巨噬细胞非编码RNA Linc00305在红系分化中的调控功能和作用机制研究

体内红系终末分化需要血岛中心巨噬细胞微环境支持。目前对中心巨噬细胞的调控了解非常有限，但已发现巨噬细胞炎症与骨髓增生异常综合症、再障和慢性病贫血等疾病红系异常分化密切相关。我们前期发现非编码RNA Linc00305在单核/巨噬细胞中富集，并在原代人骨髓巨噬细胞中表达，其在THP-1中过表达显著促进炎性通路的激活。本项目计划在骨髓巨噬细胞中明确Linc00305的炎症调控功能，通过巨噬-红系祖细胞共培养和巨噬细胞特异转基因小鼠等方法明确Linc00305在常规和贫血刺激条件下对红系终末分化的调节作用。同时通过RNA-Pulldown、RIP、Co-IP等技术阐明Linc00305参与炎症调节的具体机制，并用贫血患者骨髓切片分析中心巨噬细胞Linc00305表达与贫血发生的相关性。研究可为深入了解红系造血微环境、炎性贫血的治疗、以及体外高效红细胞分化等重大需求提供理论依据和新的作用靶点。

Terminal erythroid differentiation in vivo requires intimate interactions of the erythroblasts to the central macrophage they surrounding. Although current knowledge to the gene regulatory network of central macrophage is very limited. Increasing evidences support the association of dysregulated macrophage inflammation to the abnormal erythroid differentiation in multiple diseases like myelodysplastic syndrome, aplastic anemia and chronic disease anemia. We have previously identified a human monocyte/macrophage-enriched long non-coding RNA Linc00305, and detected its expression also in the primary human bone marrow macrophages. Overexpression of Linc00305 in THP-1 cells significantly promotes NF-κB activation and upregulates inflammatory genes. In this project, we will first confirm the role of Linc00305 in inflammatory regulation in primary human bone marrow macrophages. The potential effects of linc00305 on terminal erythroid differentiation will be assessed by co-culture of genetic modified macrophages with the cord blood-derived erythroblasts. Macrophage-specific Linc00305 transgenic mice will be established to examine the in vivo effect of Linc00305 on erythropoiesis, and on stress-induced erythropoiesis. RNA-Pulldown, RIP and Co-IP will be applied to elucidate the molecular mechanism through which Linc00305 regulates inflammation. Finally, the level of Linc00305 within the erythroid central macrophage will be examined in human bone marrow biopsies to show the potential correlation of Linc00305 expression and anemia severity. The study would provide new insight into the micro-environment of erythropoiesis and holds promising potential in inflammatory anemia therapy and in the efficient in vitro differentiation to produce mature erythrocytes.