Meth Increases miR-146a Expression and Down-Regulates TRAF6
IL-1β signaling can participate in a positive auto-regulatory loop, resulting in increased transcription of its gene (43). In addition, it has been reported that IL-1β can induce NFκB-dependent miR-146a expression to interfere with innate immune functions (31). Non-coding RNAs play important roles in regulating cellular activities and stress responses. Furthermore, Meth is known to induce activation and nuclear translocation of NFκB (44). Thus, we used RT-qPCR to identify Meth-related changes in miR-146a and IL-1β mRNA in primary CD4+ T lymphocytes.
Healthy donor CD4+ T-cells were treated daily with 100 μM Meth, and miR-146a expression was assessed. We observed that Meth significantly up-regulated miR-146a on day 3 of treatment (Figure 2A). Likewise, we assessed IL-1β mRNA levels in untreated and Meth treated cells. Unlike extracellular IL-1β, which increased after 1 day of Meth treatment, IL-1β mRNA showed increased expression only on day 3 (Figure 2A). Notably, IL-1β release and mRNA expression are controlled by distinct mechanisms (45). In addition, CD4+ T-cells constitutively express pro-IL-1β in their cytoplasm (46). As such, our results indicate that Meth first enhances release of mature IL-1β, followed by increased IL-1β gene transcription and miR-146a expression.
Figure 2  Meth increases miR-146a expression and downregulates of TRAF6. CD4+ T-cells were treated daily with or without Meth. (A) Expression of miR-146a and IL-1β mRNA was determined by RT-qPCR. Fold change was calculated by normalizing the Meth treated cells to untreated cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to untreated controls (*p ≤ 0.05, **p ≤ 0.01). (B) Cells treated or untreated with Meth were lysed and protein extracts were analyzed for expression of TRAF6 and IRAK1 by Western Blotting. GAPDH was used as a loading control. Relative band intensity was calculated using ImageJ software. (C) Expression of TRAF6 mRNA was determined by RT-qPCR. Fold change was calculated by normalizing the Meth treated cells to untreated cells. Data represent the mean ± SD of 3 independent experiments. We next explored the effects of Meth on TRAF6 and IRAK1, known targets of miR-146a linked to IL-1β and innate immune signaling, and implicated in HIV-1 pathobiology (31, 47). Specifically, IRAK1 plays a key role in mediating IL-1β-induced NFκB activation, and decreased expression of TRAF6 has been implicated in increased HIV-1 replication (17, 47, 48).
By Western Blot analysis, we observed decreased TRAF6 protein expression on day 3 of Meth exposure, but no significant change in IRAK1 protein expression (Figure 2B). Through RT-qPCR, we found no significant change in TRAF6 mRNA expression (Figure 2C).
Collectively, our results— specifically decreased levels of TRAF6 protein and unchanged levels of TRAF6 mRNA— are consistent with fine tuning by miRNA, whereby translational repression precedes mRNA decay (49). Notably, decreased TRAF6 expression observed on day 3 correlated with increased miR-146a levels. These findings suggest a role for miR-146a in regulation of innate immune signaling pathways through repression of TRAF6 during Meth treatment.
We hypothesized that IL-1β signaling is responsible for the increased IL-1β mRNA levels, as well as increased miR-146a expression observed on day 3 of Meth treatment; we further hypothesized that IL-1β-dependent induction of miR-146a would target key innate immune pathways via TRAF6 inhibition.