Fig. 4

Overexpression of miRNA-203 induced neuronal cell death and caused cognitive impairment. A and B Representative immunofluorescence images showing ectopic expression of miR-203 (green) induced elevation of caspase 8 (red) in the hippocampal CA3. The expression of Caspase 8 was primarily distributed around miR-203 overexpressing cells. Quantification of Caspase 8 positive areas of three consecutive sections consisting of four views of bilateral CA3 and DG of three independent biological replicates were presented, with the height of columns representing the mean and the error bars showing the S.D. C and F Immunofluorescent images of TUNEL assay representing the increased apoptosis in the CA3 regions of mouse hippocampus with ectopic expression of miR-203 (green). Quantification of apoptotic cells (red) of three consecutive sections consisting of views of bilateral CA3 of three independent biological replicates were presented, with the height of columns representing the mean and the error bars showing the S.D. D, E, G Western blots showing the upregulated caspase 8 and downregulated PSD95 in mouse hippocampus with ectopic expression of miR-203, compared with that of mouse hippocampus expressing nonsense scrambles. Luminescence-based relative quantification of proteins was expressed as mean ± S.D. of three biological replicates. H, I, J Decreased nest building qualities were observed in mice with hippocampal overexpression of miR-203 (I), compared with controls overexpressing nonsense scrambles (H). Results of scoring of a 5-point rating scale were expressed as mean ± S.D. of five biological replicates, with the score of nest quality of each mouse indicated (J). K Fear conditioning test showed decreased freezing behavior in mice with hippocampal overexpression of miR-203 in the context-related test, compared with controls overexpressing nonsense scrambles. No difference in the freezing behavior in the tone-related fear conditioning test was observed (n = 8). L The representative tracks of a blank control mouse, a mouse overexpressing scramble microRNA and a mouse overexpressing miR-203 in the hippocampus on day 1 and day 8 of the probe trails in Barnes maze. M Latency to identify the target holes in the training sessions of Barnes maze. Results were presented as the group mean ± S.D. N The time spent to find the target holes. The results of each tested mouse were present, with the center line represents the mean, and error bars show mean ± S.D. (one-way ANOVA, Turkey’s post hoc). Nuclei were stained with Hoechst 33342 (blue) and the scale bar was 150 μm. Asterisks indicate statistical significance between samples (*P < 0.05; ****P < 0.0001)