Tumor specimens were obtained from patients who underwent positive debulking surgery in the Neurosurgery Department of the The First Affiliated Hospital of Soochow University (Suzhou, China) between 2011 and 2013. The diagnosed gliomas were reviewed by an experienced neuropathologist, using histological slides, according to the 2007 WHO classification. The present study comprised 20 grade I–II, 20 grade III and 20 grade IV glioma samples. In addition, 10 normal brain tissue samples were obtained from internal decompression of patients with cerebral injury. This study complied with the requirements of the ethics committee of The First Affiliated Hospital of Soochow University and informed consent was obtained from all participants.

Primary normal human astrocytes (NHA) were purchased from Sciencell Research Laboratories (Carlsbad, CA, USA). The U251, U87, SNB19 and LN229 glioma cell lines were obtained from the Institute of Biochemistry and Cell Biology (Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China). The cells were maintained in RPMI-l640 medium containing 10% FBS, 50 units/ml penicillin G, and 250 μg/ml streptomycin (all purchased from Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO₂, at 37°C. Transfections with miR-218 were performed in serum-free medium 24 h subsequent to plating, using Lipofectamine 2000 (Invitrogen Life Technologies). After 6 h, the cells were placed in complete medium and maintained at 37°C in a 5% CO₂ atmosphere.

Robo1-specific siRNA (forward, 5′-GGAUGUAUUUGCAAC AAGATT-3′ and reverse, 5′-UCUUGUUGCAAAUACAUC CTT-3′) was chemically synthesized by Qiagen (Hilden, Germany). The U87 cells were transfected with siRNA (HiPerFect Transfection Reagent; Qiagen) according to the manufacturer’s instructions. Briefly, the original stock of the siRNA was suspended in the siRNA suspension buffer provided by the manufacturer and stored at −20°C until use. On the day of transfection, 1×10⁵ cells were seeded in six-well plates (Corning Inc., Corning, NY, USA) with a total volume of 1,000 μl per well. siRNA was then gently introduced into the cells by adding 4 μl Oligofectamine™ Transfection Reagent (Invitrogen Life Technologies) and 5 μl siRNA (20 nM) per well. Non-silencing siRNA (GE Dharmacon, Lafayette, CO, USA) (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′) was used to control any effects of the transfection reagent and siRNA. The in vitro assays described herein were performed 24 h after transfection.

Total RNA, including miRNA, was extracted from the cultured cells or fresh glioma cancer tissues using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. The expression level of miR-218 was quantified using an miRNA-specific TaqMan miRNA Assay kit (Applied Biosystems, Foster City, CA, USA). U6 small nuclear RNA was used as an internal control. The mRNA expression of Slit2 and Robo1 was analyzed by qPCR, using the SYBR-Green method. All protocols were performed according to the manufacturer’s instructions and the results were normalized to the expression of GAPDH. The primer sequences were as follows: Slit2 forward, 5′-ACCTCT TGGCCAATCCTTTT-3′ and reverse, 5′-GAAGTCCTGAAT GGCCACAT-3′; Robo1 forward, 5′-GCCACCAGCAAGGAT GTATT-3′ and reverse, 5′-CCTGTAACATGGGCTGGAGT-3′; and GAPDH forward, 5′-TCGGAGTCAACGGATTTGG-3′ and reverse, 5′-CATGGGTGGAATCATATTGGA-3′.

The cells were lysed in 1% Nonidet P-40 lysis buffer for 48 h following exposure to LY294002 or vehicle treatment. The homogenates were clarified by centrifugation at 20,000 × g for 15 min, at 4°C, and the protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). SDS-PAGE was performed on 40 μg of protein from each sample, the gels were transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) and incubated with primary polyclonal goat anti-human Slit2 and rabbit anti-human Robo1 antibodies (1:200 dilution; sc-26599 and sc-25672, respectively, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) followed by incubation with horseradish peroxidase-conjugated monoclonal goat anti-rabbit and donkey anti-goat secondary antibodies (1:1,000 dilution; cat nos. sc-2004 and sc-2020, respectively; Zymed Life Technologies, Carlsbad, CA, USA). The membranes were stripped using 1X phosphate-buffered saline with Tween 20 buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China) and reprobed with a primary monoclonal mouse anti-rabbit antibody against GAPDH (1:1,000 dilution; Bioworld, Nanjing, China). The protein bands were quantitated by densitometry using the gel analysis software ImageJ (National Institutes of Health, Bethesda, MA, USA. The values were normalized to GAPDH expression.

