Fig. 3.

MiR-197 is upregulated by MeCP2 to downregulate human ADAM10 while ncRNA Gm28836 is upregulated by MeCP2 to downregulate mouse ADAM10. a, b Human glioblastoma cells U251 were transfected with empty control or plasmid to overexpress MeCP2 (N ≥ 5), and subjected to either western blotting or qRT-PCR to detect ADAM10 protein (a) or mRNA (b). c U251 cells were transfected with MeCP2 and a miR-197 inhibitor (i-197) or scramble control, and subjected to Western blotting for ADAM10 protein (N ≥ 3). GAPDH was used as loading control in both experiments. d–f U251 cells were transfected with either empty vector or MeCP2 expressing plasmids and cultured for 24 h (N = 9). RNA from these cells were extracted and subjected for qRT-PCR. The levels of pri-miR-197, pre-miR-197, and mature miR-197 were shown in d–f, respectively. g Biotin-labeled miR-197 and control scramble were transfected into U251 cells and the human ADAM10 3′-UTR pulled down by miR-197 or control scramble were quantified by qRT-PCR (N = 4). h A scheme illustration for MeCP2 regulation on miR-197 and human ADAM10. i A 7mer-m8 miR-197 binding site predicted by TargetscanHuman. The alignment of miR-197 to human and mouse ADAM10 3′-UTR and the point mutation G>A in A10-I-Mut are illustrated. j A miR-197 binding site predicted by RNAhybrid is highly conserved between human and mouse. The deletion mutation in A10-II-Mut was also illustrated. k Luciferase reporter plasmids for different regions of ADAM10 3′-UTR were transfected into U251 cells with miR-197 mimics or control (N ≥ 8). miR-197 only downregulates the A10-II-WT which contains the region illustrated in j. l Mouse neuroblastoma cell line Neuro-2a (N2a) cells were transfected with negative control or i-197, empty vector or MeCP2 expressing plasmid. Twenty-four hours later, total RNA was extracted and subjected to qRT-PCR to determine the level of Gm28836 RNA. Mouse Gapdh was used as internal control. (N = 3) m Mouse N2a cells were transfected empty vector, Gm28836 plus i-197 or its negative control. The ratio of i-197 to Gm28836 was 1:1. Twenty-four hours later, total RNA was extracted and subjected to qRT-PCR to determine the level of mouse Adam10. Mouse Gapdh was used as internal control. (N = 4) n A scheme illustration for MeCP2 regulation on mouse ADAM10 via Gm28836. All statistical data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001