Figure 7
The miR-23b-27b transgenic mice show more resistance to hypoxia-induced apoptosis. (a) A cartoon of generation of the miR-23b-27b cluster transgenic mice. The Tubb3 gene promoter was employed because of its high expression capability and specificity in neurons in the early embryonic stage. Quantitative RT-PCR detection of miR-23b and miR-27b in primary cortical neurons 24 h after transfection with the constructed plasmid. (b) Quantitative RT-PCR detection of miR-23b-27b (top) and western blot analysis of Apaf-1 protein levels (bottom) in cultured primary cortical neurons from wild-type (WT) mice and transgenic (TG) mice (n=3, unpaired t-test, *P<0.05 and **P<0.01). (c) Cultured cortical neurons isolated from E15.5 pups were stained with Cleaved-Casp3 antibody after hypoxia treatment (24 h) at DIV5 and counterstained with DAPI. Statistical analysis of the percentage of Cleaved-Casp3-positive neurons to DAPI-stained cells (cell numbers=400–500, unpaired t-test, ***P<0.001). Scale bar represents 100 μm. (d) Cultured cortical neurons were stained with TUNEL staining kit after hypoxia treatment. Statistical analysis of the percentage of TUNEL-positive neurons to DAPI-stained cells (cell numbers=400–500, unpaired t-test, **P<0.01). (e) Quantitative RT-PCR detection of miR-23b-27b (top) and western blot analysis of Apaf-1 protein levels (bottom) in the cerebral cortices from E19.5 mice (TG mice n=4 and WT mice n=6, unpaired t-test, *P<0.05 and **P<0.01). (f) Coronal sections of WT and transgenic neocortex at E19.5 were stained with Cleaved-Casp3 antibody after 6 h of hypoxia followed by a 6-h recovery or with TUNEL staining kit after a 18-h recovery. Scale bar represents 100 μm
