Normal brain tissue and human GBM samples were obtained from the Department of Neurosurgery at the Tumor Hospital (Harbin Medical University Harbin, Heilongjiang, China). None of the patients had received radiotherapy or chemotherapy prior to surgery. Samples were snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction. Study approval was obtained from the Ethics Committee of The Tumor Hospital, Harbin Medical University and all patients involved provided written informed consent.

Two human glioma cell lines, U87 and U251, purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were included in this study. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) containing 100 mg/ml streptomycin (Beyotime Institute of Biotechnology, Haimen, China), 100 U/ml penicillin ((Beyotime Institute of Biotechnology) and 10% fetal bovine serum (FBS; Hyclone) in a humidified atmosphere containing 5% CO₂ at 37°C. On the day prior to transfection, the cells were seeded onto 6-well plates (5×10⁵ cells/well) and inoculated in complete medium without antibiotics (2 ml/well).

Transfections with mature miR-133b mimics, negative control (NC) and luciferase reporter plasmid were carried out using 50 nm Lipofectamine 2000 (Shanghai GenePharma Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The sequences were as follows: hsa-miR-133b mimic, 5′-UUUGGUCCCCUUCAACCAGCUA-3′; and NC, 5′-UUCUCCGAACGUGUCACGUTT-3.

The U87 and U251 cells were plated onto 96-well plates and treated with miR-133b mimics and NC for 24 h. A 100 μl plastic pipette tip was then dragged across the plate to create a wound line that is a cell-free area. Images (magnification, ×40) of wound lines for each treatment were taken at 0 and 24 h using a digital camera (Nikon, Tokyo, Japan). The distance of wound closure (compared with the control at 0 h) was measured. These experiments were repeated three times.

A Transwell invasion assay was performed using a 24-well Transwell chamber with polycarbonate membranes with 8-μm pores (Corning-Costar Inc., Corning, NY, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Transfected cells (miR-133 mimics or NC) were loaded onto the upper filter (0.2 ml) at a density of 5×10⁵ cells/ml of serum-free medium. DMEM/F12 medium containing 15% FBS (0.5 ml) was added to the lower chamber. The cells were incubated for 24 h at 37°C in a humidified incubator containing 5% CO₂. A cotton swab was used to scrape off cells and Matrigel on the upper side of the Transwell insert filter. Invading cells were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology, Haimen, China) for 10 min, stained with hematoxylin for 2 min, rinsed in running tap water for 10 min, differentiated with 0.3% acid alcohol for 1 min, rinsed in running tap water for 10 min, stained with eosin for 2 min, and counted by capturing images in three independent fields for each Transwell insert filter (magnification, ×200). The numbers of invading cells were averaged. A migration assay was performed following the same procedure, except that the cells were incubated for 12 h and the polycarbonate filters were not coated with Matrigel. These experiments were repeated three times.

The cells were washed twice with phosphate-buffered saline and lysed directly in lysis buffer (1 mmol/l PMSF, 1 mmol/l EDTA, 40 mmol/l Tris-HCl, 100 mmol/l NaVO3, 150 mmol/l KCl and 1% Triton X-100) on ice for 15 min. Subsequent to centrifugation (12,000 × g for 20 min at 4°C), the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the protein concentrations of the lysates. Equal amounts of proteins were separated on 10% SDS-PAGE gels and then electrotransferred onto a polyvinylidene difluoride membrane. The membranes were blocked in 5% skimmed milk-Tris-buffered saline plus Tween-20 solution and incubated with rabbit MMP14 monoclonal antibody (1:1,000; Abcam, Cambridge, UK) or rabbit polyclonal β-actin antibody (1:1,000; Bioss, Beijing, China). Following incubation with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:2,000; ZSGB-BIO, Beijing, China), protein bands were detected with Fujifilm Las-4000 (Fujifilm, Tokyo, Japan).

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA (~1 μg) was reverse transcribed using the miR-133b qPCR Quantitation kit (Shanghai GenePharma Co., Ltd.). The primers of miR-133b and U6 were synthesized by Shanghai Genepharma Co., Ltd., and RT-qPCR was performed on a 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems). The reaction conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min. The mRNA expression levels of miR-133b were normalized to U6 using the standard ΔΔCt method. Each assay was performed in triplicate and all experiments were repeated at least three times.

The following target prediction software was used to identify miRNA-133b that could potentially bind MMP14: Pictar (http://www.pictar.mdc-berlin.de/), TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/) and miRWalk (http://www.umm.uni-heidelberg.de/). The U87 and U251 cells were seeded in 24-well plates. One day later, the cells were co-transfected with luciferase vectors, either the wild-type MMP14 3′-untranslated region (3′UTR) reporter plasmid or mutated MMP14 3′UTR reporter plasmid together with miR-133b mimics or miRNA-NC, and the Renilla control. After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) and normalized to Renilla luciferase activity.

SPSS statistical software, version 19.0 (IBM, Armonk, NY, USA) was used for statistical analysis. The expression of miR-133b in the tissue samples was analyzed using the Mann-Whitney U test for comparing the results of two groups. Other data were expressed as the mean ± standard deviation, and a two-sided Student's t-test was used for the statistical analysis. P<0.05 was used to indicate a statistically significant difference.

The expression levels of miR-133b were analyzed by RT-qPCR in 21 GBM specimens and 9 normal brain tissue samples. As shown in Fig. 1, the expression of miR-133b was significantly lower in the GBM specimens than in the normal brain tissues (P<0.05).

miR-133b or NC mimics were transfected into the U87 and U251 cells. After 24 h, the ectopic expression of miR-133b in the cells was analyzed by RT-qPCR. Compared with the NC group, the miR-133b mimic group showed significantly upregulated miR-133b expression in the two cell lines (P<0.05; Fig. 2).

To determine whether the overexpression of miR-133b has an effect on the migration and invasion of GBM cells, wound healing and Transwell assays were performed. As demonstrated in Fig. 3A and B (wound healing assays), the overexpression of miR-133b using mimics significantly inhibited the migratory ability of the U87 and U251 cells (P<0.05). As shown in Fig. 3C and D, the ability of the U87 and U251 cells to migrate through a Transwell insert filter was clearly inhibited by miR-133b (P<0.05). The Transwell invasion assay also showed that the number of invasive cells transfected with miR-133b mimics was significantly lower compared with the NC transfected cells (P<0.05). Therefore, these findings indicated that miR-133b acts as an invasive suppressor in GBM cells.

To investigate the mechanism by which miR-133b regulates cell invasion, an miRNA target search was performed with four target prediction databases (miRanda, TargetScan, PicTar and miRWalk), and all four databases showed that the MMP14 mRNA contained miR-133b binding sites (Fig. 4A). To examine the hypothesis that miR-133b targeted MMP14, a luciferase reporter assay was performed. The wild-type MMP-9 3′UTR reporter plasmid or mutated MMP-9 3′UTR reporter plasmid was cotransfected into the U87 and U251 cells with miR-377 mimic or NC. As shown in Fig. 4B, the U87 and U251 cell lines cotransfected with miR-133b mimic and wild-type MMP14 3′UTR reporter plasmid showed a significant decrease in reporter activity (P<0.05). To confirm that the expression of MMP14 is regulated by miR-133b, western blot analysis was performed. As shown in Fig. 4C, MMP14 was significantly downregulated in the U87 and U251 GBM cell lines following the overexpression of miR-131b (P<0.05). These findings demonstrated that the 3′UTR of MMP14 was the directed target of miR-133b.