Immune regulation of miR-30 on the Mycobacterium tuberculosis-induced TLR/MyD88 signaling pathway in THP-1 cells

Abstract

The present study aimed to examine the expression of microRNA (miR)-30 family members in THP-1 human monocytes cells during Mycobacterium tuberculosis (MTB) H37Rv infection, and to investigate the role of miR-30 in the regulation of MTB-induced Toll-like receptor (TLR)/myeloid differentiation factor 88 (MyD88) activation and cytokine expression. The THP-1 cells were infected with MTB H37Rv and the expression of miR-30 family members was determined by reverse transcription-quantitative polymerase chain reaction analysis. In addition, miR-30a and miR-30e mimics were transfected into THP-1 cells to overexpress miR-30a and miR-30e. The expression of TLR2, TLR4 and MyD88 was determined by western blot analysis, and the expression of the cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-8 was determined using ELISA assays. A luciferase reporter assay was used to identify the target gene of miR-30a. MTB infection was demonstrated to significantly induce miR-30a and miR-30e expression in THP-1 cells in a time-dependent manner. Forced overexpression of miR-30a, but not miR-30e, exhibited an inhibitory effect on TLR/MyD88 activation and cytokine expression in the uninfected and MTB-infected THP-1 cells. The luciferase reporter assay demonstrated that miR-30a directly regulates the transcriptional activity of the MyD88 3'-untranslated region. In conclusion, the present study, to the best of our knowledge, is the first to demonstrate that miR-30a suppresses TLR/MyD88 activation and cytokine expression in THP-1 cells during MTB H37Rv infection, and that MyD88 is a direct target of miR-30a. The current study may aid in the development of novel therapeutic approaches for treating MTB.

Keywords: Mycobacterium tuberculosis; immune response; miR-30; myeloid differentiation factor 88; toll-like receptor.

Figure 1.

Expression of miR-30 family members in THP-1 cells infected with Mycobacterium tuberculosis. *P<0.05 and ^#P<0.01 vs. 0 h. mRNA/miR, microRNA.

Figure 2.

Effect of miR-30a and miR-30e mimics on the expression of miR-30a and miR-30e in THP-1 cells. Relative expression of (A) miR-30a and (B) miR-30e in THP-1 cells following transfection with miR-30a and miR-30e mimics. ^#P<0.01 vs. CTRL; ^ΔP<0.01 vs. MTB. mRNA/miR, microRNA; MTB, Mycobacterium tuberculosis; CTRL, control.

Figure 3.

Expression of TLR2, TLR4 and MyD88 in MTB-infected THP-1 cells following transfection with miR-30a or miR-30e mimics assessed using western blot analysis. Quantification of expression of (A) TLR2, (B) TLR4 and (C) MyD88. (D) Western blot of TLR2, TLR4 and MyD88 expression. *P<0.05, ^#P<0.01 vs. CTRL; ^∆P<0.01 vs. MTB. miR, microRNA; MTB, Mycobacterium tuberculosis; CTRL, control; TLR, Toll-like receptor; MyD88, myeloid differentiation factor 88; lane 1, CTRL; lane 2, MTB; lane 3, miR-30a; lane 4, miR-30a+MTB; lane 5, miR-30e; lane 6, miR-30e+MTB.

Figure 4.

Expression of TNF-α, IL-6 and IL-8 in MTB-infected THP-1 cells following transfection with miR-30a or miR-30e mimics. ELISA analysis of (A) TNF-α, (B) IL-6 and (C) IL-8 levels. *P<0.05, ^#P<0.01 vs. CTRL; ^∆P<0.01 vs. MTB. miR, microRNA; MTB, Mycobacterium tuberculosis; CTRL, control; TNF-α, tumor necrosis factor α; IL, interleukin.

Figure 5.

MyD88 is a direct target of miR-30a. ^#P<0.01 vs. miR-NC. miR, microRNA; NC, negative control; MyD88, myeloid differentiation factor 88; WT, wild-type; MUT, mutant.