Long noncoding RNA MALAT1 ablation reverses sepsis in mouse: epitranscriptomic mechanisms and therapeutic application

长非编码 RNA MALAT1 消融逆转小鼠败血症：表观转录组机制和治疗应用

• 批准号: 10058019
• 负责人: Yanan Tian
• 金额: $ 23.3万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-02 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058019
• 关键词: 3' Untranslated Regions; Ablation; Anabolism; Animals; Anti-Inflammatory Agents; Antioxidants; Antisense Oligonucleotides; Antisense RNA; Biological Assay; Cells; Clinic; Complex; DNA Methylation; Development; Disease; Endotoxemia; Enzymes; Gene Expression; Genetic Transcription; Glutathione; Glutathione Disulfide; Histones; Human; Immune; Incidence; Inflammation; Inflammatory; Inflammatory Response; Introns; Knockout Mice; MALAT1 gene; Mediating; Messenger RNA; Metabolic; Metabolism; Methionine; Methionine Metabolism Pathway; Methylation; Modeling; Morbidity - disease rate; Mus; Oxidation-Reduction; Oxidative Stress; Peritoneal Macrophages; Positioning Attribute; RNA; RNA Splicing; RNA Stability; RNA methylation; Reader; Reporting; Resistance; Resolution; Role; S-Adenosylmethionine; Sepsis; Septic Shock; Site; Structure; Supplementation; System; Terminator Codon; Testing; Therapeutic; Treatment Efficacy; Untranslated RNA; cecal ligation puncture; cytokine; epitranscriptomics; improved; inhibitor/antagonist; mRNA Decay; macrophage; methionine adenosyltransferase; mouse model; therapeutic evaluation; therapeutic target

Sepsis is a disease with high incidence and lethality and is accompanied by profound metabolic disturbances of the methionine metabolism cycle. In mammalian methionine metabolism, S-adenosylmethionine (SAM) is produced, which occupies a central position in the metabolism of all cells as an essential methyl donor to maintain normal methylation of DNA, RNA, histones as well glutathione which is important for cellular redox system. Methionine adenosyltransferase (MAT2A) is essential for the biosynthesis of SAM and the gene expression of MAT2A is regulated at transcriptional and post-transcriptional levels through m6A RNA methylation of the 3’UTR of MAT2A mRNA. Post-transcriptionally, the mRNA of MAT2A stability is regulated by the m6A methylation mediated by RNA methylation writer METTL16 which regulates MAT2A mRNA by methylating 3’UTR of the MAT2A mRNA. METTL16 forms a complex with a lncRNA MALAT1. The role of MALAT1 in regulating MAT2A however, is not clear. In genetically ablated MALAT1 mice, MAT2A gene expression is up-regulated and LPS-induce ROS was significantly suppressed with increases in the levels of glutathione. MALAT1 null mice are highly resistant to the septic shock induced by endotoxemia with increased global m6A methylation. Our central hypothesis is MALAT1 regulates SAM biosynthesis by controlling MAT2A gene expression and MALAT1-regulated methionine metabolism pathway is a therapeutic target for sepsis. The specific aims are: Aim 1. Epitranscriptomic mechanisms of endotoxemia-induced inflammation through regulating m6A RNA methylation METTL16 which is the key enzyme for methylating 3’UTR of MAT2A, resulting in either intron retention or decay of the mRNA. The potential regulatory effects of lncRNA MALAT1 on MATTL16 will be analyzed using macrophage from MALAT1 null mice in comparison with the wild type control. The m6A-seq analysis will be performed to analyze the effects of endotoxemia-induced alteration of global m6A methylation and identify the critical methylation sites. The regulatory effect of MALAT1 on the gene expression of MAT2A will be analyzed. The effects of supplementation of methyl donor (SAM, Methionine) counteracting LPS-induced inflammatory cytokines secretion in peritoneal macrophage, while the inhibition of SAM synthesis by competitive inhibitor of MAT2A cLEU exacerbates the inflammatory responses. The experimental results in this aim will provide basis for therapeutic strategy development . Aim 2. Therapeutic targeting MALAT1 for improving methionine metabolic functions and anti- inflammatory function in mouse models. Motivated by the strong evidence of MALAT1 in regulating methionine cycle, concurrently with Aim 1, the antisense RNA approach with a highly effective antisense oligonucleotide, LNA GapmeR against MALAT1 will be tested for treatment of sepsis in mouse model in combination with methionine/SAM.

脓毒症是一种发病率高、致死率高、并伴有严重代谢紊乱的疾病 蛋氨酸代谢周期。在哺乳动物蛋氨酸代谢中，S-腺苷甲硫氨酸（SAM）是 产生，作为重要的甲基供体，在所有细胞的代谢中占据中心地位 维持 DNA、RNA、组蛋白以及谷胱甘肽的正常甲基化，这对细胞氧化还原很重要 系统。蛋氨酸腺苷转移酶 (MAT2A) 对于 SAM 和基因的生物合成至关重要 MAT2A 的表达通过 m6A RNA 甲基化在转录和转录后水平进行调节 MAT2A mRNA 的 3’UTR。 转录后，MAT2A mRNA 稳定性受 RNA 介导的 m6A 甲基化调节 甲基化写入器 METTL16，通过甲基化 MAT2A mRNA 的 3'UTR 来调节 MAT2A mRNA。 METTL16 与 lncRNA MALAT1 形成复合物。然而，MALAT1 在调节 MAT2A 中的作用并不 清除。在基因消除的 MALAT1 小鼠中，MAT2A 基因表达上调，并且 LPS 诱导的 ROS 水平降低 随着谷胱甘肽水平的增加而显着抑制。 MALAT1 缺失小鼠对 内毒素血症引起的败血性休克，整体 m6A 甲基化增加。我们的中心假设是 MALAT1通过控制MAT2A基因表达和MALAT1调节来调节SAM生物合成 蛋氨酸代谢途径是脓毒症的治疗靶点。具体目标是： 目标 1. 通过调节 m6A 内毒素血症诱导炎症的表观转录组机制 RNA 甲基化 METTL16 是甲基化 MAT2A 3'UTR 的关键酶，导致 mRNA 的内含子保留或衰变。 lncRNA MALAT1 对 MATTL16 的潜在调控作用将 与野生型对照相比，使用 MALAT1 缺失小鼠的巨噬细胞进行分析。 m6A 序列 将进行分析以分析内毒素血症诱导的整体 m6A 甲基化改变的影响 并确定关键的甲基化位点。 MALAT1对MAT2A基因表达的调控作用 将被分析。补充甲基供体（SAM、蛋氨酸）对抗 LPS 诱导的影响 腹腔巨噬细胞分泌炎性细胞因子，而竞争性抑制 SAM 合成 MAT2A cLEU 抑制剂会加剧炎症反应。该目标的实验结果将 为治疗策略的制定提供依据。 目标 2. 治疗靶向 MALAT1 以改善蛋氨酸代谢功能并抗- 小鼠模型中的炎症功能。受到 MALAT1 调节作用的有力证据的推动 蛋氨酸循环，与目标 1 同时进行，具有高效反义的反义 RNA 方法 针对 MALAT1 的寡核苷酸 LNA GapmeR 将在小鼠模型中测试其败血症的治疗效果 与蛋氨酸/SAM 组合。