Roles of Malat1 in response to hypoxia

点击翻译按钮获取中文摘要

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.