MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC Takashi Sasayama * , Masamitsu Nishihara, Takeshi Kondoh, Kohkichi Hosoda and Eiji Kohmura Department of Neurosurgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, Japan MicroRNAs (miRNAs) are effective post-transcriptional regula- tors of gene expression and are important in many biological proc- esses. Although the oncogenic and tumor suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumor invasion and migration remains largely unex- plored. Recently, miR-10b was identified as an miRNA highly expressed in metastatic breast cancer, promoting cell migration and invasion. Here, we performed real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays on 43 glioma samples (17 glioblastoma, 6 anaplastic astrocytoma, 10 low-grade astrocy- toma, 6 oligodendroglioma and 4 ependymoma) and 6 glioma cell lines. We found that miR-10b expression was upregulated in all glioma samples compared to non-neoplastic brain tissues. The expression levels of miR-10b were associated with higher grade glioma. In addition, mRNA expressions of RhoC and urokinase- type plasminogen activator receptor (uPAR), which were thought to be regulated by miR-10b via HOXD10, were statistically signifi- cantly correlated with the expression of miR-10b (p < 0.001, p 5 0.001, respectively). Also, protein expression levels of RhoC and uPAR were associated with expression levels of miR-10b (p 5 0.009, p 5 0.014, respectively). Finally, multifocal lesions on enhanced MRI of 7 malignant gliomas were associated with higher expression levels of miR-10b (p 5 0.02). Our data indicated that miR-10b might play some role in the invasion of glioma cells. ' 2009 UICC Key words: miR-10b; glioma; invasion; uPAR; RhoC Gliomas are the most common brain tumors of the adult central nervous system. One of the insidious biological features of glioma is the potential of single cells to invade normal brain tissue. Inva- sive tumor cells escape surgical removal and radiation exposure. The mechanism of invasion is quite complex and involves inte- grated biochemical processes requiring a coordinated effort man- aging a number of intracellular and extracellular interactions. 1 Tu- mor cells achieve this by excessive production of several proteases and by modifying ECM, creating local access through surrounding tissue and migrating into other parts of the brain. 2,3 Also, members of the Ras superfamily of GTPases, most notably the Rho proteins, plays a prominent role in cell migration. 4 MicroRNAs (miRNAs), which encode small non-coding RNAs of approximately 22 nucleotides, are now recognized as a very large gene family present throughout the genomes of plants and metazoans. 5–7 Evolutionally conserved miRNAs have stem-loop structures and are often found as clustered sequences. Mature miRNAs processed by two-step cleavage involving Drosha and Dicer are thought to regulate the negative expression of a large number of genes carrying target sites within 3’ untranslated regions, whereas recent evidence points to miRNA utilization of multiple mechanisms for gene silencing. Recent work has revealed important roles of miRNAs and miRNA processing in tumorigene- sis. 8,9 A large set of miRNAs is overexpressed in human tumors compared to normal tissues, 10,11 and gene silencing by miRNAs enhances tumor cell growth. 12 These findings suggest that this class of regulators includes enhancers of tumor progression. The miR-10b gene is located in the middle of the HOXD cluster on chromosome 2q31, near HOXD4. 13 A combination of mouse and human cells exhibited that miR-10b is highly expressed in metastatic breast cancer cells and positively regulates cell migra- tion and invasion. 13 Overexpression of miR-10b in otherwise non- metastatic breast tumors initiates robust invasion and metastasis. The miR-10b proceeds to inhibit translation of HOXD10, resulting in increased expression of a well-characterized prometastatic gene, RhoC. In addition, the level of miR-10b expression in pri- mary breast carcinomas correlates with clinical progression. Recently, miR-10b was found to be overexpressed in hepatocellu- lar carcinoma compared to benign hepatic tumors and nontumor liver tissues. 14 These findings suggested a novel pathway which regulates tumor cell invasion and migration by miR-10b. miR-10b was previously reported to be upregulated in glioblas- toma tissues. 15 Our preliminary miRNA microarray studies also showed miR-10b upregulation in glioblastoma; therefore, we examined the expression level of miR-10b in 43 cases of various gliomas and found that miR-10b was upregulated in all gliomas compared to nontumor brain tissues. Overexpression levels of miR-10b were highly associated with higher grade gliomas. Also, expression levels of invasive factors RhoC and urokinase-type plasminogen activator receptor (uPAR) were associated with expression levels of miR-10b. In addition, high expression levels of miR-10b were associated with multifocal tumors and dissemi- nation. From these findings, it is possible that miR-10b might play some role in the invasion of glioma. Material and methods Clinical specimens and cell lines Glioma tissues were obtained from therapeutic procedures per- formed as routine clinical management at our institution. Tissue samples were resected during surgery and immediately frozen in liquid nitrogen for subsequent total RNA extraction. A total of 43 gliomas (glioblastoma (GBM) 5 17, anaplastic astrocytoma (AA) 5 6, low-grade glioma (LGA) 5 10 (diffuse astrocytoma 5 7, pilocytic astrocytoma 5 3), oligodendroglioma (OG) 5 6, epen- dymoma (EP) 5 4), 5 meningiomas, 3 schwannomas and 5 non- neoplastic brain specimens were included in our study. Six GBM cell lines (A172, U87MG, T98G, U251, YH13 and SF126) were also used. GBM cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing glutamine, 10% fetal bovine serum and penicillin/streptomycin. Cells were grown at 37°C in a 5% CO 2 incubator. RNA isolation and fluorescent labeling Total RNA for miRNA microarray was obtained from tissue samples using Trizol reagent (Invitrogen, Carlsbad, CA). To pre- pare cellular miRNA, small RNA containing miRNA was isolated from total RNA using the RNeasy MinElute Cleanup Kit (QIAGEN Inc., Mississauga, Ontario, Canada), as described previ- ously. The isolated small RNA (1 lg) was subjected to direct labeling with a fluorescent dye using the PlatinumBright 647 Infrared nucleic acid labeling kit (KREATECH, Amsterdam, The Additional Supporting Information may be found in the online version of this article. Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology; Grant numbers: 15659337, 19790998, 17790968. *Correspondence to: Department of Neurosurgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650- 0017 Japan. Fax: 181-78-382-5979. E-mail: takasasa@med.kobe-u.ac.jp Received 15 July 2008; Accepted after revision 7 April 2009 DOI 10.1002/ijc.24522 Published online 23 April 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 1407–1413 (2009) ' 2009 UICC Publication of the International Union Against Cancer