Altering β-cell number through stable alteration of miR-21 and miR-34a expression

Figures & data

Figure 1. Overexpression of miR-21 reduces cell number, while knockdown of miR-34a increases cell number in the absence of cytokines. (A) Cells (50 000 cells) were cultured for 72 h prior to trypsinization and manual counting in a counting chamber. Results of 5 independent experiments are shown as means + SEM. Statistical significance was determined using a 2-way paired t test. * P < 0.05 vs. miR-neg. (B) 20 000 cells were seeded pr well in the xCELLigence E-plate and cell index measured in real-time for 24 h. The graph depicts the average cell index ± SEM determined from triplicates from each time point from a single experiment.

Figure 2. Early β-cell apoptosis is potentiated by overexpression of miR-21, but reduced by knockdown of miR-34a both in the absence and the presence of cytokines. Cells (50 000 cells) were left untreated (A) or were exposed to IL-1β and IFN-γ for 24 h (B). Nucleosomal fragments in adherent cell lysates were detected by ELISA (Cell Death Detection ELISA). Results of 10 independent experiments are shown as means + SEM. Statistical significance was determined using a 2-way paired t test. *** P < 0.001 vs. miR-neg.

Figure 3. Synthesis of NO is potentiated by overexpression of miR-21, and reduced by knockdown of miR-34a in the presence of cytokines. Cells (50 000 cells) were left untreated (A) or were exposed to IL-1β and IFN-γ for 24 h (B). Culture medium was retrieved and accumulated nitric oxide was determined using the Griess reaction. Results of 9 independent experiments are shown as means + SEM. Statistical significance was determined using a 2-way paired t test. ** P < 0.01 vs. miR-neg, *** P < 0.001 vs. miR-neg.

Figure 4. miR-21 overexpression and miR-34a knockdown affect β-cell proliferation. (A and B) Cells (25 000 cells) were left untreated (A) or were exposed to IL-1β and IFN-γ for 24 h (B). Nuclei were stained using EdU and HCS Blue, and fixed with mounting reagent. Nuclei were counted by 2 blinded observers as total number of cells (blue nuclei) and proliferating cells (green nuclei). The ratio of proliferating cells was calculated. Results of 4 independent experiments are shown as means + SEM. Statistical significance was determined using a 2-way paired t test. * P < 0.05 vs. miR-neg, ** P < 0.01 vs. miR-neg. (C) Representative photomicrographs of β-cell proliferation. Panel I shows proliferating cells identified by EdU staining, panel II shows a merge of total number of cells (blue nuclei) and proliferating cells (green nuclei), and panel III shows a merge between panel I and II and an image taken using digital interference contrast microscopy (DIC). Panel IV shows a close-up of panel III (arrows indicate proliferating cells).