Cell invasion was determined using Transwell and scratch-wound assays. For the Transwell assay, the appropriate oligonucleotides were transfected into the cells according to the aforementioned protocol. Subsequent to incubation for 48 h, 3×10⁴ cells were transferred to the top of the Matrigel-coated invasion chambers (BD Biosciences, San Jose, CA, USA) in a serum-free Dulbecco’s modified Eagle’s medium (DMEM). DMEM containing 10% fetal bovine serum was added to the lower chamber. After 24 h, the non-invading cells were removed, and the invading cells were fixed using 95% ethanol, stained with 0.1% crystal violet and images were captured under a ×100 magnification. The tests were repeated in three independent experiments. For the scratch-wound assay, the appropriate oligonucleotides were transfected into the cells in six-well plates. The cell layers were then scratched using a 200 μl sterile pipette tip to form wound gaps. The wound location in the six-well plates was marked. Images of the cells were captured to record the wound width at 0 and 24 h, and the images were captured at the marked wound location to measure the migratory ability of the cells.

The 3′-UTR sequence of Robo1 predicted to interact with miR-218 or a mutated sequence with the predicted target sites was synthesized and inserted into the XbaI and FseI sites of a pGL3 control vector (Promega Corporation, Madison, WI, USA). These constructs were named pGL3-Robo1–3′-UTR and pGL3-Robo1–3′-UTR-mut, respectively. For the reporter assay, the U87 cells were plated onto 24-well plates and transfected with pGL3-Robo1–3′-UTR or pGL3-Robo1–3′-UTR-mut and P-miR-218 or P-miR-control vectors using the FuGENE HD transfection reagent (Promega Corporation). A Renilla luciferase vector pRL-SV50 (Promega Corporation) was cotransfected to normalize the differences in transfection efficiency. Subsequent to 48 h transfection, the cells were harvested and assayed using the Dual-Luciferase Reporter Assay System (Promega Corporation), according to the manufacturer’s instructions. Transfection was repeated in triplicate in three independent experiments.

Robo1 was identified as a notable novel target of miR-218 using the conventional prediction tool, TargetScan (www.targetscan.org; Whitehead Institute for Biomedical Research, Cambridge, MA, USA).

Experimental data were presented as the mean ± standard deviation. All analyses were performed using a two-tailed Student’s t-test performed on SPSS software, version 12.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To analyze the expression levels of miR-218, a qPCR analysis of miR-218 expression was conducted in the U251, U87, SNB19 and LN229 glioma cell lines. The present results revealed that miR-218 is downregulated in all glioma cell lines compared with the NHA cells (P<0.05). In addition, when miR-218 expression was measured in 10 normal brain tissue and 60 glioma tissue samples, it was observed that the expression level of miR-218 was significantly decreased in the glioma tissues, particularly in grade III/IV glioma tissues, compared with the normal brain tissues (Fig. 1). This result demonstrates that miR-218 expression decreases markedly between normal brain tissue and low grade to GBM tissue. Overall, the present results indicate that miR-218 is downregulated in glioma cell lines and glioma tissues.

As invasiveness is one of the pathophysiological features of human malignant gliomas, the effects of miR-218 on the invasiveness and migration of glioma cells were checked by Transwell and scratch-wound assays. An in vitro Matrigel invasion assay revealed that the invasiveness of U87 cells transfected with the miR-218 mimic was suppressed compared with control and NC groups (Fig. 2A and B). The results of the in vitro wound healing assay revealed that miR-218 significantly attenuated the migration of U87 cells compared with control and NC groups (Fig. 2C and D). This finding indicates that the upregulation of miR-218 inhibits the invasive ability of glioma cells in vitro.

Development of invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways. Numerous studies have reported that the Slit2-Robo1 signaling channels can inhibit glioma invasion and migration. However, the specific roles of Slit2/Robo1 in cancer cell invasion have not yet been completely elucidated in vivo. The present study also observed, using qPCR and western blot analysis, that treatment with the miR-218 mimic for 24 h significantly upregulated the expression of Slit2, which was followed by a decrease in Robo1 (Fig. 3). These results indicate that miR-218 reduced the expression of Robo1 via the inactivation of Slit2-Robo1 signaling.

A luciferase reporter assay further confirmed the direct interaction between miR-218 and the 3′-UTR of Robo1 mRNA (Fig. 4). The luciferase activity for the wild-type 3′-UTR of Robo1 was significantly inhibited by co-transfection with miR-218 mimics compared with constructs containing mutated 3′-UTRs. The present study demonstrated that Robo1 is a direct target of miR-218.

To assess the role of Robo1 in the miR-218-dependent inhibition of cell migration and invasion, miR-218 inhibitor was transfected into U87 cells treated with Robo1 siRNA. As expected, Robo1 protein expression was significantly reduced by the specific Robo1 siRNA (Fig. 5). The enhanced invasive ability of miR-218 inhibitor-transfected U87 cells declined when Robo1 siRNA was co-transfected with the miR-218 inhibitor (Fig. 6). These results indicate that Robo1 is essential for miR-218-dependent cell migration and invasion.