lncRNA AK085865调控巨噬细胞极化在CVB3诱导的病毒性心肌炎中的作用及其机制研究

Viral myocarditis (VM) is a kind of clinical common infectious disease of cardiovascular system. Accumulating documents indicated that cardiac injury was associated with heart-infiltrating macrophages and differentially polarizated macrophages played a role in VM. However, mechanisms that underlie macrophage polarization remain largely undefined. Our preliminary experiments found that differentially expressed lncRNA profiles existed in in heart infiltrated macrophages from CVB3 infected male and female mice, indicating that individual lncRNA may play a role in macrophage polarization. By the advanced bioinformatics analysis and reverse prediction of lncRNA target gene, combined with in vitro functional experiments, we found that AK085865 expression difference was most obvious, and high expression in M2 macrophages. AK085865 Knockdown could inhibit BMDMs to M2 polarization, suggesting that AK085865 is involved in the macrophage polarization process, but its mechanism is largely unknown..In the present study, we aim to explore the role of AK085865 in the regulation of macrophage polarization by gain or loss of functions. Meanwhile, we will identify protein targets binding to AK085865 by RNA pull-down and mass spectrometry analysis and explore the molecular mechanism of lncRNA-protein complex in regulating macrophage polarization. Besides, we will explore the possible mechanisms of AK085865 in regulating macrophage polarization and its potential role in VM by constructing a knockout mouse model. Our study will help to further understand the mechanism of VM and provide a novel therapeutic strategy in treating inflammatory cardiac disease.

申请人前期发现，CVB3感染的雌雄小鼠心脏浸润巨噬细胞lncRNA表达谱存在显著差异。通过生物信息学分析及lncRNA靶基因反向预测，结合体外功能实验，发现AK085865差异最为明显，且高表达于M2型巨噬细胞，敲减AK085865可抑制BMDMs向M2极化，提示AK085865参与了巨噬细胞极化，然而其机制不明。本研究中，我们拟通过阻断或过表达等方法，探讨AK085865在巨噬细胞极化中的调控作用；通过RNA pulldown及质谱分析鉴定AK085865结合蛋白，进而深入研究AK085865与结合蛋白的表达调控关系及AK085865-蛋白质复合体调控巨噬细胞极化的下游分子机制；通过构建基因敲除小鼠模型，观察AK085865对病毒性心肌炎发病的影响，试图阐明AK085865调控巨噬细胞极化在病毒性心肌炎中的作用及其分子机制，从而为病毒性心肌炎发病机制研究和生物治疗提供新的理论和实验依据。

积累的证据显示，巨噬细胞极化在柯萨奇病毒 b3(CVB3)诱导的病毒性心肌炎发生发展过程中起着重要作用。然而，巨噬细胞极化的机制仍有待探索。在这里，我们试图确定新的和功能重要的长非编码RNA(lncRNAs)在巨噬细胞极化和调控病毒性心肌炎中的作用机制。我们发现 lncRNA AK085865是巨噬细胞极化的重要调节因子。抑制lncRNA AK085865可降低M2巨噬细胞的表型表达，同时促进M1表型的极化。此外，lncRNA AK085865-/-小鼠对CVB3诱导的病毒性心肌炎的敏感性增加。我们观察到巨噬细胞明显偏向M1，而lncRNA AK085865-/-心肌炎小鼠M2数量显著减少。此外，我们发现 lncRNA AK085865特异性地与白细胞介素增强因子结合因子2(ILF2)相互作用，并通过在 ILF2-ILF3复合物介导的微小RNA(miRNA)加工途径中作为负调节因子促进M2巨噬细胞极化。与野生型(wt)小鼠相比，lncRNA AK085865-/-小鼠骨髓源性巨噬细胞(BMDMs)的 miR-192水平显著下调，而 pri-mir-192水平显著升高。相反，敲除ILF2导致lncRNA AK085865-/-小鼠BMDMs中成熟 miR-192水平升高，减少了 pri-mir-192的表达。此外，miR-192过表达促进了体外巨噬细胞M2极化，白细胞介素-1受体相关激酶1(Irak1)被确定为直接靶点。Mir-192过表达有效地挽救了小鼠由 CVB3感染引起的致死性心肌炎，并将心肌浸润的巨噬细胞转变为M2表型。总的来说，我们的发现揭示了lncRNA AK085865在体外和体内调节巨噬细胞极化的关键机制，并提供了一个潜在的病毒性心肌炎的治疗靶点。

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