miR-222通过作用于ETS1调节鼻息肉组织中Th17细胞分化

Numerous studies have shown that nasal polyps in Chinese patients is characterized by Th17 cell differentiation and subsequent neutrophilia. MicroRNAs are known to be involved in Th17 differention, and ETS1 has been regarded as a negative regulator of Th17 differentiation, while microRNA-222 has been proven to downregulate the level of ETS1. Therefore, we hypothesize that microRNA-222 is able to regulate Th17 differention by targeting ETS1 in Chinese patients with neutrophilic nasal polyps. Our study plan to focus on the difference of microRNA expressions in neutrophilic nasal polyps by testing microRNA profiles, neutrophil infiltration，Th17 cytokines and ETS1 levels, and analyze their correlations, next we plan to identify the immunological relationship of these factors in nasal ployps by focusing on microRNA-222 as the target. In doing so, ETS1 is expected to be the target gene of mircroRNA-222 and then mediate Th17 differentiation in nasal polyps. After that, we plan to determine this regulatory effect of microRNA-222 on ETS1/Th17 cells in nasal polyps cell and Jurkat cell line culture by using microRNA-222 lentivirus vectors and cytokine antagonists, and in mice model. Finally we will screen the possible signal pathways by take advatage of gene chip technology, and confirm the related signal mechanisms using specific inhibitor. Our findings on the regulatory effect of microRNA-222 on ETS1/Th17 subset in neutrophic nasal polyps are expected to be benefical for the improvement clinical efficacy of nasal polyps.

我们及国内外其他学者研究证实，国人鼻息肉存在特征性的Th17细胞优势分化，鉴于miRNA被证实参与调控Th17细胞分化，ETS1又是Th17细胞分化的负调控因素，而miR-222可下调ETS1，因此我们假设国人鼻息肉患者的Th17细胞的优势分化可由miR-222通过ETS1进行调控。为此，本研究拟首先检测鼻息肉组织中miRNA差异表达及中性粒细胞浸润、Th17细胞因子和ETS1水平并进行关联分析和组织定位，确定miR-222和其靶向ETS1，然后通过离体鼻息肉细胞以及Jurkat细胞株的培养，在构建miR-222慢病毒载体转染和ETS1拮抗等干预检测其对于CD4+细胞分化的影响，并采用transwell技术评估Th17细胞分化对于中性粒细胞的浸润趋化作用；最后通过基因芯片技术筛选可能的信号通路，通过慢病毒载体动物实验证实该过程及相关信号机制，为剖析鼻息肉的发病机制提供依据并筛选干预靶点。

慢性鼻窦炎、鼻息肉发病过程中Th17细胞的分化机制和促炎效应尚不清楚。本研究检测了检测了国人鼻息肉的临床表型特征，评估了嗜酸性粒细胞化和中性粒细胞化的鼻息肉组织中差异表达的miRNA与Th17相关细胞因子相关性；并重点以miR-222、miR-223、miR-17等为研究靶点，通过体外各种实验手段探索了其靶基因，检测了Th17细胞和Treg细胞类型的细胞因子如IL-17A、IL-21等在炎症反应中的作用并验证了其作用通路；研究发现，中国发现慢性鼻窦炎患者鼻息肉组织中抗菌蛋白如SPLUNC1等较对照鼻黏膜组织降低，证实非嗜酸性鼻息肉组织中SPLUNC1的表达更高并且可以被Th17细胞的IL-17A细胞因子上调；检测了鼻息肉组织中Foxp3蛋白的表达情况和GSK-3β之间的关联性，证实息肉组织中GSK-3β表达与Foxp3存在负相关；检测了鼻息肉患者组织中IL-17A、SAA的表达情况，证实Th17细胞的主要因子IL-17A与SAA表达增高存在关联，通过细胞培养的体外实验，证实该过程与IL-17A诱导的ERK信号通路活化有关；另外检测了鼻息肉患者组织中IL-17A和MUC5AC的表达情况，证实IL-17A与MUC5AC高表达和杯状细胞过度化生有密切关联。在miRNA在慢性鼻窦炎发病中的作用研究方面，通过组织学分析结合体外实验，系统性检测了嗜酸性和非嗜酸性慢性鼻窦炎患者鼻息肉组织的组织学特征和miRNA表达谱的差异；发现慢性鼻窦炎鼻息肉组织中存在miR-146a等的下调，miR-222、miR-223、miR-17表达的上调；发现miR-222的表达增高并且与Th17细胞因子的表达存在相关性；另外通过体外转染miR-222能够调控ETS-1的表达，提示ETS-1可能是miR-222调控鼻息肉中Th17优势分化的靶点；通过体外实验证实IL-17A、IL-13等可以刺激Ets样因子诱导的MUC5AC的表达，但IL-17A诱导的Ets样因子和MUC5AC高表达对激素治疗相对不敏感；证实了炎症条件下由IL-6/ miR-17信号通路介导的一种非Foxp3依赖性Treg细胞免疫活性的调控机制，即miR-17通过负调控Eos在内的Foxp3相关共调节分子的表达而抑制Treg细胞抑制功能的发挥。这些研究为阐明Th17在慢性鼻窦炎鼻息肉发病中的作用，探索更有效的干预靶点提供了基础。