Figure 3: MicroRNA miR-9 reduces TTP expression in neural cells.

(a) Immunoblot analysis demonstrating a decrease in TTP expression in P19 cells undergoing neural differentiation. Antibody against β-tubulin (Tubβ) is used to control lane loading. (b) Top, interaction between miR-9 and the cognate target sequence in the mouse TTP 3′ UTR predicted using RNAhybrid⁶². Middle, interspecies alignment of the target sequences with invariant nucleotides shown in upper case Nucleotides mutagenized to inactivate miR-9-binding are marked by asterisks. Bottom, PhastCons score reflecting probability of sequence conservation across vertebrates⁶³. (c) Northern blot showing that mature miR-9 levels dramatically increase in P19 cells following EB/RA-induced neural differentiation. U6 RNA is used as a loading control. (d) RLuc-3′TTP-wt and RLuc-3′TTP-miR9TSmut Renilla luciferase reporter constructs used in this study. (e) HEK293T cells were co-transfected with RLuc-3′TTP-wt or RLuc-3′TTP-miR9TSmut and miR-9 expression plasmid or the corresponding empty vector. Firefly luciferase plasmid pEM231 (ref. 7) was included as a normalization control. Luciferase expression was assayed 24 h post transfection using the Dual-Glo kit (Promega) and the data were processed as recommended. Note that miR-9 dramatically inhibits the RLuc-3′TTP-wt expression while having a scientifically lesser effect on the RLuc-3′TTP-miR9TSmut construct lacking the conserved miR-9 target site. Expression levels of the corresponding miR-vector samples were set to 1. Data are averaged from three experiments±s.d. and compared by t-test. (f) Following the EB/RA steps, differentiating P19 cells were plated for 12 h and then transfected with a 2′OMe-RNA antisense oligonucleotide against miR-9. Samples were collected 48 h post transfection and the effects of the antisense treatment on the levels of mature miR-9 were analysed with northern blot analysis using U6 RNA as a loading control. (g) Immunoblot analysis showing that knockdown of miR-9 carried out as described in f leads to noticeable upregulation of the TTP protein.