Inducible microRNA-214 contributes to the suppression of NF-κB-mediated inflammatory response via targeting myd88 gene in fish

Abstract

Upon recognition of bacterial pathogens by pattern recognition receptors, cells are activated to produce pro-inflammatory cytokines and type I IFN by multiple signaling pathways. Every step of the process must be precisely regulated to prevent dysregulation. MicroRNAs (miRNAs) have been shown to be important regulators with profound effects on inflammatory response. Nevertheless, the miRNA-mediated regulatory mechanism remains unclear in fish species. Here, we addressed the role of miiuy croaker miR-214 in the bacteria triggered inflammatory response. miR-214 could significantly be up-regulated by Vibro harveyi and LPS stimulation. Up-regulating miR-214 subsequently inhibits the production of inflammatory cytokines by targeting myd88 to avoid excessive inflammation. Moreover, the negative regulatory mechanism of miR-214 has been demonstrated to be via the myd88-mediated NF-κB pathway. This is the first to focus on miR-214 acting as the negative regulator involved in the bacteria-triggered inflammatory response and thus may provide knowledge on the host-cell regulator responses to microbial infection.

Keywords: MyD88; NF-κB; cellular immune response; excessive inflammation; gene regulation; microRNA (miRNA); microRNA mechanism; microRNA-214; myeloid differentiation primary response gene (88) (MYD88); negatively regulation.

FIGURE 1.

The expression profiles of miR-214 detected by qPCR. A, expression profiles of miR-214 after V. harveyi infection in liver. B, expression profiles of miR-214 after LPS stimulation in liver. The data were normalized to 5.8S rRNA. Results are standardized to 1 in control cells. Data represented the mean ± S.E. from three independent triplicate experiments.

FIGURE 2.

miR-214 suppresses the mRNA expression of inflammatory cytokines in LPS-stimulated macrophages. A, the macrophages were transfected with control miRNA (miR-Ctrl) or miR-214 (left) and control inhibitor (Ctrl-inhibitor) or miR-214 inhibitor (right) within a final concentration of 100 nm. At 48 h post-transfection, miR-214 expression was measured by qPCR and normalized to 5.8S rRNA. B, the macrophages were transfected with miR-214 mimics or miR-Ctrl. After 24 h, the cells were stimulated with LPS and the expression levels of TNF-α, IL-6, IL-1β, and IL-10 were analyzed by qPCR after another 24 h. C, the macrophages were transfected with miR-214 inhibitor or Ctrl-inhibitor. After 24 h, the cells were stimulated with LPS and the expression levels of TNF-α, IL-6, IL-1β, and IL-10 were analyzed by qPCR after another 24 h. Results are standardized to 1 in control cells. Data are presented as the mean ± S.E. from three independent triplicate experiments. **, p < 0.01; *, p < 0.05 versus the controls.

FIGURE 3.

miR-214 regulates the expression of myd88, IRAK1, IRAK4, and TRAF6. The macrophages were transfected with miR-214 or miR-Ctrl and miR-214 inhibitor or Ctrl-inhibitor (final concentration, 100 nm). After 48 h, myd88 (A), IRAK1 (B), IRAK4 (C), and TRAF6 (D) mRNA levels were determined by qPCR and RT-PCR (E). Results are standardized to 1 in control cells, and mRNA levels are normalized to β-actin. Data are presented as the mean ± S.E. from three independent triplicate experiments. *, p < 0.05 versus the controls.

