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Figure 4 | Molecular Cancer

Figure 4

From: microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

Figure 4

CARD10 and COPS8 are direct miR-146a targets. (A) mRNA expression of the two new miR-146a targets, CARD10 and COPS8, and IRAK1, a known miR_146a target, was determined by qPCR in control- and miR-146a-transfected SNU638 cells. Expression of each transcript is shown relative to the expression in control-transfected cells. CARD10, COPS8 and IRAK1 expression was down-regulated following miR-146a over-expression. Data are shown as mean ± S.D. of four biological replicates. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. (B) miR-146a transfection also reduced the protein levels of CARD10, COPS8 and IRAK1 in miR-146a-transfected SNU638 cells. The expression of each protein is shown relative to the expression in control-transfected cells. Bands were quantified relative to β-actin. Data are shown as mean ± S.D. of four biological replicates. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. (C) Verification of direct and functional target binding using luciferase constructs holding WT 3’UTRs and mutated (Mut) 3’UTRs (the central 4 nucleotide were replaced in miR-146a binding site) for CARD10, COPS8 and IRAK1. Constructs containing either WT or mutated (Mut) 3’UTRs downstream to the firefly luciferase gene were co-transfected into HEK293 cells together with Renilla luciferase control plasmid and either miR-146a or control (siGlo). Left, luciferase activity is given relative to activity in control-transfected cells. miR-146a reduced luciferase activity of constructs with WT CARD10, COPS8 and IRAK1 3’UTRs. Right, sequence alignments of miR-146a and potential WT and Mut 3’UTR target sites are shown, mutated bases are shown in red. Data are shown as mean ± S.D. often biological replicates. *** = P < 0.001. Sequence alignments are adapted from DIANALab (2009).

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