MiR-128 Targets p70S6K1 eight normal brain tissues, glioma tissues at Grades II, III, and IV with thirteen cases each, and normalized to the U6 levels. The levels of miR-128 expression in glioma tissues were significantly decreased when compared with normal tissues (Figure 1B). There was no significant difference of miR-128 expression levels in glioma tissues between Grade III and Grade IV, which was significantly lower than that in Grade II (Figure 1B). This result indicates that miR-128 is down-regulated in glioma. P70S6K1 was a direct target of miR-128 To better understand molecular action of miR-128 in glioma, we searched for potential targets of miR-128 by TargetScan, and tested the potential targets using bioinformatic database. We found that miR-128 has a putative binding site to the 39-UTR region of p70S6K1 with 100% conserved sequence in human, mouse, and rabbit (Figure 2A). The p70S6K1 39-UTR was cloned into pMIR- REPORT miRNA reporter vector. Overexpression of miR-128 decreased luciferase activity of this reporter to 70% of the control level (Figure 2B), suggesting that miR-128 inhibits the 39-UTR function of p70S6K1. To test whether miR-128 specifically inhibited p70S6K1 by its potential binding site of seed sequence, the mutated reporter at miR-128 binding site was constructed. Forced expression of miR-128 did not affect the mutant p70S6K1 reporter activities (Figure 2B). These results indicate that p70S6K1 Figure 2. MiR-128 directly targeted p70S6K1. (A) Schematic diagram of putative miR-128 binding site in the 39-UTR regions of p70S6K1 in human, mouse and rabbit. The seed sequence of miR-128 matches 39-UTR regions of p70S6K1 (in bold). The mutated nucleotides of the p70S6K1 39- UTR was labeled by asterisk. (B) Relative luciferase activities of p70S6K1 wild type (WT) and mutant (Mut) 39-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 mean6SD 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 mean6SD 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. doi:10.1371/journal.pone.0032709.g002 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32709