Roles of Malat1 in response to hypoxia

我们的团队先前通过NSERC资助的项目发现了长非编码RNA Malat 1，该项目使用基因表达的平行修饰来筛选对脂质代谢至关重要的组织（即肝脏和脂肪）中的衰老和饮食调节基因。我们最近证明，低氧条件下，通过刺激AMPK/HIF-1？轴线基于这些新的发现，我们建议研究Malat 1在调节细胞和生理适应中的作用，这些适应是在低氧条件下触发的，或者是通过激活脂多糖（LPS）诱导的炎症。使用体外和体内模型，我们将：** 1-确定控制缺氧时Malat 1表达水平的分子途径。这将通过表达谱分析（北方印迹和qPCR）报告基因试验、染色质免疫沉淀（ChIP）和BRIC衰变试验进行。将在野生型小鼠中确认Malat 1在缺氧时的表达谱。** 2-确定细胞和小鼠中Malat 1缺失对急性和慢性缺氧的影响。* 将细胞和小鼠置于低氧室中或用LPS处理以研究氧消耗以及细胞和组织适应，例如生长、组织学、线粒体融合/分裂、低氧基因表达特征、糖酵解酶表达、能量代谢的变化。将特别关注肺部、肌肉、大脑和外周血管系统，并将通过基于示踪剂/造影剂的成像技术进行评估。* 3-确定Malat 1在缺氧期间发挥作用所需的功能域。*在体外，暴露于正常或低氧条件或用LPS处理的Malat 1-/-成纤维细胞将被小mascRNA结构域（被切割并留在细胞质中的61 bp序列）或全长Malat 1减去mascRNA结构域感染。或者，野生型肺和肌肉细胞将用靶向这些结构域的反义寡核苷酸处理。将测量对代谢、呼吸和生长的影响。将测试mascRNA作为miRNA样分子海绵的潜在作用，因为我们怀疑这是ChREBP的情况。Malat 1结合伴侣在缺氧过程中的表征将表明其在低氧环境中的增加是否驱动细胞适应或作为负反馈回路。* * 这是一个非常创新的研究计划，建立在我们以前的NSERC资助的Malat1项目的基础上。该计划将导致非编码RNA对细胞生物学的重要性和贡献的新兴研究取得重大进展。考虑到拟议实验的性质，它完全可以为培养在自然科学这一新领域具有高素质的人才做出积极贡献。

Our team previously uncovered the long non-coding RNA Malat1 through NSERC-funded programs using parallel modifications in gene expression to screen for genes modulated upon aging and diet in tissues critical for lipid metabolism, namely liver and adipose. We recently demonstrated that low oxygen conditions robustly increase Malat1 gene expression through the stimulation of an AMPK/HIF-1? axis. Based on these novel findings, we propose to study the contribution of Malat1 in the modulation of cellular and physiological adaptations that take place in response to hypoxia, either triggered in low oxygen conditions, or through activation of lipopolysaccharide (LPS)-induced inflammation. Using both in vitro and in vivo models, we will:******1 – Determine the molecular pathways that control the expression levels of Malat1 upon hypoxia.***This will be performed through expression profiling (Northern blotting and qPCR) reporter gene assays, chromatin immunoprecipitation (ChIP), and BRIC decay assays. Expression profiles of Malat1 upon hypoxia will be confirmed in wild-type mice.******2 – Determine the impact of Malat1 deletion in cells and mice upon acute and chronic hypoxia. ***Cells and mice will be placed in hypoxic chambers or treated with LPS to study oxygen consumption as well as cell and tissue adaptations such as changes in growth, histology, mitochondrial fusion/fission, hypoxic gene expression signature, glycolytic enzyme expression, energy metabolism. Special attention will be given to the lungs, muscles, brain, and peripheral vasculature, which will be assessed through tracer/contrast agent-based imaging techniques.******3- Determine the functional domains of Malat1 required for its effects during hypoxia.***In vitro, Malat1-/- fibroblasts exposed to normal or low oxygen conditions or treated with LPS will be infected with the small mascRNA domain (a 61 bp sequence that is cleaved and leaves to the cytoplasm) or the full-length Malat1 minus mascRNA domain. Alternatively, wild-type lung and muscle cells will be treated with anti-sense oligonucleotides targeting these domains. Effects on metabolism, respiration, and growth will be measured. The mascRNA will be tested for potential roles as an miRNA-like molecular sponge, as we suspect it is the case for ChREBP. Characterization of Malat1 binding partners over the course of hypoxia will indicate whether its increase in low oxygen settings drives cellular adaptations or serve as a negative feedback loop. ******This is a very innovative research plan that builds upon our previous NSERC-funded project on Malat1. The proposed program will lead to significant advances in the burgeoning research on the importance and contribution of non-coding RNAs to cell biology. Given the nature of the proposed experiments, it is perfectly positioned to contribute positively to the formation of people highly qualified in this novel aspect of natural sciences.