Fig. 2: Loss of miR-135a-5p in AD is caused by the reduction of Foxd3.

a The degradation curve of miR-135a-5p, miR-132-3p, and U6 were measured by qRT-PCR in primary hippocampal neurons at DIV 7 after treated with 10 μg/mL actinomycin D for 0–12 h (n = 4 for each group). b The degradation curve of miR-135a-5p in primary neurons after infection of hTau (Tau) or control virus (Con) (n = 4 for each group). c, d Alterations of pri-miR-135a-1/2 in primary neurons after infection of hTau (Tau) and control virus (Con) (c) or in hippocampus of 9 months APP/PS1 and control mice (WT) (d) (n = 4 for each group). e A diagram for the construct of pri-miR-135a-1 promoter-luciferase reporter vectors with different lengths of promoter regions (1–3 kb). f pri-miR-135a-1 promoter vectors were co-transfected into HEK293T cells with pRL-TK vector (Renilla luciferase reporter vector). After 48 h, the luciferase activity was analyzed (n = 3 for each group, one-way ANOVA, p < 0.0001, Dunnett’s post hoc p < 0.0001 vs pGL3). g The pGL3-1kb was co-transfected into HEK293T with transcriptional factors (Gata-1, Foxd3, Foxo1), and the luciferase activity was analyzed at 48 h later (n = 3 for each group, one-way ANOVA, p < 0.0001, Dunnett’s post hoc p < 0.0001 vs. Con). h ChIP-seq peaks showed the binding site of Foxd3 in the promoter of pri-miR-135a-1 through Integrative Genomics Viewer (IGV) for genomic regions chr9: 106151716-106155805. i, j Primary hippocampal neurons at DIV 5 was infected with AAV-hTau (Tau) or control virus (Con). One week later, neurons were infected with AAV-Foxd3-Flag. The cell lyses were immunoprecipitated with anti-Flag and then primers of the pri-miR-135a-1 promoter (P-135a) were used for PCR assay. The representative DNA gels and blots were shown in (i) and quantitative analysis was shown in (j) (n = 3 for each group, p = 0.0016). k The protein level of Foxd3 from hippocampus of 9 months APP/PS1 and control mice (WT) was measured by western blotting (upper panel) and quantitative analysis was performed (lower panel) (n = 4 for each group, p = 0.0019). l The expression of miR-135a-5p in primary neurons infected with AAV-Foxd3, AAV-hTau or control virus was detected by qRT-PCR (n = 3 for each group, one-way ANOVA, p = 0.0003, Tukey’s post hoc, Tau vs. Con p = 0.0004, Tau + Foxd3 vs. Tau p = 0.0007) (Data are presented as mean ± S.E.M. and two-tailed t tests were used unless otherwise specified. Source data are provided as a Source Data file. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT/Con if without specific explain.).