lncRNA-mtb在结核分枝杆菌感染巨噬细胞中的功能研究

Role of lncRNA-mtb in Mycobacterium tuberculosis-infected macrophages

目的研究lncRNA-mtb在结核分枝杆菌感染巨噬细胞中的作用机制。方法软件预测、Western blot验证lncRNA-mtb的非编码性质及在不同分枝杆菌感染巨噬细胞中的表达差异;RNA干扰技术(RNA interference,RNAi)沉默巨噬细胞lncRNA-mtb表达,ELISA检测细胞因子的分泌情况;流式细胞术、Western blot、RT-qPCR技术进一步确认结核分枝杆菌感染可通过lncRNA-mtb诱导巨噬细胞炎症活化。干扰lncRNA-mtb后,检测结核分枝杆菌感染巨噬细胞后对杀菌功能的影响。结果结核分枝杆菌H37Rv和H37Ra感染巨噬细胞后,可导致lncRNA-mtb的表达增加。干扰lncRNA-mtb的表达后,H37Rv刺激导致巨噬细胞炎症因子IL-1β的分泌显著降低,进一步研究发现,lncRNA-mtb可能参与结核分枝杆菌诱导巨噬细胞活化NLRP3炎症小体。沉默lncRNA-mtb的巨噬细胞感染H37Ra后,结核分枝杆菌清除能力显著下降。结论H37Rv和H37Ra感染巨噬细胞可促进lncRNA-mtb表达上调,该lncRNA-mtb参与结核菌诱导细胞的炎症,沉默lncRNA-mtb可减弱巨噬细胞对结核分枝杆菌清除能力。

Objective To study the mechanism of lncRNA-mtb in Mycobacterium tuberculosis(M.tuberculosis)-infected macrophages.Methods A predicting software and Western blot assay were used to verify the non-coding nature of lncRNA-mtb.RT-qPCR was performed to detect lncRNA-mtb expression in macrophages infected by different Mycobacteria.Cytokine secretion was detected with ELISA after silencing the expression of lncRNA-mtb in macrophages with RNA interference(RNAi)technology.Western blot and RT-qPCR were performed to analyze whether the activation of macrophage inflammation could be induced by lncRNA-mtb during M.tuberculosis infection.Effects of lncRNA-mtb silencing on the bactericidal activity of M.tuberculosis-infected macrophages were evaluated.Results Increased expression of lncRNA-mtb was observed in macrophages infected with M.tuberculosis H37Rv or H37Ra.After silencing the expression of lncRNA-mtb,IL-1βsecretion in H37Rv infected-macrophages was significantly decreased.Further studies revealed that lncRNA-mtb might be involved in the activation of NLRP3 in M.tuberculosis-infected macrophages.The bactericidal activity of macrophages against H37Ra was impaired after silencing the expression of lncRNA-mtb.Conclusions Enhanced expression of lncRNA-mtb could be induced in macrophages infected with H37Rv or H37Ra.Moreover,lncRNA-mtb was involved in M.tuberculosis-induced inflammation in cells.Silencing lncRNA-mtb wolud attenuate the ability of macrophages to clear M.tuberculosis.