Table 4_Elevated miR-17-5p facilitates mycobacterial immune evasion by targeting MAP3K2 in macrophages.doc

IntroductionTuberculosis (TB) rema\ins a major global health challenge. Mycobacterium avium (M. avium), a non-tuberculosis mycobacterium, causes pulmonary infections and can evade host immune surveillance by persisting within macrophages. MicroRNAs (miRNAs) are key regulators of host immunity; however, their roles in mycobacterial pathogenesis are not fully understood. This study investigated the role of miR-17-5p in macrophage-mediated immune responses during M. avium infection, with a focus on MAP3K2-mediated MAPK signaling.
MethodsDifferentially expressed miRNAs were identified through small RNA sequencing of exosomes from M. avium-infected THP-1 macrophages. Candidate miRNAs were validated by RT-qPCR in THP-1 derived exosomes and serum samples from TB patients. MAP3K2 was evaluated as a miR-17-5p target using bioinformatics prediction, dual-luciferase reporter assays, and expression analysis. Effects on immune responses and MAPK signaling were assessed using qPCR, ELISA, Western blotting, ROS measurement, and CFU assays.
ResultsmiR-17-5p expression was significantly elevated in M. avium–infected macrophages, as well as in serum and peripheral blood mononuclear cells (PBMCs) from TB patients. Increased miR-17-5p suppressed MAP3K2 expression and attenuated MAPK signaling, reducing phosphorylation of ERK, JNK, and p38. This resulted in decreased production of inflammatory mediators (TNF-α, IL-6, IL-1β), reduced iNOS and ROS levels, and impaired bacterial clearance.
DiscussionmiR-17-5p promotes M. avium survival by targeting MAP3K2 and suppressing MAPK-dependent immune functions in macrophages. These findings highlight miR-17-5p as a potential diagnostic biomarker and therapeutic target in TB and related mycobacterial infections.

引言：结核病（Tuberculosis, TB）仍是全球主要的公共卫生挑战。鸟分枝杆菌（Mycobacterium avium, M. avium）作为一种非结核分枝杆菌，可引发肺部感染，并通过在巨噬细胞内持续存活逃避免疫监视。微小RNA（MicroRNAs, miRNAs）是宿主免疫的关键调控因子，但其在分枝杆菌致病过程中的作用尚未完全阐明。本研究探讨了miR-17-5p在鸟分枝杆菌感染期间巨噬细胞介导的免疫应答中的作用，重点关注MAP3K2介导的丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）信号通路。
方法：通过对鸟分枝杆菌感染的THP-1巨噬细胞来源的外泌体进行小RNA测序，筛选差异表达的微小RNA。采用实时荧光定量PCR（RT-qPCR）对THP-1细胞来源外泌体及结核病患者血清样本中的候选微小RNA进行验证。通过生物信息学预测、双荧光素酶报告基因实验及表达分析，验证MAP3K2作为miR-17-5p的靶基因。采用qPCR、酶联免疫吸附试验（ELISA）、蛋白质印迹（Western blotting）、活性氧（ROS）检测及菌落形成单位（CFU）实验，评估其对免疫应答及MAPK信号通路的影响。
结果：miR-17-5p在鸟分枝杆菌感染的巨噬细胞及结核病患者血清、外周血单个核细胞（PBMCs）中均显著高表达。上调的miR-17-5p可抑制MAP3K2表达并减弱MAPK信号通路活性，降低细胞外调节蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）及p38的磷酸化水平。这可导致炎症介质（TNF-α、IL-6、IL-1β）生成减少，诱导型一氧化氮合酶（iNOS）及ROS水平降低，并削弱细菌清除能力。
讨论：miR-17-5p通过靶向MAP3K2并抑制巨噬细胞中依赖MAPK的免疫功能，促进鸟分枝杆菌的存活。本研究结果表明，miR-17-5p可作为结核病及相关分枝杆菌感染的潜在诊断生物标志物与治疗靶点。