肺炎支原体相关非编码RNA的筛选和鉴定

It was suggested by early research works that: ①Mycoplasma pneumoniae P1 gene harbored several variations; ②M. pneumoniae group is genetically highly diverse and the resistance of macrolides was spread by multiple clone dissemination; ③mRNA Expression and morphology were identified to change by subinhibitory concentrations. Epigenetic features such as biological control of Mycoplasma pneumonia itself as well as the interaction between hosts and strains will became the basis of the next phase of work. Consecutive application will study on a non-coding RNA at transcriptome level which preliminary research is carried on Mycoplasma pneumoniae self regulation as well as with a host of reciprocal control mechanism.The first part of current application is ①Identification of sRNAs in Mycoplasma pneumoniae by a bioinformatics based approach; ②Screening and identification of natural antisense transcripts(NATs) in Mycoplasma pneumoniae by a novel approach based on RNase I protection assay. The second part of current application is ①Establishment of normal human lung epithelial cell infection model of Mycoplasma pneumoniae; ②Screening and identification of obviously altered miRNAs in response to mycoplasma pneumoniae infections using miRNA chips; ③Identification of regulatory role of miRNA in the inflammatory response. This study is very helpful for us to further clarify the basic biological characteristics of Mycoplasma pneumoniae and genetic variation.

申请人前期工作结果显示：一、肺炎支原体p1基因变异度较大；二、肺炎支原体对大环内酯类耐药高度流行与克隆传播无关；三、肺炎支原体在环境因素改变时致病基因存在转录组水平的调控。上述研究结果提示针对肺炎支原体自身生物学调控以及与宿主间相互作用等表观遗传学特征的基础研究成为下阶段工作的重点。本次申请的连续课题拟从肺炎支原体转录组水平着手，以非编码RNA为切入点，初步探索肺炎支原体自身调控以及与宿主间相互调控的机制。研究第一部分拟对肺炎支原体自身小RNA（sRNAs）进行生物学预测、筛选、鉴定及初步功能研究，将有助于更全面和系统的认识肺炎支原体自身基因表达调控网络；第二部分拟构建肺炎支原体感染正常人肺上皮细胞模型，利用微小RNA（miRNAs）芯片筛选并检测感染前后肺上皮细胞的miRNA表达谱变化，并探索差异表达的作用机制。本研究对于进一步阐明肺炎支原体基本生物学特性和遗传变异规律具有重要意义。

肺炎支原体在自身进化以及与人类宿主相互作用的过程中，相互构成了一个复杂的网络，以精细调控肺炎支原体感染的免疫反应，但确切免疫调控机制仍不清楚。肺炎支原体全基因测序已完成，为进一步进行上述研究提供了良好基础。本课题从肺炎支原体转录组水平着手，以非编码RNA为切入点，初步探索肺炎支原体自身调控以及与宿主间相互调控的机制，对于进一步阐明肺炎支原体基本生物学特性和遗传变异规律具有重要意义。目前已利用RNAseq及生物信息学分析软件两种不同的方法对P1 型Ⅰ和Ⅱ型肺炎支原体代表菌株( MP M129和MP FH) 的sRNAs、tRNA及rRNA进行预测和筛选，并对sRNA的靶标mRNA进行相关预测，并设计引物开展对其进行鉴定，有助于更全面和系统的认识肺炎支原体自身基因表达调控网络。目前已完成人正常肺上皮细胞BEAS-2B感染肺炎支原体模型的建立，并建立了提纯RNA的方法，样本已进行RNAseq；与国内世界领先的基因组学中心生物信息学分析单位深圳华大基因研究院建立合作，对人正常肺上皮细胞BEAS-2B感染肺炎支原体模型后细胞、肺炎支原体相关部分信号通路分析进行研究价值评估；筛选出研究价值较大的信号通路分子，参考国内外文献，并对肺炎支原体感染人支气管上皮细胞BEAS-2B中P53/RGS13通路的作用机制展开研究。研究表明，肺炎支原体感染可能诱发非凋亡途径伤害，肺上皮细胞细胞采用降低凋亡的保护策略来对抗肺炎支原体的感染；肺上皮细胞可通过下调 P53 并进一步上调 RGS13 的方式在一定程度上减弱 Mpn 导致的炎症反应，起到对细胞和组织的保护作用。