有免疫中分子机制的理解。
采用微胶质细胞基因表达谱分析和 qRT-PCR 鉴定新型长基因间非编码 RNA Nostrill，该 RNA 在 LPS 刺激的小胶质细胞系、LPS 刺激的原代小胶质细胞和 LPS 注射的小鼠皮质组织中均上调。采用沉默和过表达研究、RNA 免疫沉淀、染色质免疫沉淀、染色质分离 RNA 纯化分析和 qRT-PCR 研究该长非编码 RNA 在小胶质细胞中的功能。体外实验研究沉默 LPS 刺激的小胶质细胞中的新型长非编码 RNA 对神经毒性的影响。
我们在此报告了基因间 lncRNA NR_126553 或 2500002B13Rik 的特征，现称为 Nostrill（诱导型一氧化氮合酶转录调控基因间 lncRNA 基因座）。Nostrill 在 LPS 刺激的 BV2 细胞、原代小鼠小胶质细胞和 LPS 注射小鼠的皮质组织中诱导表达。Nostrill 的诱导表达依赖于 NF-κB，沉默 Nostrill 可降低 LPS 刺激的 BV2 和原代小胶质细胞中诱导型一氧化氮合酶（iNOS）的表达和一氧化氮（NO）的产生。过表达 Nostrill 可增加 iNOS 的表达和 NO 的产生。RNA 免疫沉淀实验表明，Nostrill 在 LPS 刺激后与 NF-κB 亚基 p65 发生物理结合。沉默 Nostrill 可显著减少 NF-κB p65 和 RNA 聚合酶 II 向 iNOS 启动子的募集，并降低 iNOS 基因座处 iNOS 基因的 H3K4me3 激活组蛋白修饰。体外研究表明，沉默 Nostrill 可降低 LPS 刺激的小胶质细胞中的小胶质细胞神经毒性。
我们的数据表明 NF-κB 诱导的 Nostrill 具有新的调节作用，并表明 Nostrill 作为 iNOS 转录的共激活因子发挥作用，通过调节表观遗传染色质重塑导致小胶质细胞中一氧化氮的产生。Nostrill 可能是减少神经退行性变中小胶质细胞中 iNOS 介导的炎症过程相关神经毒性的靶点。
