MicroRNA‑502‑3p promotes Mycobacterium tuberculosis survival in macrophages by modulating the inflammatory response by targeting ROCK1

Abstract

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tuberculosis) infection and has the highest mortality rate of any single infectious disease worldwide. The aim of the present study was to investigate the function of microRNA (miR)‑502‑3p in M. tuberculosis‑infected macrophages. The Gene Expression Omnibus database was used to analyze miR‑502‑3p expression in patients with TB and healthy individuals. THP‑1 and RAW 264.7 cells were transfected with miR‑502‑3p mimic, miR‑502‑3p inhibitor, pcDNA3.1‑ROCK1 or their negative controls. The expression levels of miR‑502‑3p and inflammatory cytokines were evaluated using reverse transcription‑quantitative PCR. The colony‑forming unit assay was performed to assess the survival of M. tuberculosis in macrophages, and Toll‑like receptor (TLR)4/NF‑κB signaling pathway‑associated protein expression levels were detected by western blotting. The nuclear translocation of NF‑κB p65 was detected via immunocytochemistry. TargetScan was used to predict the binding sites between miR‑502‑3p and ROCK1. The interaction between miR‑502‑3p and Rho‑associated coiled‑coil‑forming protein kinase 1 (ROCK1) was confirmed using a dual‑luciferase reporter assay; ROCK1 was demonstrated to be a direct target gene of miR‑502‑3p. Results from the present study demonstrated that miR‑502‑3p expression was significantly increased during M. tuberculosis infection in macrophages. Upregulation of miR‑502‑3p expression levels significantly enhanced the survival of intracellular M. tuberculosis. IL‑6, TNF‑α, and IL‑1β mRNA expression levels were significantly upregulated during M. tuberculosis infection but were downregulated by miR‑502‑3p overexpression. Moreover, miR‑502‑3p mimics transfection significantly downregulated TLR4/NF‑κB signaling pathway‑associated protein expression and significantly reduced nuclear transcription of NF‑κB in M. tuberculosis‑infected macrophages. ROCK1 overexpression reversed the miR‑502‑3p inhibitory effect on cytokine production in M. tuberculosis‑infected macrophages. In conclusion, miR‑502‑3p/ROCK1 may serve an anti‑inflammatory role and may improve the survival of M. tuberculosis within macrophages, which may provide a promising therapeutic target for TB.

Keywords: Mycobacterium tuberculosis; Rho‑associated coiled-coil-forming protein kinase 1; inflammation; macrophages; microRNA‑502‑3p.

Figure 1.

M. tuberculosis infection induces miR-502-3p expression in patients with TB. (A) Seven miRNAs overlapped between the GSE34608 and GSE116542 databases. (B) miR-502-3p expression levels were elevated in patients with TB compared to healthy patients analyzed using the GSE116542 databases. (C) miR-502-3p expression in M. tuberculosis-infected THP-1 and RAW 264.7 cells at an MOI of 10 for the indicated time was determined via reverse transcription-quantitative PCR. (D) miR-502-3p expression in M. tuberculosis-infected THP-1 and RAW 264.7 cells at indicated MOI for 48 h was determined by reverse transcription-quantitative PCR. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 h or MOI of 0. miR/miRNA, microRNA; MOI, multiplicity of infection; TB, tuberculosis; M., Mycobacterium.

Figure 2.

miR-502-3p facilitates Mycobacterium tuberculosis survival in macrophages. (A) miR-502-3p expression levels in THP-1 and RAW 264.7 cells were measured by reverse transcription-quantitative-PCR following transfection. (B) M. tuberculosis survival was measured using the colony-forming unit assay. ***P<0.001 vs. mimic NC; ^###P<0.001 vs. inhibitor NC. miR, microRNA; NC, negative control.

Figure 3.

miR-502-3p suppresses cytokine mRNA expression levels in Mycobacterium tuberculosis-infected macrophages. (A) IL-6, (B) TNF-α and (C) IL-1β mRNA expression levels were measured by reverse transcription-quantitative PCR following transfections and infection. Untreated macrophages were used as the control. **P<0.01 and ***P<0.001 vs. control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. M.tb. miR, microRNA; M.tb, Mycobacterium tuberculosis; NC, negative control.

Figure 4.

miR-502-3p directly targets ROCK1. (A) Predicted target site of miR-502-3p in the ROCK1 3′UTR. (B) Dual-luciferase reporter assay verified the targeting relationship between miR-502-3p and ROCK1. **P<0.01 vs. mimic NC. (C) ROCK1 protein expression levels of ROCK1 were measured by western blotting following transfections and infection. Untreated macrophages were used as the control. ***P<0.001 vs. control; ^###P<0.001 vs. M.tb. Hsa, Homo sapiens; miR, microRNA; M.tb, Mycobacterium tuberculosis; MUT, mutant; NC, negative control; ROCK1, Rho-associated coiled-coil-forming protein kinase 1; UTR, untranslated region; WT, wild-type.

Figure 5.

miR-502-3p regulates the TLR4/NF-κB signaling pathway in M. tuberculosis-infected macrophages. TLR4/NF-κB signaling pathway-associated proteins were assessed using western blotting. ***P<0.001 vs. control; ^##P<0.01 and ^###P<0.001 vs. M.tb. miR, microRNA; TLR, toll-like receptor; M.tb, Mycobacterium tuberculosis; p, phosphorylated; NC, negative control.

Figure 6.

miR-502-3p inhibits the expression of NF-κB p-p65 in nucleus. NF-κB p-p65 expression was evaluated by immunofluorescence staining. Scale bar, 50 µm. Untreated macrophages were used as the control. ***P<0.001 vs. control; ^##P<0.01 and ^###P<0.001 vs. M.tb. miR, microRNA; M.tb, Mycobacterium tuberculosis; NC, negative control.

Figure 7.

ROCK1 overexpression reverses the miR-502-3p inhibitory effect on cytokine mRNA expression levels in M. tuberculosis-infected macrophages. (A) ROCK1 protein expression levels were measured by western blotting. ***P<0.001. (B) IL-6, TNF-α and IL-1β mRNA expression levels were measured by reverse transcription-quantitative PCR following transfection and infection. ***P<0.001 vs. control; ^##P<0.01 and ^###P<0.001 vs. M.tb; ^{^}P<0.05, ^{^^}P<0.01 and ^{^^^}P<0.001 vs. M.tb + miR-502-3p mimic + pcDNA3.1. miR, microRNA; M.tb, Mycobacterium tuberculosis; NC, negative control; ROCK1, Rho-associated coiled-coil-forming protein kinase 1.