MiR-128 directly targeted p70S6K1.

<p>(A) Schematic diagram of putative miR-128 binding site in the 3′-UTR regions of p70S6K1 in human, mouse and rabbit. The seed sequence of miR-128 matches 3′-UTR regions of p70S6K1 (in bold). The mutated nucleotides of the p70S6K1 3′-UTR was labeled by asterisk. (B) Relative luciferase activities of p70S6K1 wild type (WT) and mutant (Mut) 3′-prime UTR regions were obtained by co-transfection of scrambled control miRNA or miR-128 precursor, and pRL-TK plasmid; and calculated as the ratio of firefly/renilla activities in the cells and normalized to those of the control. The results were presented as mean±SD from three independent experiments with each experiment in triplicate. Asterisk indicates significant difference when compared to control (P<0.05). (C) U87 and U251 cells stably expressing scrambled control (SCR) and miR-128 were subjected to RT-PCR analysis for miR-128 and U6 expression levels. (D) Overexpression of miR-128 inhibited p70S6K1 expression at protein level. The expression levels of total p70S6K1, phospho-p70S6K1 (Ser389), and GAPDH protein in U87 and U251 cells stably expressing miR-SCR and miR-128 were analyzed by immunoblotting. (E) Relative signals were quantified using VisionworksLS Acquisition and analysis software. Results are presented as mean±SD from three independent experiments. Double asterisks indicate significant difference when compared to the control (P<0.01). (F) miR-128 overexpression did not change the p70S6K1 mRNA level detected by RT-PCR. Relative densities were quantified using VisionworksLS Acquisition and analysis software.