miR-146a-5p expression in α- and β-cell lines.

(A) INS1 cells were exposed to IL-1β (160 pg/ml) for 6 h, 12 h and 24 h. The expression of miR-146a-5p normalized to the internal control let-7c was quantified with qRT-PCR. Mean ± SEM (n = 5). (B) INS1 cells were exposed to IL-1β (160 pg/ml), IFN-γ (5ng/ml) or TNF-α (10 ng/ml) or a mixture of all three cytokines for 8 h. The expression of miR-146a-5p normalized to the internal control let-7c was quantified with qRT-PCR. Mean ± SEM (n = 3). (C) The Luciferase promoter assay was conducted in INS1 cells exposed to IL-1β (160 pg/ml) for 6 h or IL-1β (160 pg/ml) and IFN-γ (5 ng/ml) for 6 h. The cells were pre-treated with transient transfection mix containing both miR-146a-5p promoter construct and a Renilla construct. Mean ± SEM (n = 4). (D) miR-146a-5p expression in the INSrαβ cell line with or without induced Pdx-1 by addition of doxycycline for 24 h prior to cytokine exposure to IL-1β (150 pg/ml) for 2 h, 12 h, or 24 h. The expression of miR-146a-5p and let-7c was quantified using qRT-PCR. Mean ± SEM (n = 4–5). (E) α-TC1 and β-TC3 cells were exposed for 24 h to IL-1β (160 pg/ml) or IL-1β (160 pg/ml) and IFN-γ (5 ng/ml). The expressions of both miR-146a-5p and the internal control let-7c were quantified by qRT-PCR. Mean ± SEM (n = 6–8). *p<0.05; ** p<0.01; *** p<0.001.