Roles of Malat1 in response to hypoxia

我们的团队之前通过NSERC资助的计划发现了长的非编码RNA MALAT1，使用基因表达的平行修改来筛选对脂肪代谢至关重要的组织(即肝脏和脂肪)中随年龄和饮食调节的基因。我们最近证实，低氧条件通过刺激AMPK/HIF-1？轴心。基于这些新的发现，我们建议研究MALAT1在细胞和生理适应的调节中的作用，这些适应发生在低氧条件下触发的低氧反应中，或者通过激活脂多糖(LPS)诱导的炎症来实现。使用体外和体内模型，我们将：*1确定在低氧条件下控制MALAT1表达水平的分子途径。*这将通过表达谱(Northern blotting和qPCR)报告基因分析、染色质免疫沉淀(ChIP)和BRIC衰退分析来实现。野生型小鼠缺氧时MALAT1的表达谱将得到证实。*2确定细胞和小鼠中MALAT1缺失对急性和慢性缺氧的影响。*细胞和小鼠将被放置在缺氧室或内毒素治疗中，以研究氧气消耗以及细胞和组织适应的变化，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、肌肉、脑和外周血管系统，将通过基于示踪剂/造影剂的成像技术进行评估。*3-确定MALAT1在低氧条件下的作用所需的功能结构域。*在体外，暴露于正常或低氧条件下或经脂多糖处理的MALAT1-/-成纤维细胞将感染小mascRNA结构域(被切割的61bp序列，并进入细胞质)或全长MALAT1减去mascRNA结构域。或者，野生型肺和肌肉细胞将被针对这些区域的反义寡核苷酸处理。将测量对新陈代谢、呼吸和生长的影响。MascRNA将被测试作为类似miRNA的分子海绵的潜在作用，因为我们怀疑ChREBP就是这种情况。MALAT1结合伙伴在缺氧过程中的特征将表明其在低氧环境中的增加是驱动细胞适应还是作为负反馈循环。*这是一个非常创新的研究计划，它建立在我们之前由NSERC资助的MALAT1项目的基础上。拟议的计划将导致关于非编码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。鉴于拟议中的实验的性质，它处于完美的地位，可以为形成在自然科学这一新颖方面高度合格的人做出积极贡献。

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.