Fig. 3

miR-7 and miR-504 regulate dendritic spines of cultured hippocampal neurons in a Shank3-dependent manner. a miR-7, miR-34a, or miR-504 overexpression decreased the luciferase activity of wild-type SHANK3 3′UTR in cultured mouse hippocampal neurons. b miR-7, miR-34a, and miR-504 did not affect the luciferase activity of the mutant SHANK3 3′UTR constructs in cultured neurons. c Overexpression of miR-7 or miR-504 decreased the density of dendritic spines in cultured mouse hippocampal neurons (n = 16–30). d Representative western blot images show that overexpression of miR-7 or miR-504 decreased the expression of the rat Shank3 construct with the 3′UTR, but not of that without the 3′UTR, in HEK293T cells. EGFP expressing plasmid was co-transfected as an internal control. Notably, we observed decreased expression of EGFP when miR-7 was cotransfected. Nevertheless, after normalization with EGFP, the expression of Shank3 with 3′UTR, but not that without 3′UTR, was significantly decreased by miR-7. e Co-transfection of the Shank3 construct without the 3′UTR, but not that with the 3′UTR, rescued the decreased dendritic spine density by miR-7 or miR-504 overexpression (n = 20–30). f LNA-inhibitor against miR-7, miR-34a, or miR-504 increased the expression of co-transfected SHANK3 3′UTR in cultured mouse hippocampal neurons. g LNA-inhibitor against miR-7, miR-34a, and miR-504 did not affect the expression of the mutant SHANK3 3′UTR in cultured neurons. h LNA-inhibitor against miR-7 or miR-504 increased dendritic spines in cultured neurons (n = 18–20). i Co-transfection of Shank3 siRNA blocked the increase of dendritic spines in response to miR-7 or miR-504 inhibition (n = 16–20). j ciRS-7 partially blocked the repressive effect of miR-7 on the expression of wild-type SHANK3 3′UTR in HEK293T cells. Neither miR-7 nor ciRS-7 changed the expression of miR-7 binding-mutant SHANK3 3′UTR. ciRS-7-ir is a control plasmid that does not express the circular RNA. k Overexpression of ciRS-7 increased dendritic spines in cultured mouse hippocampal neurons (n = 15–20). Scale bar, 10 μm. All data are presented as mean ± SEM. Statistical analyses are in Additional file 1: Table S3