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. 2021 Nov 29;27:256–275. doi: 10.1016/j.omtn.2021.11.019

Figure 3.

Figure 3

miR-148a-3p attenuates tau hyperphosphorylation by the direct targeting of p35

(A) Venn diagram of predicted targets of miR-148a-3p as defined using three databases: TargetScan, miRDB, and Tarbase. (B and C) Correlation analysis of miR-148a-3p with p35 (CDK5R1) (B) and PTEN (C) mRNA in APP/PS1 mouse cortex at different stages of AD. (D) Potential binding site for miR-148a-3p in the 3′ UTR of p35 mRNA as suggested by bioinformatics analysis. (E) Construction of wild-type (Wt) and mutant (Mut) 3′ UTR of p35 within a pGL3 vector. (F) Relative luciferase activity after co-transfection with a plasmid constructed with a Wt or Mut p35 3′ UTR with scrambled control (Scr) or miR-148a-3p mimic (148a) in HEK293 cells. (G) Representative western blot images of p35 expression in APPswe cells after transfection with miR-148a-3p mimic (148a), anti-miR-148a-3p (Anti-148a), and scrambled controls (Scr/Anti-scr). (H) Quantification of p35 protein expression. (I) p35 mRNA gene expression levels quantified by qRT-PCR after transfection of APPswe cells with miR-148a-3p mimic (148a), anti-miR-148a-3p (Anti-148a), and scrambled controls (Scr/Anti-scr). (J and K) Representative western blot images of p35 and CDK5 expression in APPswe cells (J) and quantification (K) after overexpression of p35 (exclusion of nonspecific band of p35). (L) Binding of p35 and CDK5 using an immunoprecipitation assay in APPswe cell homogenates immunoprecipitated with anti-p35 antibody. (M and N) Representative western blot images of p35, p25, and CDK5 expression (M) and quantification (N) in APPswe cells transfected with miR-148a-3p mimic (148a), anti-miR-148a-3p (Anti-148a), and scrambled controls (Scr/Anti-scr). (O and P) Representative western blot images of the phosphorylation of tau protein at AT8, S199, S396, and S404 sites (O) and quantification (P) after transfection of APPswe cells with scrambled control (Scr), miR-148a-3p mimic (148a), and p35. Results represent means ± SEM, n = 5. ∗∗p < 0.01, ∗∗∗p < 0.001 versus Scr or Anti-scr; $p < 0.05, $$p < 0.01, $$$p < 0.001 versus 148a. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.