Dontula Ranadheer, Dinasarapu Ashok, Chetty Chandramu, Pannuru Padmavathi, Herbert Engelhard, Ozer Howard, Lakka Sajani S
Section of Hematology/Oncology, University of Illinois Cancer Center, College of Medicine at Chicago, IL, USA.
Genes Cancer. 2013 Jul;4(7-8):285-96. doi: 10.1177/1947601913500141.
Glioblastoma (GBM) is the most common and malignant primary adult brain cancer. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM, and this site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). In this study, we sought to identify the role of miR-203 as a tumor suppressor in the pathogenesis of GBM. We analyzed the miR-203 expression data of GBM patients in 10 normal and 495 tumor tissue samples derived from The Cancer Genome Atlas data set. Quantitative real-time PCR and in situ hybridization in 10 high-grade GBM and 10 low-grade anaplastic astrocytoma tumor samples showed decreased levels of miR-203 expression in anaplastic astrocytoma and GBM tissues and cell lines. Exogenous expression of miR-203 using a plasmid expressing miR-203 precursor (pmiR-203) suppressed glioma cell proliferation, migration, and invasion. We determined that one relevant target of miR-203 was Robo1, given that miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and Western blot analysis. Moreover, cotransfection experiments using a luciferase-based transcription reporter assay have shown direct regulation of Robo1 by miR-203. We also show that Robo1 mediates miR-203 mediated antimigratory functions as up-regulation of Robo1 abrogates miR-203 mediated antimigratory effects. We also show that miR-203 expression suppressed ERK phosphorylation and MMP-9 expression in glioma cells. Furthermore, we demonstrate that miR-203 inhibits migration of the glioma cells by disrupting the Robo1/ERK/MMP-9 signaling axis. Taken together, these studies demonstrate that up-regulation of Robo1 in response to the decrease in miR-203 in glioma cells is responsible for glioma tumor cell migration and invasion.
胶质母细胞瘤（GBM）是最常见且恶性程度最高的原发性成人大脑肿瘤。14号染色体长臂上的等位基因缺失在GBM的发病机制中起着重要作用，该位点被认为含有多个与GBM相关的肿瘤抑制基因，该区域还编码微小RNA-203（miR-203）。在本研究中，我们试图确定miR-203作为肿瘤抑制因子在GBM发病机制中的作用。我们分析了来自癌症基因组图谱数据集的10份正常组织样本和495份肿瘤组织样本中GBM患者的miR-203表达数据。对10份高级别GBM和10份低级别间变性星形细胞瘤肿瘤样本进行的定量实时聚合酶链反应和原位杂交显示，间变性星形细胞瘤、GBM组织及细胞系中miR-203表达水平降低。使用表达miR-203前体的质粒（pmiR-203）对外源表达miR-203可抑制胶质瘤细胞的增殖、迁移和侵袭。鉴于通过逆转录聚合酶链反应和蛋白质免疫印迹分析确定miR-203表达降低了Robo1的mRNA和蛋白质水平，我们确定miR-203的一个相关靶点是Robo1。此外，使用基于荧光素酶的转录报告基因检测的共转染实验表明miR-203可直接调控Robo1。我们还表明，Robo1介导miR-203介导的抗迁移功能，因为Robo1的上调消除了miR-203介导的抗迁移作用。我们还表明，miR-203表达抑制了胶质瘤细胞中的细胞外信号调节激酶磷酸化和基质金属蛋白酶-9表达。此外，我们证明miR-203通过破坏Robo1/细胞外信号调节激酶/基质金属蛋白酶-9信号轴抑制胶质瘤细胞的迁移。综上所述，这些研究表明，胶质瘤细胞中miR-203减少导致Robo1上调，这与胶质瘤肿瘤细胞的迁移和侵袭有关。
