The present study was approved by the Ethical Committee of Shenzhen Second People's Hospital (Shenzen, China) and written informed consent was obtained from all subjects prior to enrollment in the present study. A total of 19 glioma tissue samples were obtained from patients (age, range 31–69 years, median 45 years; 12 male and 7 female subjects) undergoing tumor resection surgery at the Department of Neurosurgery, Shenzhen Second People's Hospital between February 2013 and May 2015. A total of 8 healthy brain tissue samples were obtained from patients with cerebral trauma (age, range 24–57 years, median 36 years; 6 male and 2 female subjects) that underwent brain surgery between February 2013 and May 2015 at the Department of Neurosurgery, Shenzhen Second People's Hospital. None of the patients had received prior treatments, including radiation or chemotherapy. The tissues were immediately frozen and stored at −80°C until use.

U87, U251, LN229, LN18 human glioma cell lines and HEB normal human glial cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), in a humidified chamber supplemented with 5% CO₂ at 37°C.

miR-205 mimics (5′-UCCUUCAUUCCACCGGAGUCUG-3′) and negative control (NC) miRNA mimics (5′-UUCUCCGAACGUGUCACGUTT-3′) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). YAP1 small interfering RNA (siRNA; 5′-GGUGAUACUAUCAACCAAATT-3′) and NC siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were synthesized and purified by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). According to the manufacturer's protocol, cells were transfected with miRNA mimics or siRNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 24 h, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and a Cell Counting kit 8 (CCK8) assay were performed. Transwell migration and invasion assays, and western blot analysis were performed at 48 and 72 h post-transfection, respectively.

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using a First-Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using the SYBR Green PCR mixture (Applied Biosystems; Thermo Fisher Scientific, Inc.) with an ABI Prism 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Thermocycling conditions were as follows: Initial 1 step at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. U6 and GADPH were used as internal standards to normalize the expression of miR-205 and YAP1 mRNA, respectively. The primer sequences are presented in Table I. Relative gene expression was calculated using the 2^−ΔΔCq method (16).

Cell proliferation was evaluated by performing a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Transfected cells were harvested, counted and seeded into a 96-well plate at a density of 2,000 cells/well. The CCK-8 assay was performed at 24, 48, 72, and 96 h following incubation. At each time point, 10 µl CCK-8 solution was added to each well and incubated at 37°C for a further 2 h. The absorbance was detected at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each assay was performed in triplicate.

Cellular migration and invasion was examined using a Transwell migration and invasion assay with Transwell chambers (8 mm pores; Costar; Corning Incorporated, Corning, NY, USA). For the Transwell invasion assay, the Transwell chambers were coated with Matrigel (BD Biosciences, San Jose, CA, USA). For the migration and invasion assays, transfected cells were harvested, counted and re-suspended. A total of 5×10⁴ cells in 200 µl FBS-free culture medium was added to the upper chamber, while 500 µl culture medium supplemented with 20% FBS was added to the lower chamber. Following incubation for 48 h at 37°C, cells that had migrated or invaded through the Transwell chamber were fixed with 100% methanol at room temperature for 10 min, stained with 0.1% crystal violet at room temperature for 10 min, and counted in five random areas of each Transwell chamber using an inverted microscope (Olympus Corporation, Tokyo, Japan).

The direct target genes of miR-205 were analyzed using TargetScan (targetscan.org) and miRanda (microrna.org).

Luciferase reporter plasmids, psiCHECK2-YAP1-3′UTR wild type (Wt) and psiCHECK2-YAP1-3′UTR mutant (Mut), were synthesized by Shanghai GenePharma Co., Ltd. HEK293T cells (ATCC) were seeded in 24-well plates at 50–60% confluence. Following incubation overnight, cells were transfected with psiCHECK2-YAP1-3′UTR Wt or psiCHECK2-YAP1-3′UTR Mut, and miR-205 mimics or NC, using Lipofectamine 2000. Following incubation for 48 h at 37°C, cells were collected, and firefly and Renilla luciferase activities were detected using a Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol.

