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. 2018 Mar 1;11(3):1218-1227.
eCollection 2018.

MicroRNA-26b Inhibits the Immune Response to Mycobacterium Tuberculosis (M.tb) Infection in THP-1 Cells via Targeting TGFβ-activated kinase-1 (TAK1), a Promoter of the NF-κB Pathway

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Free PMC article

MicroRNA-26b Inhibits the Immune Response to Mycobacterium Tuberculosis (M.tb) Infection in THP-1 Cells via Targeting TGFβ-activated kinase-1 (TAK1), a Promoter of the NF-κB Pathway

Hongwei Li et al. Int J Clin Exp Pathol. .
Free PMC article

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has led to many clinical disorders and remains a major global health problem, leading to great morbidity and mortality worldwide. Previous studies reported that Mycobacterium tuberculosis (M.tb) has evolved to utilize various mechanisms to evade or attenuate the immune response, such as dysregulation of miRNAs. However, reports concerning the role of miRNAs in M.tb infection are limited. Here, we report that a host microRNA, miR-26b, was significantly down-regulated by M.tb infection in THP-1 cells. Subsequently, our results of in vitro experiments demonstrate that miR-26b is a negative regulator of the NF-κB pathway by directly targeting TAK1, resulting in the inhibition of immune response, and promotion of M.tb replication and gene expression. Taken together, our findings provide detailed molecular mechanisms for how miR-26 inhibits inflammatory cytokine secretion during M.tb infection in THP-1 cells, and these results suggest anti-M.tb as a promising therapy.

Keywords: Mycobacterium tuberculosis; NF-κB; TAK1; miR-26b.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
miR-26b is markedly down-regulated in M.tb infected THP-1. (A-D) THP-1 cells were infected with H37Rv at MOI 10. Supernatant was harvested at different time point and ELISA assay was conducted for measuring IFN-γ (A), IL-1β (B), IL-6 (C) and TNF-α (D) secretion. (E) Heat-map of microRNA array. (F-I) Cells were treated as (A-D) and were harvested at indicated time for RNA extraction. Real-time PCR assay was conducted to measure the levels of miR-26b (F), miR-124 (G), miR-16 (H) and miR-451 (I). Results represent of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01 vs uninfected groups.
Figure 2
Figure 2
miR-26b suppresses M.tb induced inflammatory cytokines secretion. THP-1 cells were transfected with miR-26b mimics or mimics NC. 24 h post-transfection, cells were infected with M.tb H37Rv strain at MOI of 10. (A) The miR-26b expression levels at 48 hpi were quantified by qRT-PCR. (B-E) 24 hr post infection, supernatant was harvested. ELISA assay were performed to measure IFN-γ (B), IL-1β (C) IL-6 (D) and TNF-α (E) secretion. Results represent of three independent experiments (mean ± SD). **P < 0.01, ##P < 0.01 vs mimics NC groups.
Figure 3
Figure 3
miR-26b suppresses TNFα-induced NF-κB signaling in THP-1 cell. (A, B) THP-1 cells were first transfected with mimics NC or miR-26b mimics (A), inhibitor NC or miR-26b inhibitor (B), followed by co-transfection of pRL-TK and pNF-κB-Luc, treatment with 20 ng/ml TNFα, and analysis for luciferase activity. **P < 0.01 vs mimics NC group or inhibitor NC group. (C, D) THP-1 cells transfected with mimics NC or miR-26b mimics (C), inhibitor NC or miR-26b inhibitor (D) were treated with 2 ng/ml TNFα for the 5 min. Cells were harvested for immunoblotting analysis. All data represent the mean value ± SD of at least three independent experiments.
Figure 4
Figure 4
TAK1 is directly targeted by miR-26b. (A) Schematic diagram of the predicted target sites of miR-26b in TAK1 3’UTRs. The predicted target sites are underlined and mutated as indicated. (B) THP1 cells were co-transfected with TAK1 WT or TAK1 mutant luciferase reporter vector (100 ng), and miR-26b mimics or NC, miR-26b inhibitor or NC inhibitor for 36 h and then harvested for luciferase assay. **P < 0.01 vs mimics NC group or inhibitor NC group. (C, D) THP-1 cells were transfected with miR-26b mimics or NC mimics (C), miR-26b inhibitor or NC inhibitor (D) for 36 h and harvested for Western blot analysis of TAK1 expression. All data represent the mean value ± SD of at least three independent experiments.
Figure 5
Figure 5
miR-26b regulates host immune response towards M.tb through targeting TAK1. THP-1 cells were transfected with miR-26b mimics, pcDNA-TAK1, or both. 24 hr after transfection, cells were infected with H37Rv at MOI 10. 24 hr post-infection, supernatant were harvested for ELISA assay to detect IFN-γ (A), IL-1β (B), IL-6 (C) and TNF-α (D) secretion. All data represent the mean value ± SD of at least three independent experiments. *P < 0.05, **P < 0.01 vs non-transfected groups; ##P < 0.01 vs miR-26b-transfected groups.

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