Abstract:

Objective ·To explore the regulatory effect of miR-185-5p on autophagy in Mycobacterium-infected macrophages and its influence on the survival of intracellular Mycobacterium. Methods ·Macrophages derived from human monocytic-leukemia (THP-1) cells were infected with Bacillus Calmette-Guérin (BCG) strain. Exosomes from cell culture supernatant were isolated by ultracentrifugation, and exosome surface marker proteins, size and morphology were detected by Western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively. The expression level of miR-185-5p in both macrophages and exosomes was detected by real time quantitative polymerase chain reaction (qPCR). The exosomes were labeled with the fluorescent dye PKH26 and co-cultured with macrophages to observe the phagocytosis of exosomes by macrophages. Subsequently, the overexpression and inhibition of miR-185-5p in macrophages were achieved by transfecting cells with miR-185-5p mimic and inhibitor, and the influence of miR-185-5p on intracellular BCG survival was detected by colony-forming units (CFU) assay. Autophagy marker microtubule-associated protein 1 light chain 3 (LC3) protein was detected by Western blotting.The effects of overexpression and inhibition of miR-185-5p and the use of autophagy inhibitor chloroquine (CQ) on macrophage autophagy were studied. The SensGFP-StubRFP-LC3 lentvirus was further employed to detect the effect of miR-185-5p on autophagic flux. Finally, TargetScan, miRDB and Starbase databases were used to predict the target genes of miR-185-5p, and the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of these predicted target genes. Results ·The macrophage exosomes were isolated by ultracentrifugation. Exosome marker proteins cluster differentiation 9 (CD9) and tumor susceptibility gene 101 (TSG101) were successfully detected by Western blotting. The exosomes had a bilayer membrane with a diameter of approximately 150 nm through TEM and NTA identification. After BCG infection, the expression of miR-185-5p was up-regulated in both macrophages and exosomes (P=0.000), and showed a time-dependent increase in macrophages. Exosomes, which were co-cultured with macrophages, can be phagocytized by macrophages. Compared with respective control of mimic and inhibitor, mimic significantly up-regulated miR-185-5p expression (P=0.000), while inhibitor inhibited miR-185-5p expression (P=0.002). miR-185-5p mimic significantly increased the number of CFU of intracellular BCG (P=0.000), while miR-185-5p inhibitor decreased the number of CFU (P=0.041). BCG infection up-regulated the expression of autophagy protein LC3Ⅱ in macrophages, while LC3Ⅱ expression was decreased by miR-185-5p mimic and increased by miR-185-5p inhibitor. CQ treatment combined with miR-185-5p inhibitor transfection still significantly increased LC3Ⅱ expression in macrophages. The SensGFP-StubRFP-LC3 lentivirus was further used to detect autophagy flux, and it was suggested that, compared with that in the respective control group, mimic significantly reduced the formation of autophagosomes (P=0.034), while inhibitor increased the formation of autophagosomes (P=0.042). Through target genes prediction and GO and KEGG functional enrichment analysis, it was found that miR-185-5p might play a role in autophagy regulation by targeting nuclear receptor subfamily 1 group D member 1 (NR1D1), Ras-related protein Rab-35 (RAB35), cell division control protein 42 homolog (CDC42), etc. Conclusion ·Mycobacterium infection can induce the up-regulation of miR-185-5p in both macrophages and exosomes, and miR-185-5p can inhibit the autophagy process of macrophages by inhibiting the formation of autophagosomes, which can promote the growth and survival of intracellular Mycobacterium.

Key words: Mycobacterium infection, macrophage, miR-185-5p, autophagy, exosome