MiR-128 Targets p70S6K1 correlation of the miR-128 and p70S6K1 expression levels in glioma tissues with Spearman’s correlation, r = 20.741 (P,0.001), confirming that lower expression levels of miR-128 were significantly associated with higher levels of p70S6K1 protein expression in the same set of glioma tissues. Overexpression of miR-128 inhibited HIF-1a and VEGF expression Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric tran- scription factor composed of HIF-1a and HIF-1b subunits [28]. HIF-1b protein is constitutively expressed in most human cells. In contrast, HIF-1a expression is induced in the cells by hypoxia, growth factors, and oncogenes [29,30]. High levels of HIF-1a expression are observed in many human cancers, and correlated with tumor growth. Previous results showed that p70S6K1 was the upstream regulator of HIF-1a expression [15,17,22]. To deter- mine whether suppression of p70S6K1 expression due to miR-128 overexpression affects HIF-1 expression in glioma, we found that HIF-1a but not HIF-1b protein levels were inhibited in U87 and U251 cells stably expressing miR-128 when compared to cells stably expressing miR-SCR (Figure 4A). VEGF, the critical regulator of angiogenesis, is mainly regulated by HIF-1a through the binding to the hypoxia-responsive element of VEGF promoter region. In consistent with decreased HIF-1a levels, the VEGF levels in miR-128-expressing U87 and U251 cells were down- regulated to 55% and 30%, respectively (Figure 4B). Moreover, VEGF protein levels in the medium were tested by ELISA as previously described [15], and the results showed that overex- pression of miR-128 inhibited VEGF protein production (Figure 4C). These results suggest that forced expression of miR- 128 inhibited HIF-1a and VEGF expression via p70S6K1 signaling. Figure 5. Overexpression of p70S6K1 restored the inhibitory effects of miR-128 in HIF-1 and VEGF expression. U87 and U251 cells stably expressing miR-SCR or miR-128 were transfected with p70S6K1 cDNA plasmid without 39 UTR at 2 mg per well. After transfection for 48 h, the levels of specific proteins were analyzed by immunoblotting. (A) Overexpression of p70S6K1 increased levels of p70S6K1, phospho-p70S6K1, HIF-1a and HIF-1b protein expression. (B) Forced expression of p70S6K1 restored VEGF mRNA expression inhibited by miR-128. The levels of VEGF and GAPDH were analyzed by semi-quantitative RT-PCR and normalized to the control level. Double asterisks indicate significant difference of VEGF levels between the control and miR-128 treatment (P,0.01). Double hashes indicate significant difference of VEGF in the cells with versus without p70S6K1 overexpression (P,0.01). doi:10.1371/journal.pone.0032709.g005 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32709