LncRNA-Gm26809在沙门菌鞭毛蛋白诱导TLR5和NLRC4炎性通路中的调节作用研究

Activation of the TLR5 and/or NLRC4 signaling pathways by flagellin and the resulting immune responses play important roles in host anti-bacterial immunity. And the regulation of signaling and the corresponding mechanisms are important topics in immunology. LncRNA can regulate gene expression at epigenetic, transcriptional, and post-transcriptional levels. And the regulation role of lncRNA in the innate immune response have attracted much attention. As far, the regulation of LncRNA on flagellin-induced inflammatory responses has not been reported. Thus, in this project, we identified that the expression level of lncRNA-Gm26809 was up-regulated in flagellin-treated mouse peritoneal macrophages by RNA-seq and qRT-PCR. The adenovirus overexpression or gene silencing system are used to evaluate the function of Gm26809 in inflammatory response induced by flagellin in vitro and in vivo. We will identify target proteins of Gm26809 by RNA pull down, RIP, RNA and protein colocalization techniques based on TLR5 and NLRC4 signaling pathways. The function and molecular mechanism research of Gm26809 in regulation of inflammatory response induced by flagellin will provide a new theoretical basis for the regulation of immune response by non-coding RNA, and provide new target and method for the treatment of bacterial infection and inflammatory diseases.

沙门菌鞭毛蛋白活化TLR5和NLRC4通路诱导的炎性应答在机体抗菌感染中发挥重要作用，其调节机制是重要科学问题。LncRNA可在表观遗传、转录及转录后水平调控基因表达，其对免疫应答的调控受到广泛关注。LncRNA对鞭毛蛋白诱导炎性应答的调控鲜有报道。基于此，本研究以小鼠巨噬细胞为模型，通过RNA-seq技术筛选获得了鞭毛蛋白诱导炎性过程中显著上调的lncRNA-Gm26809；拟利用腺病毒过表达或基因沉默系统通过体内外实验评价Gm26809在鞭毛蛋白诱导炎性应答过程中的功能；以TLR5和NLRC4信号通路为切入点，利用RNA pull down、RIP、RNA与蛋白质共定位技术鉴定Gm26809的靶蛋白，初步阐明lncRNA对鞭毛蛋白诱导炎性应答的调控作用，并揭示其作用分子机制，以期为非编码RNA对免疫应答的调控提供新的理论基础，为机体的抗菌感染研究和炎性疾病的治疗提供新的靶点和思路。

沙门菌鞭毛蛋白活化TLR5和NLRC4通路诱导的炎性应答在机体抗菌感染中发挥重要作用，其调节机制是重要科学问题。LncRNA可在表观遗传、转录及转录后水平调控基因表达，其对免疫应答的调控受到广泛关注。然而LncRNA对鞭毛蛋白诱导炎性应答的调控作用研究目前尚处于空白状态。本项目以巨噬细胞为模型，通过RNA-seq技术筛选获得了鞭毛蛋白诱导炎性过程中显著上调的lncRNA-Gm24562；发现lncRNA-Gm24562的表达量在鞭毛蛋白诱导巨噬细胞炎性应答过程中随着刺激时间的增加而增加，同时存在剂量依赖关系。利用肠道类器官、小鼠等体内外模型进一步表明了鞭毛蛋白可以诱导lncRNA-Gm24562表达水平的升高。FISH试验的定位结果显示lncRNA-Gm24562可分布在细胞核和细胞质中，但是主要以分布在细胞质中为主，预示着lncRNA-Gm24562可能主要在细胞质中发挥调节鞭毛蛋白诱导的炎性应答功能。利用慢病毒干扰和过表达技术通过体内外实验发现lncRNA-Gm24562可以抑制鞭毛蛋白诱导的IL-1β、IL-6和TNF-α等炎性因子的表达，具有抑制炎性应答的功能。Western blotting和间接免疫荧光试验表明lncRNA-Gm24562可以抑制NF-κB p65的磷酸化和入核，进而影响炎性因子的表达；通过RNA pull down技术筛选获得了一批lncRNA-Gm24562潜在的互作蛋白，初步探究了lncRNA-Gm24562发挥功能的分子机制。本项目为lncRNA在宿主炎性应答中发挥调控作用提供新的数据支持，也为相关炎性疾病的治疗提供了潜在的靶点。