FIGURE 4.

miR-214 targets miiuy croaker myd88. A, sequence alignment of miR-214 and pre-miR-214 and its construction plasmids. B, HEK293 cells were cotransfected with pmirGLO empty plasmid, wild-type myd88-3′UTR (WT), and the mutant type of myd88-3′UTR (MT), together with miR-214 or miR-Ctrl and pre-miR-214 plasmid or its control. After 48 h, luciferase activity was determined and normalized to Renilla luciferase activity. C, HEK293 cells were cotransfected with myd88-3′UTR (WT), together with miR-214 or miR-Ctrl and miR-214 inhibitor or Ctrl-inhibitor. Each assay was transfected with equal amounts of oligonucleotides (final concentration, 100 nm). After 24 h, luciferase activity was determined and normalized to Renilla luciferase activity. D, HEK293 cells were cotransfected with myd88-3′UTR (WT), together with miR-214 and miR-Ctrl in concentration (left) and time (right) gradient manners. E, HEK293 cells were cotransfected with myd88-3′UTR (WT), together with pre-miR-214 plasmid and control plasmid in concentration (left) and time (right) gradient manners. The luciferase activity value was achieved against the Renilla luciferase activity. F, HEK293 cells were cotransfected with pIZ/EGFP-myd88-3′UTR or empty vector, together with miR-214 or miR-Ctrl and pre-miR-214 or pcDNA6.2 plasmid. At 48 h post-transfection, the fluorescence intensity was evaluated. Data are presented as the mean ± S.E. from three independent triplicate experiments. **, p < 0.01 versus the controls.

FIGURE 5.

miR-214 treatment decreases the abundance of myd88 at both mRNA and protein level. A, the macrophages were transfected with miR-214 or miR-Ctrl within the concentration gradient. After 48 h, myd88 mRNA levels were determined by qPCR and normalized to β-actin (left), and myd88 protein levels were determined by Western blot and normalized to β-actin (right). B, the macrophages were transfected with miR-214 inhibitor or Ctrl-inhibitor within concentration gradient. After 48 h, myd88 mRNA levels were determined by qPCR and normalized to β-actin (left), and myd88 protein levels were determined by Western blot and normalized to β-actin (right). C, HEK293 cells were cotransfected with myd88 expression plasmid, together with pre-miR-214 and control plasmid. After 48 h, myd88 mRNA levels were determined by qPCR and normalized to β-actin (left), and myd88 protein levels were determined by Western blot and normalized to β-actin (right). Data are presented as the mean ± S.E. from three independent triplicate experiments. **, p < 0.01; *, p < 0.05 versus the controls.

FIGURE 6.

The expression levels of myd88 and inflammatory cytokines after myd88 interference. Miiuy croker macrophages were transfected with control siRNA (si-Ctrl) or siRNA against myd88 (si-MyD88). A, after 48 or 72 h, myd88 protein levels were determined by Western blot and normalized to β-actin. B, after 48, myd88 mRNA levels were determined by qPCR and normalized to β-actin. C and D, after 24 h transfected with si-Ctrl or si-MyD88, macrophages were then stimulated with LPS for another 6 or 12 h and the expression of TNF-α (C), IL-6 (D), IL-1β (E), and IL-10 (F) were determined. Data are presented as the mean ± S.E. from three independent triplicate experiments. **, p < 0.01; *, p < 0.05 versus the controls.

FIGURE 7.

Overexpression of miR-214 suppresses myd88-mediated NF-κB pathway. A, HEK293 cells were cotransfectd with myd88 expression plasmid, pRL-TK Renilla luciferase plasmid, together with NF-κB reporter genes. After 48 h, the luciferase activity was measured and results presented relative to the luciferase activity in control cell. B, miR-214 or miR-Ctrl and miR-214 inhibitor or Ctrl-inhibitor were cotransfected with myd88 expression plasmid, pRL-TK Renilla luciferase plasmid, together with luciferase reporter NF-κB into HEK293 cells. After 24 h, luciferase activity was measured and results presented relative to the luciferase activity in the control cell. C, the concentration gradient experiment of miR-214 (left) or pre-miR-214 plasmid (right) was conducted. After 24 h, the luciferase activity was measured. D, the luciferase activity was measured after 24 or 48 h cotransfection with miR-214 (left) or pre-miR-214 plasmid (right). E, proposed model for miR-214 of inflammatory cytokine secretion via myd88. Data are presented as the mean ± S.E. from three independent triplicate experiments. **, p < 0.01; *, p < 0.05 versus the controls.