MiR-128 Targets p70S6K1 Forced expression of p70S6K1 restored the inhibitory effects of miR-128 As we showed above, p70S6K1 is a direct target of miR-128. Therefore, we wondered whether forced expression of p70S6K1 is enough to reverse the expression of miR-128-inhibited down- stream molecules. P70S6K1 cDNA plasmid without 39UTR region was transfected into miR-128- or miR-SCR-expressing cells. As shown in Figure 5A, the decreased level of p70S6K1 by miR-128 expression was rescued via the introduction of p70S6K1 cDNA. Similarly, ectopic expression of p70S6K1 also restored miR-128-inhibited HIF-1a and VEGF expression in both U87 and U251 cells (Figures 5A and 5B), indicating that miR-128 inhibits HIF-1a and VEGF expression by targeting p70S6K1. MiR-128 attenuated cell proliferation and tumor growth, and decreased p70S6K1 protein level in tumor xenografts P70S6K1 signaling pathway plays an important role in regulating cell cycle and cell proliferation [31,32]. To study whether miR-128 can regulate cell growth by targeting p70S6K1, cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. When compared to miR-SCR treatment, overexpression of miR-128 in U87 and U251 cells inhibited cell proliferation after the culture for 3–4 days, suggesting that miR-128 affects cell growth in vitro (Figure 6A). To further clarify the inhibitory effect of miR-128 on tumor growth in vivo, U87 cells stably expressing miR- 128 were injected subcutaneously into both flanks of the nude mice. Cells stably expressing miR-SCR were used as negative control. The lengths and widths of tumors were measured when the xenografts were visible on Day 18, and the volumes of tumors were calculated. As shown in Figure 6B, the size of xenografts from miR-128-expressing cells was smaller than that from miR-SCR control group after 20 days of implantation. After 28 days, the xenografts were trimmed out, and tumor weight was measured. Forced expression of miR-128 in U87 cells attenuated tumor growth by 75% when compared to miR-SCR control group (Figures 6C and 6D). Furthermore, the protein levels of p70S6K1 in xenografts from miR-128-expressing cells were much lower Figure 6. Overexpression of miR-128 inhibited cell proliferation and tumor growth. (A) U87 and U251 cells were transiently transfected with miR-SCR or pre-miR-128 precursor. After the transfection for 24 h, cells were trypsinzed and seed into 96-well plate (2000 cells/well). Cell proliferation was determined after 24, 48, 72, and 96 h by using Cell Counting Kit-8 (CCK-8) to detect the absorbance at 450 nm. Values represent means 6 standard deviation (SD). Double asterisks indicate significant difference when compared to the miR-SCR group (P,0.01). (B) Nude mice were injected subcutaneously with 56106 U87 cells stably expressing miR-128 (miR-128) or miR-SCR. Each treatment group contained 8 tumors. When the xenografts were visible, the width and length of tumors were measured. Asterisk indicates significant difference when compared to the miR-SCR group (P,0.05). (C) The mice were euthanized on Day 28 and the xenografts were trimmed out. Representative tumors from each group were shown. Scale bar, 2 mm. (D) Overexpression of miR-128 significantly inhibited tumor growth in vivo. The tumor weight was measured for each xenograft. After 28 days, the weight of tumors from miR-128 group decreased by 75% compared to SCR group. Double asterisks indicate significant difference when compared to the miR-SCR group (P,0.01). (E) The levels of p70S6K1 from the tumor tissues of miR-128-expressing group were much higher than that of SCR control group by immunoblotting assay. (F) The expression levels of HIF-1a and VEGF were analyzed in tumor tissues by immunohistochemistry with representative images showed. Magnification, 6200. doi:10.1371/journal.pone.0032709.g006 PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e32709