MiR-128 Targets p70S6K1 Figure 3. The p70S6K1 protein levels were negatively correlated with miR-128 levels in glioma tissues. (A) The expressions of p70S6K1 in human glioma specimens and normal brain tissues were determined by immunoblotting and the fold changes were obtained by the ratio of p70S6K1 to GAPDH level. Double asterisks indicate significant difference when compared to normal brain tissues (P,0.01). (B) Spearman’s correlation analysis was used to determine the correlation between the expression levels of p70S6K1 and miR-128 by SPSS software (Spearman’s correlation analysis, r = 20.741; P,0.001). doi:10.1371/journal.pone.0032709.g003 is a direct target of miR-128 with the specific binding site at the seed sequence. Next, we established cell lines stably expressing miR-128 and its scrambled control (miR-SCR) using human glioma cells U87 and U251 by lentiviral transduction. The expression levels of miR-128 were analyzed in these stable cell lines using RT-PCR. As shown in Figure 2C, the expression levels of miR-128 were increased in pLe-miR-128-expressing U87 and U251 cells, respectively, demonstrating that these stable cells successfully expressed miR- 128. The cells with miR-128 overexpression showed low levels of p70S6K1 and p-p70S6K1 proteins by 30–50% reduction when compared to those of scrambled control cells (Figures 2D and 2E). In addition, forced expression of miR-128 did not change p70S6K1 mRNA expression levels (Figure 2F), showing that miR-128 specifically regulates p70S6K1 protein expression at posttranscriptional levels. P70S6K1 levels inversely related to the levels of miR-128 expression in glioma tissues To determine whether the reduced miR-128 expression correlates with the levels of p70S6K1 expression in tumor tissues, protein expression of p70S6K1 in glioma and normal brain tissues was analyzed by immunoblotting. The results revealed that the protein levels of p70S6K1 in glioma tissues were dramatically higher than those in normal brain tissues (Figure 3A). As shown in Figure 3B, Spearman’s correlation test showed a significant inverse Figure 4. Overexpression of miR-128 inhibited HIF-1 and VEGF expression. Stable U87 and U251 cell lines expressing scrambled miRNA (SCR) or miR-128 were established. (A) Levels of HIF-1a, HIF-1b, and GAPDH protein expression were detected by immunoblotting (B) VEGF mRNA levels were detected by semi-quantitative RT-PCR assay. Asterisk and double asterisks indicate significant difference when compared to U87 cell expressing miR-SCR or U25 cell expressing miR-SCR at P,0.05 or at P,0.01, respectively. (C) VEGF protein levels were detected by ELISA assay. Double asterisks indicate significant difference when compared to the control (P,0.01). doi:10.1371/journal.pone.0032709.g004 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32709