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

脓毒症是一种发病率高、致命性强的疾病，并伴有严重的代谢紊乱。 蛋氨酸新陈代谢周期。在哺乳动物的蛋氨酸代谢中，S-腺苷蛋氨酸是 产生的，在所有细胞的代谢中占据中心位置，作为必要的甲基供体 维持DNA、RNA、组蛋白以及谷胱甘肽的正常甲基化，谷胱甘肽对细胞氧化还原至关重要 系统。蛋氨酸腺苷转移酶(MAT2A)是SAM及其基因生物合成所必需的。 通过m6A RNA甲基化在转录和转录后水平调节MAT2A的表达 MAT2AmRNA的3‘非编码区。 转录后，MAT2A稳定性的mRNA受RNA介导的m6A甲基化调节 甲基化写入者METTL16，通过甲基化MAT2AmRNA的3‘非编码区来调节MAT2AmRNA。 METTL16与lncRNA MALAT1形成络合物。然而，MALAT1在调节MAT2A中的作用不是 安全。在基因消融的MALAT1小鼠中，MAT2A基因表达上调，内毒素诱导的ROS 随着谷胱甘肽水平的升高而显著抑制。MALAT1基因缺失的小鼠对 内毒素血症致感染性休克并伴有整体m6A甲基化增加。我们的中心假设是 MALAT1通过调控MAT2A基因表达和MALAT1调节SAM的生物合成 蛋氨酸代谢途径是脓毒症的治疗靶点。具体目标是： 目的1.内毒素血症炎症的表观转录调控机制 RNA甲基化METTL16是使MAT2A的3‘非编码区甲基化的关键酶，导致 信使核糖核酸内含子的保留或衰退。LncRNA MALAT1对MATTL16的潜在调控作用 用MALAT1基因缺失小鼠的巨噬细胞与野生型对照进行比较。M6A-SEQ 将进行分析以分析内毒素血症引起的全球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 将会被分析。补充甲基供体(SAM、蛋氨酸)对内毒素诱导的影响 腹膜巨噬细胞分泌炎性细胞因子，同时竞争性抑制SAM合成 MAT2AcLEU的抑制剂可加重炎症反应。这一目标的实验结果将 为治疗策略的制定提供依据。 目的2.靶向MALAT1治疗改善蛋氨酸代谢功能和抗 对小鼠模型的炎症作用。受到MALAT1调控的强有力证据的推动 蛋氨酸循环，与AIM 1同时，反义RNA途径与高效反义 针对MALAT1的LNA GapmeR寡核苷酸将用于治疗小鼠脓毒症模型 与蛋氨酸/SAM联合应用。

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.