lincRNA-cox2在结核菌感染中调节细胞自噬功能的研究

Mycobacterium tuberculosis(MTB) is one of the most troublesome human pathogenic bacteria which could block phagolysosome biogenesis and thus establish a safe niche within host macrophages for its survival. Recently, scientists have uncovered autophagy as a new means of overcoming this block and promoting the killing of mycobacteria tuberculosis（MTB）.However，the signaling pathways governing autophagy under MTB infection remain to be fully delineated. Long noncoding RNAs (lncRNAs), tentatively defined as noncoding RNAs more than 200 nt in length, are characterized by the complexity and diversity of their sequences and mechanisms of action. Lately, sporadic pubilications reported that lncRNA might play important roles during cellular development and their misregulation has also been shown in various types of diseases. To the best of our knowledge, the relationship between lncRNA and MTB has never been reported. Our team have found that lincRNA-cox2, a recently identified long noncoding RNA located ~51kb upstream of the protein-coding gene cyclooxygenase-2 (COX-2),displayed marked upregulation in MTB infected THP-1 cells via a microarray analysis followed by bioassays.We also found that autophagy-related proteins, such as beclin-1 and LC3-II, were reduced after down-regulating lincRNA-cox2. In this project，further study will be performed to evaluate lincRNA-cox2 on the following point: 1）to compare the patterns of lincRNA-cox2 expression between MTB infection and non-infection groups in cell model, mouse model and a series of human alveolar macrophage isolated by bronchoalveolar lavage; 2) to discover the underlying mechanism leading to the lincRNA-cox2 alternation, and investigate the relationship between lincRNA-cox2 expression and MTB inhibition; 3) to identify the target of lincRNA-cox2 using CHIRP-seq, and explore the molecular machenism through which lincRNA-cox2 regulate authophagy in host defence against MTB infection.

细胞自噬过程与结核病的保护性免疫机制存在着重要的联系，但具体作用方式尚待进一步阐明。长链非编码RNA（lncRNA）作为新发现的一类内源性调控因子，被认为广泛参与调控各种应答。本课题组前期工作利用比较基因组学技术，发现lincRNA-cox2在结核菌感染巨噬细胞中显著高表达，且干扰 lincRNA-cox2可下调自噬相关基因的表达，提示lincRNA-cox2可能通过调控自噬发生而针对结核菌感染发挥保护性免疫作用。本项目拟在细胞水平、动物水平、临床标本中验证lincRNA-cox2表达特性，通过基因过表达及抑制实验探索lincRNA-cox2上游信号通路及其对细胞自噬的调控作用。 同时，利用ChIRP-seq技术发现lincRNA-cox2靶基因，希望阐明lincRNA-cox2在促进自噬发生及针对MTB免疫调控的分子机制，为寻找抗MTB感染新的治疗手段提供理论依据。

关于lncRNA的功能研究在国际上尚处于摸索阶段，很多问题亟待解决，目前国内外均未有lincRNA-cox2调控MTB感染的相关报道。本项目按计划进行了如下研究：1）证实lincRNA-cox2在结核菌患者体内高表达，其过表达会提示抗结核治疗预后较差；2）发现TLR/p38MAPK是调控lincRNA-cox2表达的关键信号通路；3）确认lincRNA-cox2是促进细胞自噬发生的关键组件，且证实了lincRNA-cox2可通过参与调控自噬来达到抗MTB感染的目的。在此基础上，本项目同时发现，尽管过度的炎性因子和自噬通常促进细胞坏死（necrosis）发生，lincRNA-cox2却可以在上调炎性因子表达水平（如TNF）及自噬发生的同时，抑制细胞的坏死，这说明lincRNA-cox2调控细胞自噬和细胞坏死是分别进行的。功能研究进一步证明，lincRNA-cox2可同时靶向iNOS和PYK2两个分子的启动子，通过与上调iNOS表达刺激炎症及自噬发生，通过上调PYK2表达抑制CYLD/RIPK/MLKL通路，从而抑制细胞程序性坏死（necroptosis）。同时，我们利用基因敲除小鼠，证实抑制necroptosis也是可以促进杀菌的，这说明自噬和necroptosis，尽管二者相互促进，却分别是免疫抵御结核和免疫保护结核的两种不同机制。lincRNA-cox2可通过不同的路径分别刺激自噬发生而抑制细胞necroptosis，从而达到尽可能多的杀灭体内结核菌的目的。这为研究结核病发生发展提供了全新的角度，lincRNA-cox2也很有可能成为新的潜在药靶。本课题组培养的硕士生已毕业2名，本项目共发表研究论文4篇，其中SCI论文2篇，另仍有3篇论文正在投稿中。