MiR-128 Targets p70S6K1 than that from miR-SCR control cells (Figure 6E), confirming that miR-128 overexpression suppressed p70S6K1 expression in vivo. The expression levels of HIF-1a and VEGF in the tumor sections were also higher in miR-SCR control group than those in miR- 128-expressing group (Figure 6F). These results indicate that miR- 128 inhibits cell proliferation and tumor growth. MiR-128 inhibited angiogenesis in nude mice In previous studies, we demonstrated that miR-128 down- regulated HIF-1a and VEGF expression by targeting p70S6K1. HIF-1a expression is frequently increased in many human cancers and is associated with angiogenesis induced by tumors and hypoxia [21]. Our group and others have demonstrated that p70S6K1 signaling is required for HIF-1-mediated VEGF expression in response to growth factor stimulation and oncogene activation [15–17]. To test whether miR-128 inhibits tumor angiogenesis, U87 cells stably expressing miR-128 or miR-SCR were mixed with Matrigel and injected into both flanks of the nude mice. The mice were sacrificed 11 days after implantation. The Matrigel plugs from the mice implanted with miR-128-expressing cells were represented in Figure 7A. The relative angiogenesis responses were assayed by measuring the hemoglobin levels. When compared to miR-SCR group, the angiogenesis responses in miR- 128 group were decreased by 50% (Figure 6B). In addition, the number of CD31-positive microvessels was much lower in the sections from xenografts of miR-128-expressing U87 cells Figure 7. Overexpression of miR-128 suppressed tumor angiogenesis in vivo. U87 cells stably expressing miR-128 or miR-SCR were trypsinized and resuspended in serum-free medium at 26106cells/ml, and aliquots of the cells (0.1 ml) were mixed with 0.2 ml of growth-factor- reduced Matrigel, and injected subcutaneously into the nude mice. The mice were euthanized after the implantation for 11 days. (A) The representative Matrigel plugs are shown. Scale bar, 2 mm. (B) The hemoglobin levels in 8 tumors from miR-128 group decreased to 50% compared to SCR control group. Asterisk indicates significant difference when compared to the miR-SCR group (P,0.05). (C) Representative tumor sections stained by antibody against human CD31. Magnification, 6200. (D) CD31-positive microvessels were counted from 8 tumors in three different fields per section at 6400 magnification. Asterisk indicates significant difference when compared to the miR-SCR group (P,0.05). (E) Representative normal and different grades of glioma tumor sections stained by antibody against human CD31. Magnification, 6200. (F) Spearman’s correlation test was used to analyze the correlation of miR-128 levels and CD31-positive microvessel densities from 15 glioma tumors using SPSS software. doi:10.1371/journal.pone.0032709.g007 PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e32709