组蛋白甲基转移酶EZH1调控登革病毒诱导细胞因子产生的机制研究

Fatal systemic inflammatory mediators, such as cytokines (CKs), contributes to its evolution from an asymptotic infection to severe forms of dengue which have high mortality. There is an unbalance between pro-inflammatory cytokines and anti-inflammatory in patients with severe dengue fever although its regulatory mechanisms are still unknown. When we studied the regulatory effects of let-7e /EZH2 axis on cytokines, we found that after EZH2 silence EZH1 expression was increased, H3K27me3 was decreased and H3K4me3 was increased, indicating that EZH1 was not a simple alternative to EZH2. According these results we hypothesize that: EZH1 could regulate cytokines expression by forming two kinds of splice variants (EZH1α and EZH1β) at different stages of virus infection. Cytoplasm EZH1β is formed in a complex with Eed and prevent Eed from entering the cell nucleus, the number of functional PRC2 complexes is decreased while H3K4me3 increases and activate transcription factors TFIID to promote genes expression. Ezh1β is targeted for degradation through ubiquitination, freeing Eed to enter the nucleus. There Eed interacts with Ezh1α-Suz12 dimers to increase the number of functional PRC2 complexes and promote the formation of H3K27me3 to inhibit gene expression. We will study the expression and distribution of EZH1, including EZH1α and EZH1β, from four research levels: molecular, cellular, animal models and clinical samples to reveal regulatory mechanisms of EZH1 on CKs expression. Acute-on-chronic liver failure (ACLF) is a syndrome characterized by acute decompensation (AD) of a patient with cirrhosis, organ failure(s), and high short-term mortality rate. It was proposed (systemic inflammation [SI] hypothesis) that ACLF is the expression of an acute exacerbation of the SI already present in decompensated cirrhosis. SI in ACLF is very similar with that in patients with severe dengue fever. We will confirm the function of EZH1 in ACLF. Study results will provide new therapeutic targets for clinical illness associated with severe inflammation.

重症登革热病死率高，致死性全身炎症与细胞因子（CK）失调相关，但机制不明。我们研究表观调控轴Let-7e/EZH2对CK的作用发现，沉默EZH2后EZH1表达上升，H3K27me3减少，而H3K4me3上升，意味着EZH1并不是简单的EZH2替代物。据此我们假设：在病毒感染不同阶段EZH1通过形成两种剪切体(Ezh1α和Ezh1β）而调节CK表达。胞浆Ezh1β与Eed结合而阻止Eed入核形成功能性PRC2–Ezh1复合物，促使H3K4me3形成激活转录因子TFIID促进基因表达；Ezh1β降解后，Eed入核与染色体上PRC2–Ezh1α复合物结合促进H3K27me3形成而抑制基因表达。我们将从分子、细胞、动物和临床样本四个层面研究EZH1剪切体的表达和分布，探明其调节CK机制，并在与重症登革热病人全身炎症非常相似的慢加急肝衰竭病人样本中验证，为临床炎症相关疾病提供新的治疗靶点。

登革病毒（DENV）引起的重症登革热死亡率高，与细胞因子（CK）失调相关，但具体机制不明。我们研究组蛋白转移酶EZH1对CK的作用发现：EZH1/2抑制剂UNC1999可抑制DENV NS1及LPS诱导人PBMC表达IFNγ、TNFa、IL-6，但促进IL-1β的表达；同时影响免疫细胞表型。为深入研究EZH1作用，制备EZH1敲除鼠（EZH1-/-），分离原代骨髓细胞并诱导成巨噬细胞（BMDM），相对正常小鼠，EZH1-/-BMDM感染DENV后病毒载量下降，IL-9和IL-17等炎性CK表达也下降。EZH1-/-小鼠脾脏细胞感染DENV后经29色流式细胞术分析发现，Treg、单核细胞和中性粒细胞的PD-1表达下调；Treg、单核细胞的LAG-3表达下调；Treg、CD4+T细胞、CD8+T细胞CD86表达下调；中性粒细胞中CD62L表达下降，但NK细胞中CD62L表达上升。通过盲肠穿刺结扎术构建脓毒症模型，发现Ezh1/H3K27me3/Cebpb轴能促进肝脏单核细胞来源巨噬细胞向Ly6C lo亚群转化，从而减轻脓毒症性肝损伤，这为脓毒症治疗提供了新思路。对于EZH1的上游调控分子我们也进行了探究，发现miR-20a可以直接抑制EZH1，miR-20a/EZH1轴可通过减少H3K27甲基化来抑制肝细胞癌（HCC）的增殖和转移，UNC1999能抑制肿瘤细胞的增殖，并增强索拉非尼的抗肿瘤作用。let-7e 可靶向EZH2，siEZH2可以通过上调H3K4me3、下调let-7e和H3K27me3来提高TNFα的产生，同时通过抑制核NF-κB p65水平来降低TNFα表达。我们关于EZH1 调控细胞因子机制、EZH1上游调控分子的研究为登革热、脓毒症引起的肝损伤和肿瘤的治疗提供新思路。