lncRNA-EPS靶向IL6基因负调控巨噬细胞自噬在活动性肺结核发病机制中的研究

Long non-coding RNA (lncRNA) plays an important role in the development of active pulmonary tuberculosis, however the exact mechanism of its pathogenesis has not yet been elucidated. In the pre-experiments of lncRNAs screening, it has been observed that the gene expression of lncRNA-EPS decreased, and its level was negatively correlated with the expression of autophagy-related protein LC3 in the patients with acute pulmonary tuberculosis. It has also been found that lncRNA-EPS may affect autophagy of M. tuberculosis (MTB) infected cells regulating IL6 gene expression through bioinformatics analysis. Meanwhile, IL6 can promote macrophage autophagy. Therefore, the experiment was designed to support the hypothesis that decrease of lncRNA-EPS expression may promote IL6 gene expression, and promote autophagy of MTB-infected macrophages, which was beneficial to MTB clearance. To confirm this hypothesis, we will explore the effects of different expression levels of lncRNA-EPS on autophagy and MTB clearance by molecular biology and electron microscopy technologies on cell and animal levels. Meanwhile, it will be observed that the molecular mechanism of lncRNA-EPS targets IL6 gene to regulate cell autophagy. In this study, we will reveal effects of lncRNA-EPS on autophagy and its molecular mechanism and will provide a new strategy for the prevention and treatment of tuberculosis.

长链非编码RNA（lncRNA）在活动性肺结核发生中发挥重要作用，其确切机制尚未阐明。前期研究发现，活动性肺结核患者单核细胞中lncRNA-EPS表达降低，且其水平与自噬相关蛋白LC3表达水平呈负相关。生物信息学分析发现，lncRNA-EPS通过靶向抑制IL6基因表达而抑制细胞自噬。同时，前期研究还发现，IL6可促进巨噬细胞自噬发生。因此，我们推测结核分枝杆菌（MTB）感染后lncRNA-EPS表达降低促进IL6基因表达，进而促进巨噬细胞自噬发生，最终有利于MTB清除。为证实这一假说，我们将在细胞和动物两个水平上验证，采用分子生物学、电镜等技术，观察不同表达水平的lncRNA-EPS对MTB感染后巨噬细胞自噬以及MTB清除等作用。同时，观察lncRNA-EPS靶向IL6基因调控巨噬细胞自噬的分子机制。本课题将揭示lncRNA-EPS调控巨噬细胞自噬作用及其分子机制，为防治结核病提供新策略。

背景：长链非编码RNA-EPS（Long intergenic noncoding RNA erythroid prosurvival，lincRNA-EPS）在调节巨噬细胞的各种生物学过程中发挥重要作用，包括炎症反应、细胞凋亡和自噬。而lincRNA-EPS在调节巨噬细胞对结核分枝杆菌的免疫反应中的作用未见报道。目的与方法：本研究采用免疫印迹、流式细胞术与免疫荧光等技术，旨在探讨活动性肺结核（Pulmonary tuberculosis，PTB）患者和健康个体单核细胞中lincRNA-EPS的表达。同时，我们还研究lincRNA-EPS对卡介苗（Bacillus Calmette-Guérin，BCG）感染的巨噬细胞凋亡和自噬的影响，为寻找治疗结核病新靶点提供新策略。结果：本研究发现，与健康组相比，活动性PTB患者的单核细胞中lincRNA-EPS表达明显下调，并伴有单核细胞凋亡较弱和自噬增强。体外研究发现，敲低lincRNAEPS抑制了BCG感染的RAW264.7巨噬细胞凋亡并促进了其自噬水平。此外，我们还发现lincRNA-EPS通过JNK/MAPK信号通路调节BCG感染的RAW264.7巨噬细胞凋亡和自噬。结论：本研究结果表明，lincRNA-EPS下调通过激活BCG感染的RAW264.7巨噬细胞的JNK/MAPK信号通路，进而抑制细胞凋亡并增强自噬。