The MEG3/miR-133b regulatory axis in exosomes influences macrophage responses to drug-resistant mycobacterium tuberculosis.

Drug-resistant tuberculosis remains a major global health challenge, with spinal involvement causing substantial morbidity. Exosomes and non-coding RNAs (lncRNAs and miRNAs) are increasingly recognized as mediators of intercellular communication and immune modulation during mycobacterial infection, yet their specific roles in drug-resistant contexts are incompletely understood. To investigate the potential interaction between exosomal lncRNA MEG3 and miR-133b and its influence on macrophage proliferation and apoptosis in an in vitro model of drug-resistant Mycobacterium tuberculosis (MTB) infection. High-throughput miRNA sequencing was performed on serum from 15 patients with spinal tuberculosis (8 drug-resistant, 7 drug-sensitive). Differential expression analysis used DESeq2 with Benjamini-Hochberg FDR correction; GO and KEGG enrichment were assessed via clusterProfiler. Exosomes were isolated from RAW264.7 macrophages infected with drug-sensitive or drug-resistant MTB (MOI = 5) by differential ultracentrifugation and characterized by transmission electron microscopy and Western blotting for CD9/CD63. MEG3 levels in exosomes were quantified by RT-qPCR. Exosome uptake was evaluated with PKH67 labeling and fluorescence microscopy. Stable miR-133b overexpression or silencing lines were infected with MTB strains and analyzed by MTT assay (proliferation), TUNEL/flow cytometry (apoptosis), Western blot/RT-qPCR (Bcl-2/Bax), and dual-luciferase reporter assay (MEG3-miR-133b binding). miR-133b was significantly downregulated in drug-resistant versus drug-sensitive serum (adjusted P = 0.0008, log₂ fold change =  - 2.13) and enriched in pathways involving enzyme activity, binding, metabolism, signal transduction, and immunity. Exosomes from drug-resistant MTB-infected macrophages contained higher MEG3 levels and were internalized by recipient macrophages. In infected cells, miR-133b overexpression inhibited proliferation, increased apoptosis, decreased Bcl-2, and increased Bax; silencing produced opposite effects. Luciferase assays confirmed direct binding of miR-133b to MEG3, while qPCR indicated a complex, drug-resistance-context-dependent regulatory relationship. These exploratory findings support a potential regulatory interaction between exosomal MEG3 and miR-133b that influences macrophage proliferation and apoptosis during drug-resistant MTB infection in vitro. Although the results provide preliminary mechanistic evidence, additional biochemical, cellular, and in vivo validation will be required before definitive mechanistic conclusions or therapeutic implications can be established. These exploratory findings support a potential regulatory interaction between exosomal MEG3 and miR-133b that modulates macrophage fate in drug-resistant MTB infection in vitro. The axis appears context-specific. Validation in primary human macrophages, animal models, and larger clinical cohorts is required before any biomarker or therapeutic implications can be considered.