MicroRNA-30c靶向LYST在成人噬血细胞综合征NK细胞损伤中的作用机制研究

Hemophagocytic Syndrome (HPS) is a relatively rare but life-threatening disease with confusing clinical manifestations and high mortality. Over the past ten years, it has garnered increased attention with the improvement of the diagnostic technologies and the increasing awareness of the disease, however, the pathogenesis of adult (acquired) HLH is not fully understood. Low/absent natural killer (NK) cell activity is the key pathogenesis underlying HPS, which results in the inappropriate activation of T-lymphocytes and macrophages and excessive cytokine production (interferon-γ, TNF-a, IL-2, IL-6, IL-8, IL-10, IL-12 and IL-4), and further results in fever and other clinical manifestations of HPS. Recent studies have shown miRNA-30c family plays an important role in regulating immunity and the function of NK cells. We detected the expression of miRNA-30c family in the NK cells from adult HPS patients and normal samples. Our results showed the expression of miRNA-30c was significantly higher in NK cells from adult HPS patients. Bioinformatics analysis shows miRNA-30c can target multiple cytokines and LYST which is a gene susceptible for HPS. Transient overexpression and silence miRNA-30c in NK92 cell line could post-transcriptionally regulate the expression of LYST protein. So, we hypothesize the mechanism of miRNA-30c participating the pathogenesis of HPS is promoting the dysregulated cytotoxicity of natural killer cells by targeting LYST. In this project, we will comprehensively investigate this mechanism and prove LYST is the targeted gene of miRNA-30c. Taken together, this project will provide the clinical and experimental evidences for miRNA-30c as a novel and valuable therapeutic target.

成人噬血细胞综合征(HPS)是一种病死率极高的免疫紊乱性疾病，深入探索其发病机制显得非常重要和迫切。NK细胞活性降低和细胞因子风暴形成是HPS病理发生过程中的关键环节。近来研究发现miRNA-30家族成员在调节NK细胞功能方面发挥重要作用，我们据此在HPS患者和正常人NK细胞中进行miRNA-30家族成员的筛查，结果显示miRNA-30c在HPS患者NK细胞中表达明显升高，生物信息学分析提示miRNA-30c能靶向多个细胞因子和噬血易感基因LYST，在NK92细胞中瞬时过表达和抑制miRNA-30c显示和LYST有相互作用的转录后调控关系。据此我们推测miRNA-30c高表达通过靶向LYST参与了HPS患者NK细胞损伤。本项目将在NK92细胞和HPS原代细胞中全面阐述其作用机制，为我们深入理解HPS的发病机制，探索以miRNA-30c及其靶基因为新靶点的生物治疗提供重要的实验和临床依据。

背景：研究非编码RNAmiR-30c在HLH中的发病机制，寻找新的HLH靶向分子预后指标。.主要研究内容：.阐明HLH患者miRNA-30c表达水平较正常人明显升高；筛选miRNA-30c作用的靶基因；miRNA-30C调节NK细胞细胞毒活性的机制是通过影响穿孔素、颗粒酶B或CD107a 的表达；在动物实验中证实miRNA-30C高表达能降低NK细胞的细胞毒活性。.研究结果和关键数据：.1. 我们对3例正常血浆和HLH患者进行基因芯片筛查，共筛选出差异性表达的miRNA有24个，与正常人比较，有20个miRNA的表达显著上调，4个miRNA的表达显著下调。.2. HLH患者和对照组血浆中miR-125a-3p的表达水平分别为7.21±1.48和1.53±0.34，与正常人相比，HLH患者血浆中miR-125a-3p的表达上调，差异有显著的统计学意义（P0.001）；miR-320a在HLH患者血浆中的表达水平较正常人明显上调，差异有统计学意义（5.83±1.59和1.33±0.31,P0.001）。.3. 血浆miR-125a-3p的表达水平与HLH患者的临床资料相关性分析显示，miR-125a-3p的表达与患者的性别、年龄、噬血现象、Fbg及sCD25等指标无显著相关性。miR-125a-3p的表达与实验室指标WBC和ANC呈负相关，相关系数分别为（r-0.463,p=0.013)和（r-0.435,p=0.021)。血浆miR-320a的表达水平与LDH（r0.531,p0.004)和SCr（r0.623,p=0.0004）呈正相关,miR-320a-3p的表达与性别、年龄、噬血现象、Fbg及sCD25等指标无相关性。 .4. ROC曲线分析显示，miR-125a-3p和miR-320a-3p运用于HLH的诊断时，其曲线下面积（AUC）分别为0.783和0.719，敏感性分别为78.6％和53.6％，特异性分别为66.7％和93.3％，最佳临界值分别为1.61和 3.03。.科学意义：.HLH患者血浆中存在特异性的miRNA表达谱，血浆miR-125a-3p和miR-320a在HLH患者中的表达明显上调，提示可能与HLH的发病密切相关。miR-125a-3p和miR-320a可能是诊断HLH的潜在生物标志物。