The glioma cell line U251 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). U87, U373, SHG44 and T98 cells were gifts from the Neuroscience Institute of Soochow University. The normal human astrocyte 1800 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Paisley, UK) supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco, USA), 100 μg/ml streptomycin and 1 U/ml penicillin.

Fresh glioma specimens were obtained with written informed consent from 7 patients with glioma who underwent surgery at the First Affiliated Hospital of Soochow University. None of the patients had received radiotherapy or chemotherapy prior to surgery. Two normal brain tissue samples were obtained from patients who underwent surgery for cerebral trauma. The resected tissue samples were immediately snap frozen in liquid nitrogen until use. All human materials were used in accordance with the policies of the local institutional review boards.

Total RNA was extracted with TRIzol (Invitrogen, Oslo, Norway). For mature microRNA expression analysis, miR-155 was detected using an All-in-One™ miRNA qRT-PCR Detection kit (GeneCopoeia, Rockville, MD, USA) according to the manufacturer’s instructions. For mRNA, total RNA isolated from glioma samples and cells were subsequently reverse transcribed to cDNA using All-in-One First-Strand cDNA Synthesis kit (GeneCopoeia). The synthesized cDNA was then used to amplify gene regions by quantitative PCR using the All-in-One qPCR mix (GeneCopoeia catalog no. AOPR-0200). The U6 small nuclear RNA and GAPDH mRNA were used as internal controls for miR-155 and FOXO3a mRNA, respectively. Relative expression was calculated using the ΔΔCt method. The reactions were placed in a 96-well plate (ABI) using the 7500 Real-Time PCR system (Applied Biosystems).

Protein of the treated cell lines was extracted by mammalian protein extraction reagent (Pierce, USA) supplemented with protease inhibitors and phosphatase inhibitor cocktail (Sigma, USA). Protein (25 μg) samples were separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes. After blocking with 1% BSA (Amresco, USA), the membranes were incubated with antibodies against FOXO3a (75D; Cell Signaling, Danvers, MA, USA) diluted 1:1000 or β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:2000 overnight at 4°C; the membrane was washed and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Dako, Glostrup, Denmark) or goat anti-mouse (Santa Cruz Biotechnology, Inc.) secondary antibody diluted 1:2000 for 2 h. The membrane was washed again, and the antigen-antibody reaction was visualized using an ECL detection system. All experiments were repeated in triplicate.

miR-155 mimics, miR-155 inhibitor and the respective negative control (miR-NC and anti-NC) were designed and synthesized by GenePharma (Shanghai, China). The miR-155 inhibitor is single-stranded RNA molecules, which can specifically knock down endogenous miR-155. Cells were transfected using Entranster-R (Engreen Biosystem Co., Ltd., China). Transfection complexes were prepared according to the manufacturer’s instructions and added directly to the cells in serum medium. miR-155 mimics and miR-NC were transfected to U251 cells, which expressed low levels of miR-155; miR-155 inhibitor (anti-miR-155), inhibitor NC (anti-NC) were transfected to U87 cells, which expressed high levels of miR-155.

Cell counts were determined using Cell Counting Kit-8 (Dojindo, Japan). Three thousand cells per well were seeded in a 96-well plate and incubated for 24 h, and the cells were transfected with miR-155 mimics, miR-155 inhibitor or NC at the final concentration of 50 nM/μl. Ten microliters of Cell Counting Kit-8 was added to 100 μl cell culture media and incubated for 2 h in CO₂ incubator at 12, 24, 48 and 72 h after transfection. Absorbance was measured at 450 nm. Three independent experiments were performed.

Cell cycle distribution was assessed using a flow cytometer (FC500 Flow Cytometer; Beckman Coulter, USA). According to the manual from the cell cycle analysis kit (Beyotime, China), cells were allowed to inoculate and adhere for 24 h in 6-well plates. Then miR-155 mimics, miR-155 inhibitor and NC were transfected into cells of the respective well, and cells were incubated for a further period of 24 h, then trypsinized, washed three times in cold phosphate-buffered saline (PBS), fixed in 70% absolute ice-cold ethanol, kept at 4°C overnight and stained in a mixture of propidium (PI) RNase A; the stained cells were analyzed using flow cytometry. This experiment was repeated three times.

