LncRNA Dnmt3aos-DNMT3A轴介导的DNA甲基化修饰在巨噬细胞极化中的作用及机制研究

Macrophage plasticity is an important feature of innate immune cells. Macrophage phenotypes are divided into two categories, the classically activated macrophages (M1) and the alternatively activated macrophages (M2). Accumulating documents indicated that macrophage polarization played an important role in vivo under physiological conditions (e.g., ontogenesis and pregnancy) and in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer). However, mechanisms that underlie macrophage polarization remain undefined. Long non-coding RNAs (lncRNA) are a class of non-protein coding transcripts with more than 200 nucleotides in size. LncRNAs have been shown to play pivotal roles in the process of human diseases and biological process such as epigenetics and cell differentiation. Our preliminary experiments found that differentially expressed lncRNAs profiles existed in M1 and M2 macrophages, indicating that individual lncRNAs may play a role in macrophage polarization. LncRNA Dnmt3aos (DNA methyltransferase 3A, opposite strand), as a known lncRNA, locates on the Dnmt3a antisense strand. In this study, we will firstly clarify the expression relationship between Dnmt3aos and DNMT3A by suppression or overexpression methods. Then we used Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) method combined with mRNA expression profile analysis to screen the Dnmt3aos-DNMT3A axis-mediated DNA methylation-modified macrophage polarization related genes, and explore the related genes roles in macrophage polarzation. Finally, Dnmt3aos-binding protein was identified by RNA pull-down, mass spectrometry and RNA immunoprecipitation (RIP), and further study the roles and molecular mechanisms of Dnmt3aos and Dnmt3aos-binding protein complex in macrophage polarization. Our study will help to enrich the mechanism of macrophage polarization and provide new immunotherapy in macrophage-associated diseases.

申请人前期发现，LncRNA Dnmt3aos在M1/M2巨噬细胞中差异表达；下调Dnmt3aos表达能抑制巨噬细胞向M2极化，且降低DNMT3A表达，提示Dnmt3aos与DNMT3A存在一定的表达调控关系；MeDIP-Seq结果显示Dnmt3aos抑制组与对照组相比，全基因组甲基化水平存在显著差异；将差异DMR相关基因与mRNA表达谱联合分析发现，差异DMR相关基因与差异mRNA存在明显相关性。本研究，我们拟用过表达或抑制等方法，明确Dnmt3aos与DNMT3A之间表达调控关系；通过MeDIP-Seq结合表达谱联合分析，鉴定Dnmt3aos-DNMT3A轴介导的DNA甲基化修饰的、调控巨噬细胞极化的关键基因；通过RNA pull-down、质谱分析和RIP鉴定Dnmt3ao互作蛋白，并深入研究Dnmt3aos-互作蛋白复合体在巨噬细胞极化中的作用，为巨噬细胞极化研究提供新的实验依据。

申请人前期研究利用lncRNA芯片检测发现，小鼠骨髓来源巨噬细胞BMDMs在体外极化条件下(IFN-γ/LPS或IL-4)分别诱导成的M1或M2巨噬细胞中lncRNA表达谱存在明显差异，结合生物信息学分析筛选出可能调控巨噬细胞极化的差异lncRNAs；real-time qPCR对候选lncRNAs进行验证后，我们将深入研究初步锁定在M2中差异表达最显著的lncRNA Dnmt3aos上。lncRNA Dnmt3aos，一个已知的lncRNA分子，位于小鼠12号染色体上的Dnmt3a基因的反义链上，PCR和RNA荧光原位杂交实验证明Dnmt3aos主要定位在细胞核中。进一步研究发现，抑制Dnmt3aos表达后，M1巨噬细胞相关标志物如iNOS，TNF-α和IL-12的表达明显增加，而M2巨噬细胞相关标志物如Arg1、YM1、和FIZZ1的表达随之降低，且降低靶基因DNMT3A表达，提示Dnmt3aos与DNMT3A存在一定的表达调控关系。MeDIP-Seq结果显示Dnmt3aos抑制组与对照组相比，全基因组甲基化水平存在显著差异；将差异DMR相关基因与mRNA表达谱联合分析发现，差异DMR相关基因与差异mRNA存在明显相关性，并鉴定到Dnmt3aos-DNMT3A轴介导的DNA甲基化修饰的，调控巨噬细胞极化的关键基因IFN-γ。本研究为巨噬细胞极化研究提供新的实验依据。