Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.
Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.
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DOI：
10.3389/fcimb.2023.1120570
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发表时间：
2023
影响因子：
5.7
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中科院分区：
文献类型：
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本研究基于前期BCG感染巨噬细胞外泌体miRNA差异表达的研究结果，旨在探讨miR-25-3p对巨噬细胞自噬的调控及其对牛分枝杆菌卡介苗（BCG）滞留巨噬细胞清除的影响。通过富集分析和Hub基因分析，筛选出关键差异表达miRNA及其靶基因。通过TargetScan数据库确定筛选的mmu-miR-25-3p与其预测靶基因DUSP10的靶向结合能力，并通过双荧光素酶报告基因检测进一步验证。 mmu-miR-25-3p模拟物、mmu-miR-25-3p抑制剂、si-DUSP10、miR-NC、si-NC和PD98059（ERK抑制剂）用于干预巨噬细胞Raw264.7。采用RT-qPCR检测mmu-miR-25-3p和DUSP10 mRNA的表达水平。 Western blot检测DUSP10、LC3-II、p-ERK1/2、beclin1、Atg5、Atg7的表达水平。采用激光共聚焦显微镜观察各组巨噬细胞Raw264.7的自噬通量，透射电镜观察DUSP10的表达分布和自噬体的结构。最后，通过集落形成单位（CFU）测定评估巨噬细胞Raw264.7的细胞内BCG负载。生物信息学分析筛选并鉴定差异表达的外泌体miRNA。结果，mmu-miR-25-3p表达显着增加，双特异性磷酸酶10（DUSP10）被预测为其靶基因，主要参与自噬调节。双荧光素酶报告基因活性测定显示 mmu-miR-25-3p 靶向 DUSP10 的 3'-非翻译区 (UTR)。 BCG感染诱导RAW264.7细胞中mmu-miR-25-3p上调和DUSP10下调，进一步增加LC3-II的表达并促进自噬。上调mmu-miR-25-3p表达会降低DUSP10的水平并增强ERK1/2的磷酸化，进而上调LC3-II、Atg5、Atg7和Beclin1的表达。免疫电镜、透射电镜和自噬流分析进一步证实，BCG感染后mmu-miR-25-3p的上调促进了巨噬细胞的自噬。 CFU 数表明，上调 mmu-miR-25-3p 表达可降低分枝杆菌负荷并加速残留分枝杆菌清除。 mmu-miR-25-3p通过抑制DUSP10的表达促进ERK1/2的磷酸化，从而增强BCG诱导的巨噬细胞自噬。这些现象减少了细胞内分枝杆菌的细菌负荷，并有利于残留分枝杆菌的清除。 mmu-miR-25-3p作为抗结核免疫治疗的靶点具有巨大的潜力，并且可以成为未来研究中加载到外泌体药物递送系统中的最佳miRNA。
The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3’-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.