Fig 1.
Analysis of the miR-155 seed sequence in the TLR3 coding region.
(a and b) The sequence logo for miR-155 was generated by Weblogo (http://weblogo.berkeley.edu/). (c) The target sites of the miR-155 sequence in the TLR3 coding regions from Homo sapiens, Gallus gallus, Danio rerio, Equus caballus, Taeniopygia guttata and Cyprinus carpio at the seed matches of the 8mer and 7mer-m8 sites. (d and e) Clustal W analysis of the miR-155 sequence with Molecular Evolutionary Genetics Analysis version 5 (MEGA5) software.
Fig 2.
Luciferase reporter assay for the interaction between miR-155 and TLR3.
(a and b) Predicted binding sites of gga-miR-155 and mdv1-mir-M4-5p in the coding region of the TLR3 mRNA. Five nucleotides were mutated in the coding region of the TLR3 mRNA. The numbers indicate the positions of the nucleotides in the wild-type sequences (NM_001011691.3). (c) Activity of the luciferase gene linked to the coding region of the TLR3 mRNA. The pGL3 firefly luciferase reporter plasmids with wild-type or mutant for the TLR3 mRNA coding regions were transiently transfected into HEK293 cells with the gga-miR-155 precursor, mdv1-mir-M4-5p or the negative control and a Renilla luciferase reporter for normalisation. Luciferase activities were measured after 48 hr. The data were presented as the means and the standard deviations (SDs) of separate transfections (n = 5). (d and e) The down-regulation of endogenous TLR3 protein expression by miR-155. CEF cells were transfected with 50–200 nM of the miR-155 mimic or the negative control mimic, and TLR3 expression in the cell lysates was analysed with western blotting. The expression of β-actin was used as a control.
Fig 3.
The regulation of TLR3 expression by miR-155.
(a) miR-155 expression in HD11 cells treated with TLR2, 4 and 7 ligand. miR-21 expression was used as the control. (b) The relative expression (fold) of TLR3 mRNA expression in HD11 cells treated with TLR2, 4 and 7 ligand. Control group = 1 (c) The expression of TLR3 protein expression in HD11 cells treated with TLR2, 4 and 7 ligand. (d) The up-regulation of TLR3 protein expression in HD11 cells treated with 50–400 nM of the gga-miR-155 antagomir. (e) The down-regulation of TLR3 protein expression in HD11 cells treated with 50–400 nM of the gga-miR-155 agomir.
Fig 4.
The regulation of IFN-β production by miR-155.
(a) miR-155 expression in CEF cells treated with TLR3 ligand. miR-21 expression was used as the control. (b) ELISA for the productions of IFN-α, IFN-β, IL1-β, IL6 and TNF-α in the CEF cells treated with TLR ligands. (c) Down-regulation of IFN-β in CEF cells transfected with 50–200 nM of the gga-miR-155 mimic. (d) Up-regulation of IFN-β in CEF cells transfected with 50–200 nM of the gga-miR-155 inhibitor. (e) Down-regulation of IFN-β in HD11 cells treated with 50–400 nM of the gga-miR-155 agomir. (f) Up-regulation of of IFN-β in HD11 cells treated with 50–400 nM of the gga-miR-155 antagomir.