miR-99a regulated expression of mTOR downstream signaling pathway genes p-4E-BP1 and p-S6K1.

(A-C) Immunoblot analyses of total and phosphorylated 4E-BP1 and S6K1 from MCF-7 breast cancer cells transfected with miR-99a mimics (A), mTOR siRNA(B), and co-transfection of both miR-99a mimics and mTOR cDNA plasmid without the 3′-UTR(C). Levels of p-4E-BP1 and p-S6K1 proteins were both markedly decreased after miR-99a mimics transfection in MCF-7 cells, Knockdown of mTOR expression using mTOR siRNA also distinctly reduced levels of p-4E-BP1 and p-S6K1 proteins, however, total 4E-BP1 and S6K1 protein showed no change; The inhibitory effects of miR-99a mimics on p-4E-BP1 and p-S6K1 could be negated by re-expression of mTOR.