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

Figure 5.

Figure 5

PTEN/Akt pathway regulates miR-148a-3p expression in a CREB-dependent manner

(A and B) Representative western blot images of p-CREB and CREB (A) and quantification of p-CREB/CREB ratio (B) after transfection of APPswe cells with PTEN-expressing plasmid (PTEN), vector control (Vector), PTEN siRNA, and scrambled siRNA (Scr siRNA) (n = 6). (C–E) Representative western blot images of p-Akt, Akt, p-CREB, and CREB (C) and quantification of ratio of p-Akt/Akt (D) and of p-CREB/CREB (E) after transfection of APPswe cells with Akt-expressing plasmid (Akt), basic vector control (Vector), Akt siRNA, and scrambled siRNA (Scr siRNA) (n = 6). (F) Potential binding sites of CREB with the miR-148a-3p promoter. (G) Binding motif of CREB transcription factor with the miR-148a-3p promoter. (H) Quantitation of ChIP assay results by qRT-PCR (n = 4). (I) ChIP assay with CREB antibody and PCR amplification of the region from the miR-148a-3p promoter. (J) Luciferase activity of wild-type miR-148a-3p promoter construct (BS Wt) or CREB-binding-site mutant construct (BS Mut) in HEK293 cells transfected with CREB-expressing plasmid (CREB) and its vector control (Vector) (n = 4). (K) Expression of miR-148a-3p using qRT-PCR after transfection of APPswe cells with CREB-overexpressing plasmid (CREB), vector control (Vector), CREB siRNA, and scrambled siRNA (Scr siRNA) (n = 4). Results represent means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus vector or Scr siRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant.