Silencing miR-125b-5p attenuates inflammatory response and apoptosis inhibition in mycobacterium tuberculosis-infected human macrophages by targeting DNA damage-regulated autophagy modulator 2 (DRAM2)

Tuberculosis is one of the most important infectious diseases worldwide and macrophage apoptosis is the major host defense mechanism against TB. We attempted to characterize the role of miRNA (miR)-125b-5p on mycobacterium tuberculosis (Mtb) infection and macrophages behaviors <i>in vitro</i>. According to fluorescence-activated cell separation (FACS), primary monocytes (CD14⁺) in TB patients were accumulated, and apoptotic monocytes were decreased. Peripheral blood mononuclear cells (PBMCs)-derived macrophages (MDMs) and monocytic cells THP-1-derived macrophage-like cells (TDMs) <i>in vitro</i> were used to be infected with H37Rv. After infection, colony-forming units assay revealed the increase of bacterial activity, FACS demonstrated the decrease of apoptosis rate of MDMs and TDMs, as well as promoted levels of IL-6, TNF-α, Bax, and Bim and suppressed levels of IL-10 and Bcl-2, examined by enzyme-linked immunosorbent assay (ELISA) and western blot assay. Expression of miR-125b-5p and DNA damage-regulated autophagy modulator 2 (DRAM2) was examined, and real-time PCR and western blot assay showed that miR-125b-5p was upregulated, whereas DRAM2 was downregulated in primary monocytes and H37Rv-infected macrophages (MDMs and TDMs). Moreover, blocking miR-125b-5p could attenuated H37Rv-induced bacterial activity and inflammatory response of MDMs and TDMs, accompanied with apoptosis inhibition. Whereas these effects of miR-125b-5p knockdown were abolished by downregulating DRAM2. In mechanism, DRAM2 was a downstream target of miR-125b-5p, as evidenced by dual-luciferase reporter assay. Collectively, silencing miR-125b-5p could protect human macrophages against Mtb infection through promoting apoptosis and inhibiting inflammatory response via targeting DRAM2, suggesting a novel target for Mtb eliminating. <b>Abbreviations:</b> TB: tuberculosis; PBMCs: peripheral blood mononuclear cells; Mtb: mycobacterium tuberculosis; AFB: acid fast bacilli; FITC: fluorescein isothiocyanate; MDMs: monocytes-derived macrophages; TDMs: THP-1-derived macrophage-like cells; ERFP: Mtb-enhanced red fluorescent protein; CFU: colony-forming units; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell separation; PI: propidium iodide; DRAM2: DNA damage-regulated autophagy modulator 2; Real-time PCR: real-time polymerase chain reaction; in-miR-125b-5p: miR-125b-5p inhibitor; si-DRAM2: siRNA against DRAM2

结核病（Tuberculosis, TB）是全球范围内最为重要的感染性疾病之一，巨噬细胞凋亡是宿主对抗结核分枝杆菌（Mycobacterium tuberculosis, Mtb）感染的核心防御机制。本研究旨在表征微小RNA（miRNA, miR）-125b-5p在结核分枝杆菌感染及巨噬细胞体外行为中的调控作用。
通过荧光激活细胞分选术（fluorescence-activated cell separation, FACS）分析发现，结核病患者体内的原代CD14⁺单核细胞出现富集，而凋亡单核细胞的比例显著降低。本研究采用外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）衍生的巨噬细胞（MDMs）以及单核细胞系THP-1衍生的巨噬样细胞（TDMs）进行体外感染实验，感染菌株为H37Rv。感染后，菌落形成单位（colony-forming units, CFU）检测结果显示细菌活性升高；FACS证实，MDMs与TDMs的凋亡率显著降低；经酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）与蛋白质印迹（western blot）检测，两组细胞中IL-6、TNF-α、Bax及Bim的表达水平上调，而IL-10与Bcl-2的表达水平受到抑制。
通过实时荧光定量聚合酶链反应与蛋白质印迹检测miR-125b-5p及DNA损伤调控自噬调节因子2（DNA damage-regulated autophagy modulator 2, DRAM2）的表达水平，结果显示，原代单核细胞及H37Rv感染的巨噬细胞（MDMs与TDMs）中，miR-125b-5p的表达显著上调，而DRAM2的表达则明显下调。
进一步研究发现，阻断miR-125b-5p可减轻H37Rv诱导的MDMs与TDMs的细菌活性升高及炎症反应，并伴随细胞凋亡的抑制。而敲低DRAM2可消除miR-125b-5p抑制剂（in-miR-125b-5p）所介导的上述保护效应。机制实验通过双荧光素酶报告基因实验证实，DRAM2是miR-125b-5p的下游直接靶基因。
综上，沉默miR-125b-5p可通过靶向DRAM2促进人巨噬细胞凋亡、抑制炎症反应，从而对抗结核分枝杆菌感染，这为结核分枝杆菌的清除提供了全新的潜在治疗靶点。
**缩写说明：**
TB：结核病（Tuberculosis）；PBMCs：外周血单个核细胞（peripheral blood mononuclear cells）；Mtb：结核分枝杆菌（Mycobacterium tuberculosis）；AFB：抗酸杆菌（acid fast bacilli）；FITC：异硫氰酸荧光素（fluorescein isothiocyanate）；MDMs：单核细胞衍生巨噬细胞（monocytes-derived macrophages）；TDMs：THP-1细胞衍生巨噬样细胞（THP-1-derived macrophage-like cells）；ERFP：结核分枝杆菌增强型红色荧光蛋白（Mtb-enhanced red fluorescent protein）；CFU：菌落形成单位（colony-forming units）；ELISA：酶联免疫吸附试验（enzyme-linked immunosorbent assay）；FACS：荧光激活细胞分选术（fluorescence-activated cell separation）；PI：碘化丙啶（propidium iodide）；DRAM2：DNA损伤调控自噬调节因子2（DNA damage-regulated autophagy modulator 2）；Real-time PCR：实时荧光定量聚合酶链反应（real-time polymerase chain reaction）；in-miR-125b-5p：miR-125b-5p抑制剂；si-DRAM2：靶向DRAM2的小干扰RNA（siRNA against DRAM2）