Overexpression and Inhibition of miR-146a Confirms Immune Signaling Molecule Targets in CD4+ T-Cells
TRAF6 and IRAK1 are known direct targets of miR-146a in several cell types, including murine macrophages and liver cells, primary human monocytes, and THP-1 monocytic cells (19, 27, 50, 51). We employed a miR-146a mimic to confirm that TRAF6 and IRAK1 are targets of the miRNA in primary CD4+ T-cells. The miR-146a mimic was transfected into CD4+ T-cells, resulting in an expected significant increase in miR-146a expression (Figure 3A). We observed no change in IL-1β mRNA expression (Figure 3B). RT-qPCR analysis showed that TRAF6 and IRAK1 mRNA levels were significantly decreased, indicating that miR-146a targeted these molecules and initiated mRNA degradation pathways (Figure 3B).
Figure 3  Overexpression and inhibition of miR-146a confirms immune signaling molecule targets in CD4+ T-cells. CD4+ T-cells were un-transfected, transfected with a negative control mimic or miR-146a mimic. (A) Successful miR-146a overexpression was confirmed by RT-qPCR. Fold change was calculated by normalizing the transfected cells to non-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to non-transfected controls (***p < 0.001). (B) Expression of IL-1β, TRAF6, and IRAK1 mRNA levels during miR-146a overexpression was determined by RT-qPCR. Fold change was calculated by normalizing miR-146a mimic-transfected cells to negative control-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to negative controls (*p < 0.05). (C) CD4+ T-cells were un-transfected, transfected with a negative control inhibitor or a miR-146a inhibitor. Successful miR-146a inhibition was confirmed by using RT-qPCR. Fold change was calculated by normalizing the transfected cells to non-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to non-transfected controls (***p < 0.001). (D) Expression of IL-1β, TRAF6, and IRAK1 mRNA levels during miR-146a inhibition was determined by RT-qPCR. Fold change was calculated by normalizing miR-146a inhibitor-transfected cells to negative control-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to negative controls (**p < 0.01). We then used a miR-146a inhibitor and analyzed changes in TRAF6, IRAK1, and IL-1β mRNA expression. Transfection of the inhibitor significantly reduced miR-146a expression (Figure 3C). IL-1β mRNA expression was unchanged, but TRAF6 and IRAK1 mRNA levels were significantly increased (Figure 3D). Taken together, these data confirm IRAK1 and TRAF6 as targets of miR-146a in CD4+ T-cells.