Figure 2: MiR-299-5p affects Atg5 levels directly and alters the levels of other autophagy-related proteins.

(a) Conservation of miR-299-5p and the predicted binding sequences between miR-299-5p and target sites in the Atg5 3′UTR. Matches within the seed sequence are indicated. The mutated sequences are showed as lower case letters. (b) Primary hippocampal neurons from AD mice were transfected with agomiR-299-5p (Ago299-5p) or antagomiR-299-5p (AM299-5p). The levels of Atg5 protein were analyzed by western blotting. (c) Luciferase vectors were generated by inserting the wild-type (WT) or mutated (MUT) 3′UTR fragments of Atg5 position 489–498 into pmirGLO plasmid. Normalized luciferase activity in lysates from Hela cells were assayed 24 h after transfection (mean ± SD of independent experiments, n = 3, *p < 0.05. ^#represents no significance). (d) AM299-5p increased luciferase activity when co-transfected with vectors containing a WT fragment (mean ± SD of independent experiments, n = 3, *p < 0.05). (e) Transfection with Ago299-5p decreased the luciferase activity of vectors containing WT fragment in a dose-dependent manner, and co-transfection with AM299-5p dose-dependently counteracted the effects of Ago299-5p in a dose-dependent manner (mean ± SD of independent experiments, n = 3, *p < 0.05). (f) In Neuro-2a (N2a) cells, Atg5 protein levels were reduced by transfection with Ago299-5p and increased by transfection with AM299-5p. Other autophagy-related proteins (LC3β and p62) were also modulated (n = 3). (g) In SH-SY5Y cells, transfection with Ago299-5p (40 nM) modulated Atg5, LC3β and p62 protein levels, and the effects were attenuated by co-transfection with AM299-5p (40 nM, n = 3). (h) Atg5 and LC3β protein levels in hippocampi of 9-month-old APPswe/PS1dE9 mice (Tg) and C57 mice (n = 4 per group).