lncRNA通过miR-381-3p调控CD1c表达在结核感染免疫中的作用与机制

CD1c plays important roles in immune responses to Mycobacterium tuberculosis. However, M. tuberculosis could significantly suppress the expression of CD1c during dendritic cell (DC) differentiation with unknown mechanism. In our previous work, we confirmed the significant down-regulation of CD1c expression in tubercuosis (TB) patients. Furthermore, in DCs infected with Bacillus Calmette-Guerin, we demonstrated that inhibition of CD1c expression is mediated by miR-381-3p. Recently, lncRNAs were found playing important roles in regulating the behaviour of microRNAs. Thus we seek to explore the possible involvement of lncRNAs in regulating miR-381-3p in TB. For this purpose, we intend to: i) confirm the regulatory effects of miR-381-3p on CD1c expression in DCs infected with M. tuberculosis; ii) screen out critical lncRNAs that could interact with miR-381-3p and are associated with M. tuberculosis infection; iii) determine the candidate lncRNAs through detecting the expression profiles of lncRNAs in clinical samples and quick loss-of-function assay in M. tuberculosis-infected DCs; iv) detect the cellular localization of lncRNAs in M. tuberculosis-infected DCs and TB pathological tissues; v) explore the regulatory effects of lncRNAs on the expression of miR-381-3p and CD1c in infected DCs as well as on the activation of T cells; vi) analyze the "sponge" effect of lncRNAs on CD1c expression through interaction with miR-381-3p. This study will elucidate the regulatory effects and mechanisms of CD1c expression by lncRNAs through interaction with miR-381-3p in the immune responses to M. tuberculosis, and establish the basis for development of novel immunotherapy for TB by targeting CD1c.

CD1c在抗结核免疫中发挥重要作用，但在DC分化中受MTB显著抑制，其机制不明。课题组前期发现结核患者CD1c表达显著下调，在BCG感染DC中证实CD1c表达抑制由miR-381-3p介导，并可能有竞争性内源lncRNA参与。在此基础上，拟：①在MTB感染DC中确证miR-381-3p对CD1c的表达调控及效应；②筛查结合miR-381-3p且与MTB感染相关的lncRNA；③临床样本表达检测结合缺失效应分析，确定候选lncRNA；④检测lncRNA在感染DC、病理组织的表达定位；⑤检测lncRNA对感染DC中miR-381-3p、CD1c表达及T细胞活性的调节；⑥检测lncRNA通过miR-381-3p调控CD1c表达的“海绵”效应。项目将阐明lncRNA通过miR-381-3p调控CD1c表达在结核感染免疫中的作用机制，为以CD1c为靶点的抗结核免疫新疗法开发提供依据。

本项目探讨了MTB感染抑制DC分化过程中脂类抗原递呈分子CD1c表达的机制。①将标准毒株H37Rv感染人原代DC，确证了miR-381-3p抑制CD1c表达、阻遏DC诱导T细胞活化的效应；②对H37Rv感染的DC进行高通量测序及数据分析，找到感染后下调并可结合miR-381-3p的lncRNA；③临床结核患者样本表达检测结合缺失效应分析，确定感染后表达水平降低且同时下调CD1c水平的候选lncRNA；④核质分离实验与免疫组化分析发现，该lncRNA在H37Rv感染DC、结核患者病理组织中主要定位于胞浆；⑤基因功能缺失与获得实验结果显示，该lncRNA不影响被感染DC中miR-381-3p的表达，但其水平与CD1c表达及DC诱导T细胞活化的效应正相关；⑥ceRNA的“海绵”效应检测结果表明，lncRNA通过结合miR-381-3p调控CD1c表达。项目初步阐明了lncRNA通过miR-381-3p调控CD1c表达在结核感染免疫中的作用机制，为以CD1c为靶点的抗结核免疫新疗法开发提供依据。