基于全基因组的ncRNA 序列分析发掘结核分枝杆菌和巨噬细胞的互作机制

Non-coding RNA(ncRNA)includes host miRNA and pathogen sRNA. It is a group of small RNAs which play a regulatory role posttranscription. Their role in pathogen infection and host anti-infection has launched a new area for study. The research on sRNA of Mycobacterium tuberculosis(MTB) and BCG during infection has just begun.This project is aimed to discover genome-wide ncRNA of MTB and macrophage and investigate the association between mRNA and ncRNA to excavate new mechanism underlying interaction between MTB and macrophage. At different timepoints after infection, the mRNAs and sRNA of the bacilli (MTB,BCG) and host cells were sequenced with RNA-seq. The integrated analysis of mRNA-sRNA for both bacilli and macrophage will be performed, and the global interaction mechanism of MTB and macrophages will be deduced. The sequences of ncRNAs (miNRAs and sRNAs) and mRNA of bacilli and host cells will be verified by qRT-PCR, Northern blot, and RNA in situ hybridization. One or two transcripts from each group of miRNAs and sRNAs potentially related to MTB persistent infection and immune evasion are selected to determine their functions. The techniques including overexpression, inhibition of expression by inhibitors or gene deletion, and co-expression of ncRNA and their deduced target genes (fragment covering interaction sites) will be used, and their parameters such as bacterial growth properties, expression of target genes, response effect on infection and anti-infection will be detected. Furthermore, the presence of miRNAs/sRNAs will be checked in sera of tuberculosis (TB) patients and the association between certain miRNAs/sRNAs and TB development or MTB persistence will be determined. In conclusion,the results will play an important role in elucidating the mechanism of MTB pathogenesis, and provide potential targets for development of novel new drugs, vaccines and diagnotic techniques for tuberculosis prevention and treatment.

非编码RNA（ncRNA）包括宿主miRNA和病原体sRNA，是一类发挥转录后调控的小RNA，其在感染和抗感染斗争中的作用是一个新型领域，感染状态下结核分枝杆菌（MTB）sRNA研究才起步。本研究利用MTB/BCG的巨噬细胞感染模型，从sRNA和miRNA鉴定、ncRNA和mRNA互作分析角度揭示MTB与巨噬细胞的作用机制。分别对感染体系中的细菌（MTB、BCG）和巨噬细胞进行mRNA和小RNA高通量测序、对mRNA和sRNA进行整合性分析，推测MTB-巨噬细胞全局性互作机制；利用qRT-PCR、Northern blot等验证序列，选择与持续感染和免疫逃避相关的1-2种miRNA和sRNA，利用过表达、抑制和共表达等手段，确定其在感染和抗感染中的作用；检测结核病人血清中的miRNA/sRNA，验证其与MTB感染发病的相关性。结果对阐明MTB致病机制和发现新型药物疫苗诊断靶标具有重要意义。

结核病是全球发病数和病死数最多的人兽共患病，探讨其致病机制意义重大。本项目在MTB/BCG感染巨噬细胞THP-1状况下，直接提取细胞和胞内菌的RNA，进行转录组平行分析和验证，为解析MTB致病机制和创制防控手段提供了新证据和新理论。主要研究内容和结果如下：.（1）感染模型研究：优化了模型参数，确定感染比10，感染12h后用庆大霉素杀死胞外菌。以此刻为0h，继续培养6h和24h。提取胞内菌，确定了纯度评价方法。分别提取细胞、胞外菌和胞内菌（MTB和BCG）RNA。.（2）高通量测序和生物信息学分析：RNAseq得到成熟的新miRNA 234个和已知miRNA 1451个。对miRNA的靶标基因进行聚类分析，发现下调表达的miRNA的靶标基因表达上调并聚类富集到炎症反应、细胞因子活性、免疫细胞活性、糖脂代谢、细胞凋亡等通路。上调表达miRNA的靶标基因下调并聚类富集到细胞周期、细胞骨架、染色体分离等通路。.测序得到MTB与BCG的sRNAs分别为1685与1010个，长度为50~200nt。数据库注释只发现15和14个已知，99.98%为未知。靶基因预测发现其潜在靶标主要与细菌生长代谢、毒力调控、耐受宿主环境和药物等相关。.（3）胞内菌主动和选择性摄取宿主RNA的研究：发现胞内菌含大量宿主各类RNA（mRNA，miRNA，lncRNA和snoRNA），证实这是一种主动和选择性摄取，其功能富集在泛素化介导的蛋白水解、溶酶体、蛋白酶体、FcγR介导的吞噬等相关通路。利用绝对荧光定量RT-PCR和原位杂交等方法验证了测序结果。对上调表达的miR-1290及4个下调表达的miRNA（miR-18b-5p、miR-378d 、miR-362-5p和miR-140-3p）进行了功能分析，确定了靶基因。.（4）miRNA作为诊断标识的研究：发现新发结核病人has-miR-374b-5p的表达水平显著高于健康对照，而has-miR-625-3p和has-miR-362-5p则相反。利用has-miR-374b-5p、has-miR-625-3p和has-miR-362-5p的组合物诊断结核病的灵敏度和特异性高于单一miRNA。.申报国家发明专利5项，获授权1项；发表论文5篇，SCI收录3篇；出版专著1部；发布国家标准1项。完成了计划目标。