Next generation sequencing reveals miR-431–3p/miR-1303 as immune-regulating microRNAs for active tuberculosis

•Whole genome sequencing identified 55 TB-related differentially expressed microRNAs.•Proteoglycan, longevity, apoptosis, central carbon metabolism, and autophagy were enriched target pathways.•miR-431–3p down-regulation and miR-1303 up-regulation were verified in vivo & vitro.•miR-431–3p over-expression and miR-1303 knock-down improved macrophage functions synergistically.•Both microRNAs regulate autophagy, apoptosis, and phagocytosis via targeting MDR1/MMP16/RIPOR2 and ATG5. RNA therapeutics is an emerging field that widens the range of treatable targets and would improve disease outcome through bypassing the antibiotic bactericidal targets to kill Mycobacterium tuberculosis (M.tb). We screened for microRNA with immune-regulatory functions against M.tb by next generation sequencing of peripheral blood mononuclear cells, followed by validation in an independent cohort. Twenty three differentially expressed microRNAs were identified between 12 active pulmonary TB patients and 4 healthy subjects, and 35 microRNAs before and after 6-month anti-TB therapy. Enriched predicted target pathways included proteoglycan, HIF-1 signaling, longevity-regulating, central carbon metabolism, and autophagy. We validated miR-431–3p down-regulation and miR-1303 up-regulation accompanied with corresponding changes in their predicted target genes in an independent validation cohort of 46 active TB patients, 30 latent TB infection subjects, and 24 non-infected healthy subjects. In vitro experiments of transfections with miR-431–3p mimic/miR-1303 short interfering RNA in THP-1 cells under ESAT-6 stimuli showed that miR-431–3p and miR-1303 were capable to augment and suppress autophagy/apoptosis/phagocytosis of macrophage via targeting MDR1/MMP16/RIPOR2 and ATG5, respectively. This study provides a proof of concept for microRNA-based host-directed immunotherapy for active TB disease. The combined miR-431–3p over-expression and miR-1303 knock-down revealed new vulnerabilities of treatment-refractory TB disease.