Cells were transfected with miR-155, anti-miR-155, or NC at 50% confluence. After 48 h, the adherent cells were harvested by trypsinization and were washed with PBS once and resuspended in 500 μl of 1X binding buffer (Annexin V/FITC kit; Sigma). Then, 10 μl Annexin V/FITC and 5 μl propidium iodide were added into the binding buffer, and the tubes were incubated at room temperature for exactly 10 min in the dark. The fluorescence of the cells was immediately determined with a flow cytometer.

Migratory ability was determined using a wound healing assay. Cells were grown in 10% FBS medium on 6-well plates. After the cells reached sub-confluence, the cells were wounded by scraping the monolayer with a 200-μl pipette tip and grown in 1% FBS medium. The floating cells were removed by gentle washes with culture medium. The healing process was dynamically examined and was recorded with a digital camera at 24 h after the wound was created.

For the Transwell assay, 2×10⁴ transfected cells were plated into 24-well Boyden chambers (Corning Costar, Cambridge, MA, USA), with an 8-μm pore polycarbonate membrane, which was coated with 30 μg of Matrigel (BD Biosciences, San Jose, CA, USA). Cells were in the upper chamber with 200 μl of serum-free medium, and medium containing 20% FBS was added to the lower chamber to serve as a chemoattractant. After 36 h, the cells were washed 3 times with PBS. Non-invasive cells were removed from the upper well by cotton swabs, and the invasive cells were then fixed with paraformaldehyde for 15 min, air-dried, and stained with 0.1% crystal violet for 15 min. The cells were recorded with a digital camera.

DNA fragments from 3′-UTR of FOXO3a that host the predicted complementary sites of miR-155 were cloned to downstream of the Renilla luciferase reporter gene in psiCHECK2 dual luciferase reporter plasmid (Promega, Madison, WI, USA). We amplified a 2192-bp FOXO3a 3′-UTR region containing four tandem repeats of miR-155 response element, which includes the artificial XhoI and NotI enzyme restriction sites with forward primer 5′-TGTTGTTCTTGTG TTTGTTTTCC-3′ and reverse primer 5′-ATTCTCCTGATC TGTTTTGTGCT-3′. The amplified fragments were then cleaved with the XhoI and NotI enzymes (New England BioLabs, Ipswich, MA, USA) as well as psiCHECK2 vector (Promega), and the above-prepared fragment and psiCHECK2 vector were then ligated by T4 DNA ligase (New England BioLabs). The recombined vector was named psi-FOXO3a.

U251 or U87 cells were seeded at 1×10⁵ cells per well in 24-well plates. At 24 h after the plating, cells were transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. In each well, 800 ng psi-FOXO3a vectors were co-transfected with 50 pmol miR-155 mimics, miR-155 inhibitors or NC accordingly. There were four replicates for each group, and the experiment was repeated at least three times. At 24 h after transfection, cells were harvested by passive adding of 100 μl buffer. Renilla luciferase activities in the cell lysate were measured with the Dual-Luciferase Reporter assay system (Promega) in TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA) and were normalized to the firefly luciferase activities.

Data shown in the graphs represent the mean values ± SD of three independent experiments performed. The difference among groups was determined by ANOVA analysis and comparison between two groups was analyzed by the Student’s t-test using GraphPad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA, USA). A value of P<0.05 was considered statistically significant.

