Luciferase reporter assay for the interaction between miR-155 and TLR3.

<p>(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.