Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival

Abstract

BackgroundThe 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.MethodsThrough 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.ResultsBioinformatics 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.Conclusionmmu-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.

Roles of autophagy in killing of mycobacterial pathogens by host macrophages – Effects of some medicinal plants

“…Infection with Mycobacterium bovis BCG was previously shown to induce the up-regulation of a miRNA called mmu-miR-25-3p and down-regulation of dual specificity phosphatase 10 (DUSP10) in RAW264.7 macrophages, which further increased the expression of LC3-II and promoted autophagy [ 38 ]. The up-regulated expression of mmu-miR-25-3p subsequently decreased DUSP10 levels and enhanced the phosphorylation of ERK1/2, which, in turn up-regulated the expression of LC3-II, Atg5, Atg7, and beclin1 [ 38 ]. Therefore, mmu-miR-25-3p appears to promote the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thereby enhancing the BCG-induced autophagy of macrophages [ 38 ].…”

“…The up-regulated expression of mmu-miR-25-3p subsequently decreased DUSP10 levels and enhanced the phosphorylation of ERK1/2, which, in turn up-regulated the expression of LC3-II, Atg5, Atg7, and beclin1 [ 38 ]. Therefore, mmu-miR-25-3p appears to promote the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thereby enhancing the BCG-induced autophagy of macrophages [ 38 ]. On the other hand, other types of microRNA have been shown to play roles in suppressing the autophagy of macrophages.…”

Hyperbaric Lidocaine Aggravates Diabetic Neuropathic Pain by Targeting p38 MAPK/ERK and PINK1/Parkin-Mediated Mitophagy Signaling Pathway

“…Meanwhile, PD98059 is an inhibitor of ERK1/2 signaling. PD98059 also inhibited autophagy induced by Mycobacterium bovis Bacillus Calmette-Guerin (BCG) [38,39]. Previous studies suggested that the activation of PINK1/Parkin is affected by ERK signaling [40].…”

Current landscape of exosomes in tuberculosis development, diagnosis, and treatment applications

“…Mechanistically, miR-18a down-regulated the ATM pathway to repress the autophagy response and promote the intracellular survival of MTB . The expression of exosomal miR-25–3p was also significantly increased in BCG-infected macrophages ( 64 ). By inhibiting the expression of DUSP10, mmu-miR-25–3p promoted the phosphorylation of ERK1/2 and thus enhanced the macrophage autophagy induced by BCG.…”