MiR-128 Inhibits Tumor Growth and Angiogenesis by Targeting p70S6K1 Zhu-mei Shi1., Jing Wang2., Zhiping Yan2, Yong-ping You 1 , Chong-yong Li2, Xu Qian2, Yu Yin2, Peng Zhao1, Ying-ying Wang 1 , Xie-feng Wang 1 , Ming-na Li3, Ling-Zhi Liu4, Ning Liu1, Bing-Hua Jiang1,2,4* 1 Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China, 2 Department of Pathology, Cancer Center, Nanjing Medical University, Nanjing, China, 3 Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China, 4 Department of Pathology, Anatomy and Cell Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America Abstract MicroRNAs are a class of small noncoding RNAs that function as critical gene regulators through targeting mRNAs for translational repression or degradation. In this study, we showed that miR-128 expression levels were decreased in glioma, and identified p70S6K1 as a novel direct target of miR-128. Overexpression of miR-128 suppressed p70S6K1 and its downstream signaling molecules such as HIF-1 and VEGF expression, and attenuated cell proliferation, tumor growth and angiogenesis. Forced expression of p70S6K1 can partly rescue the inhibitory effect of miR-128 in the cells. Taken together, these findings will shed light to the role and mechanism of miR-128 in regulating glioma tumor angiogenesis via miR-128/ p70S6K1 axis, and miR-128 may serve as a potential therapeutic target in glioma in the future. Citation: Shi Z-m, Wang J, Yan Z, You Y-p, Li C-y, et al. (2012) MiR-128 Inhibits Tumor Growth and Angiogenesis by Targeting p70S6K1. PLoS ONE 7(3): e32709. doi:10.1371/journal.pone.0032709 Editor: Jin Q. Cheng, H.Lee Moffitt Cancer Center & Research Institute, United States of America Received October 26, 2011; Accepted January 30, 2012; Published March 19, 2012 Copyright: 2012 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Natural Science Foundation of China (81071642 and 81172389), by the National Key Basic Research Program of China (2011CB504003), Key Basic Research Program of the Jiangsu Higher Education Institutions of China (09KJA310001) and by National Cancer Institute, National Institutes of Health (R01CA109460). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: binghjiang@yahoo.com . These authors contributed equally to this work. Introduction MicroRNAs (miRNAs) are a class of endogenously expressed small noncoding RNAs that are usually 18,23 nucleotides long, and regulate gene expression posttranscriptionally by targeting the untranslated regions [1]. MiRNAs have been shown to participate in a variety of cellular functions including cell apoptosis, cell proliferation, neural development, and stem cell differentiation [1– 3]. They have been identified as a new kind of gene expression regulators through targeting mRNAs for translational repression or degradation [4]. Gliomas are the most common type of primary brain tumors in adults. Among gliomas, astrocytomas have the highest incidence [5]. According to World Health Organization’s classification, astrocytomas are divided into four grades: pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme (GBM). Anaplastic astrocytoma and glioblastoma multiforme are considered malignant glioma. Recently, many miRNAs are found to play important roles in glioma. MiR-7 was down-regulated in glioblastoma which inhibited invasiveness of primary glioblastoma lines [6], while miR-26 promotes glioblastoma cell proliferation in vitro and tumor growth in vivo by targeting several tumor suppressor genes such as PTEN and RB1 [7]. MiR-128 is a brain-enriched miRNA [8]. Most of the studies of miR-128 in carcinogenesis are focusing on glioma. For example, miR-128 has been shown to be down- regulated in glioma [8–10]. Overexpression of miR-128 inhibits cell proliferation by targeting E2F3a and Bmi-1, and reduces neuroblastoma cell motility and invasiveness through inhibiting Reelin and DCX [8,10,11]. However, further investigation needs to be performed to elucidate the role of miR-128 in carcinogenesis and tumor development of glioma. P70S6K1 is one of the key downstream targets of mammalian target of rapamycin (mTOR), which plays important roles in cancerous characteristics such as cell cycle, cell apoptosis, cell growth and proliferation [12,13]. Growing evidence indicates that p70S6K1 pathway is involved in carcinogenesis, metastasis and chemotherapeutic drug resistance [12–14]. Our previous studies have demonstrated that mTOR/p70S6K1 is involved in regulat- ing tumor angiogenesis and tumorigenesis [15–17]. Tumor angiogenesis is required for tumor development and growth. Vascular endothelial growth factor (VEGF) is considered to be the most important growth factor among the angiogenic factors [18,19]. Hypoxia-inducible factor 1 (HIF-1) is one of the major regulator of VEGF [20]. High levels of HIF-1 expression are observed in many human cancers, and correlated with tumori- genesis [21]. P70S6K1 is implicated in regulating HIF-1a expression [15,16,22,23]. However, it is unclear what miRNAs directly regulate p70S6K1 in glioma. In the present study, we found that miR-128 was down- regulated in human glioma and acted as a tumor suppressor by directly targeting p70S6K1. Forced expression of miR-128 inhibited p70S6K1, HIF-1a, and VEGF expression. Overexpres- sion of p70S6K1 restored miR-128-inhibited HIF-1a and VEGF expression, confirming that p70S6K1 is the downstream target of PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32709