结核菌感染过程中LncRNA-AK140703对巨噬细胞极化改变的影响及其调控机制研究

Mycobacterium tuberculosis infection can induce the activation phenotype of macrophages changing from M1 to M2 to escape immune destruction, but the mechanism is unknown. Our previous study found that the expression of long noncoding RNA (lncRNA) AK140703 will increase in M2 macrophages and the late infection of Mycobacterium tuberculosis; Knockdown of AK140703 by siRNA significantly reduced the expression of M2 molecule; knockdown of AK140703 inmacrophage remained M1 polarization after Mycobacterium tuberculosis infection, and diminished M2 phenotype expression. Therefore we can speculate that AK140703 may play an important role in regulating macrophage polarization duing the infection of Mycobacterium tuberculosis. On this basis, we construct the model of the phenotype of macrophages carried with Mycobacterium tuberculosis and the infected mice, define the characteristic expression and regulation of which AK140703 regulate macrophage polarization in the process of tuberculosis infection; Explore that AK140703 have an effect on the polarization phenotype of macrophages in TB infection and anti-tuberculosis immune through the means of siRNA interference and gene overexpression, and illustrate the molecule mechanism of regulation in macrophage polarization through the methods of gene chip and bioinformatics to Screen target genes of AK140703. The study has important implications for understanding the pathogenetic mechanisms of tuberculosis and to explore new prevention measures.

结核菌感染可诱导巨噬细胞（MΦ）的活化表型由M1向M2改变来逃避机体免疫杀伤，但其机制不明。我们前期研究发现：①长链非编码RNA(lncRNA) AK140703在M2型MΦ和结核菌感染后期表达上调；②siRNA沉默AK140703后可显著抑制MΦ M2分子的表达；③AK140703沉默后的MΦ受结核菌感染后，其活化表型长期维持M1状态，M2极化受阻。据此推测：AK140703可能通过调控MΦ的极化改变参与结核菌的感染过程。故在此基础上构建结核菌感染MΦ模型和小鼠结核病模型，明确结核菌感染过程中AK140703在MΦ的表达特点及规律；通过RNA干扰和基因过表达等手段，探讨AK140703在结核感染中对MΦ极化表型及抗结核免疫的影响；并应用基因芯片和生物信息学方法筛选AK140703靶基因，阐明其调控MΦ极化改变的分子机制。该研究对了解结核病发病机制及探索新的防治措施具有重要意义。

巨噬细胞作为抵抗结核分枝杆菌（Mtb）主要的宿主细胞，在抗结核感染中发挥着至关重要的作用。结核菌感染可诱导巨噬细胞的活化表型由M1向M2改变来逃避机体免疫杀伤，但其相关分子机制至今未阐明。在本研究中，我们发现长链非编码RNA（lncRNA）AK140703在M2型巨噬细胞中表达显著升高，敲低AK140703可促进M2型巨噬细胞向M1极化转换。在结核菌感染的巨噬细胞和小鼠中AK140703表达水平显著升高，AK140703敲低的巨噬细胞对结核菌的杀伤活性升高，且上调了M1标志物的表达，促进炎性细胞因子的释放。这表明AK140703参与了结核菌感染巨噬细胞极化的调控过程。动物感染实验发现小鼠肺巨噬细胞敲低AK140703后，组织病理学损害减轻和肺部结核菌荷菌量降低。我们通过双荧光素酶报告基因实验及AGO2免疫共沉淀实验证实AK140703可作为ceRNA竞争性结合miR-624。进一步研究发现miR-624在结核菌感染巨噬细胞及M2型巨噬细胞中表达显著下调，上调miR-624表达可促进增加M2巨噬细胞中M1标志物表达，降低其M2标志物表达。经生物学软件预测及双荧光素酶报告基因实验证实KLF4为miR-624下游靶基因，结核菌感染中AK140703促进巨噬细胞M2极化是通过调控miR-624/KLF4实现的。我们的研究工作阐明了lncRNA AK140703在结核菌感染巨噬细胞极化中的作用及其调控机制，为结核病的预防和控制开辟新的思路，提供新的干预靶标。