Figure 5.
miR-146a negatively regulates HuR expression in lung cells. (a) A549 cells were transiently transfected with miRNA mimics at a 50 nM concentration. Cells were lysed and RNA was isolated 48 hours post-transfection. ΔΔCT qRT-PCR analysis indicated decreased HuR mRNA expression in A549 cells transfected with 50 nM of synthetic miR-146a. HuR expression was normalized to GAPDH mRNA. HuR mRNA levels were significantly different in cells transfected with synthetic miR-146a from levels in mock-treated cells and cells transfected with 50 nM of a non-targeting miRNA. (*) p < 0.01, n = 3. (b) A549 cells were transiently transfected with synthetic miR-146a at 50 and 100 nM concentrations. Cells were lysed and protein was isolated 48 hours post-transfection. Western blot analysis of cell lysates indicated decreased HuR protein expression in A549 cells transfected with 50 nM of synthetic miR-146a and 100 nM of synthetic miR-146a compared to mock-treated A549 cells. (c) Western blot analysis of cell lysates indicated decreased HuR protein expression only in H1299 Tet/TRE-miR-146a cells cultured in 1 μg/mL doxycycline.