To explore the role of FOXO3a in glioma, we used western blotting to detect the expression level of FOXO3a in glioma cell lines, human glioma and normal brain tissues. The findings showed that five carcinoma cell lines (U251, U87, U373, T98 and SHG44) presented lower expression of FOXO3a protein when compared with the normal astrocyte 1800 cell line (Fig. 1A). Among all glioma cell lines, the highest expression of FOXO3a was detected in U251 cells, while the lowest expression was observed in U87 cells. Moreover, FOXO3a levels in glioma tissue were significantly lower than those in normal brain tissues (Fig. 1B). However, no significant difference in FOXO3a mRNA was observed between glioma cell lines, and between human glioma tissues, respectively (Fig. 1C and D).

To determine whether miR-155 plays a role in glioma tumorigenesis, we conducted real-time quantitative PCR to quantify mature miR-155 in five glioma cell lines (U251, U87, U373, T98 and SHG44) and seven glioma tissue samples, using the normal human astrocyte 1800 cell line and two normal brain tissue samples as corresponding controls. As shown in Fig. 2A, miR-155 expression was notably upregulated in five glioma cell lines, when compared to that in the normal astrocyte 1800 cell line. Among all glioma cell lines, the U87 cell line expressed the highest level of miR-155 (9-fold compared to the normal control) and U251 cell line expressed the lowest level of miR-155 (3.5-fold compared to the normal control) (Fig. 2A). Higher expression of miR-155 was observed in all glioma tissue samples (Fig. 2B).

TargetScan prediction in silico and previous studies indicated that FOXO3a is the theoretical target gene of miR-155 (Fig. 3A). To test this possibility, fragments of 3′-UTR of human FOXO3a containing miR-155 complementary sites were cloned into psiCHECK2 dual luciferase reporter plasmid. When the psi-FOXO3a vector was co-transfected with miR-155, the luciferase activity of the vector was dramatically decreased (P<0.05) compared with those co-transfected with miR-NC and Mock in U251 cells (Fig. 3B). As expected, the luciferase levels of psi-FOXO3a-3′-UTR in U87 cell lines were restored after transfection with the miR-155 inhibitor (Fig. 3C).

By performing western blot analysis, a pronounced reduction in FOXO3a protein level was noted in the U251 cells in which miR-155 is overexpressed and an increase in U87 cells in which miR-155 is downregulated (Fig. 3D). However, no significant difference in FOXO3a mRNA was observed between cells overexpressing/underexpressing miR-155 and those with NC (Fig. 3E and F). This raised the possibility that miR-155 suppresses FOXO3a protein synthesis by a post-transcriptional repression mechanism, via its 3′-UTR complementary sites.

In order to explore the functional role of miR-155 on the growth of glioma cells, we performed cellular proliferation assays (CCK-8 assay) in U87 and U251 cells and found that cell proliferation was dramatically decreased in U87 cells after transfection with the miR-155 inhibitor, while proliferation was increased in U251 cells transfected with miR-155 mimics (P<0.05) (Fig. 4A and B). Then, we carried out cell apoptosis and cell cycle assays to examine whether the reduction in cell proliferation was due to increased apoptosis or attenuated cell cycle induced by downregulation of miR-155. The U87 cells transfected with the miR-155 inhibitor were found to exhibit an enhanced apoptosis rate, compared to the anti-NC and Mock groups, while U251 cells transfected with miR-155 mimics exhibited the opposite results (Fig. 4C and D), while the alteration of miR-155 expression was ineffective on the cell cycle distribution of glioma cells (Fig. 4E and F). These results suggest that miR-155 increases the cell proliferative ability by inhibiting apoptosis in glioma cells.

To investigate whether miR-155 has a direct functional role in facilitating glioma cell migration and invasion, we evaluated cancer cell invasion through Transwell assay and assessed migration through a wound healing assay. As shown in Fig. 5, inhibition of miR-155 impeded the migration of U87 cells by ~50% when compared with the control. Similarly, invasion of U87 cells was reduced by 59% following inhibition of miR-155 (Fig. 6). Conversely, transfection of U251 cells with miR-155 mimics promoted cell migration and invasion ability by 2-fold (Fig. 5 and 6). These data suggest that miR-155 is an onco-miRNA that can promote cell migration and invasion in glioma cells.