Transfected cells were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), supplemented with a protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to detect the concentration of protein. Equal amounts of protein (30 µg) were subjected to 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), and blocked in 5% skimmed milk in TBS with 0.1% Tween-20 (TBST) at room temperature for 1 h. Subsequently, the membranes were incubated with the primary antibodies mouse anti-human monoclonal YAP1 (1:1,000; cat no. ab124474; Abcam, Cambridge, UK), and mouse anti-human monoclonal GADPH antibody (1:1,000; cat no. ab125247; Abcam), at 4°C overnight. Following washing three times with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (1:5,000; cat no. ab6789; Abcam) for 1 h at room temperature. Protein bands were developed using enhanced chemiluminescence reagents (EMD Millipore). GADPH was used as an internal control for YAP1.

Data are expressed as the mean ± standard deviation of 3 independent experiments. The statistical significance of the differences between groups was assessed using a two-tailed Student's t-test for pair-wise comparisons, or a one-way analysis of variance followed by a post hoc Student-Newman-Keuls test for multiple comparisons. Statistical analysis was performed using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to determine whether miR-205 is involved in the tumorigenesis and progression of glioma, the expression of miR-205 in glioma tissues was analyzed using RT-qPCR. It was observed that miR-205 was expression was significantly reduced in glioma tissues compared with healthy brain tissues (Fig. 1A; P<0.05). The expression levels of miR-205 were additionally measured in glioma cell lines (U87, U251, LN229, LN18) and a normal human glial cell line (HEB). Compared with HEB, the expression of miR-205 was significantly reduced in glioma cell lines (Fig. 1B; P<0.05).

In order to further investigate the biological roles of miR-205 in glioma cells, U87 and LN229 cells, that express a relatively decreased level of miR-205 compared with normal glial cells, were transfected with miR-205 or NC mimics. Following transfection, RT-qPCR analysis was performed to measure miR-205 expression. As presented in Fig. 2A, miR-205 was significantly upregulated in U87 and LN229 cells transfected with miR-205 mimics compared with the NC groups (P<0.05).

The results of the CCK-8 assay demonstrated that treatment with miR-205 mimics reduced the viability of U87 and LN229 cells (Fig. 2B; P<0.05). Transwell migration and invasion assays were used to investigate the effect of miR-205 on the migratory and invasive behavior of glioma cells. The results demonstrated that upregulation of miR-205 suppressed the migratory and invasive capabilities of U87 and LN229 cells (Fig. 2C; P<0.05). The results of the present study suggested that miR-205 may function as a negative regulator of glioma progression.

In order to examine the molecular mechanism underlying the tumor suppressive roles of miR-205 in glioma, bioinformatics analysis was performed using TargetScan and miRanda. The analysis demonstrated that YAP1 was a putative target gene of miR-205 (Fig. 3A). Therefore, a luciferase reporter assay was performed to investigate whether miR-205 directly interacted with the 3′UTR of YAP1. The results of the present study demonstrated that miR-205 significantly decreased the luciferase activity of psiCHECK2-YAP1-3′UTR Wt (Fig. 3B; P<0.05). However, upregulation of miR-205 failed to affect the luciferase activities of psiCHECK2-YAP1-3′UTR Mut. In order to determine if miR-205 decreased YAP1 expression, RT-qPCR analysis and western blotting were performed, and the results demonstrated that restoring miR-205 expression significantly decreased YAP1 expression at the mRNA (Fig. 3C; P<0.05) and protein levels (Fig. 3D; P<0.05), in U87 and LN229 cells. The results of the present study demonstrated that miR-205 directly targeted the 3′UTR of YAP1 and negatively regulated its expression.

In the present study, YAP1 was identified to be a direct target of miR-205 in glioma. Therefore, it was subsequently investigated whether YAP1 acts as a downstream effector of miR-205, mediating its tumor suppressive functions. U87 and LN229 cells were transfected with YAP1 or NC siRNA. Following transfection, RT-qPCR analysis and western blotting was performed to measure YAP1 mRNA and protein expression, respectively. The results demonstrated that YAP1 was significantly downregulated at the mRNA (Fig. 4A; P<0.05) and protein levels (Fig. 4B; P<0.05), in YAP1 siRNA-transfected U87 and LN229 cells.

Functional assays demonstrated that YAP1 knockdown decreased U87 and LN229 cellular viability (Fig. 4C; P<0.05), migration and invasion (Fig. 4D; P<0.05) in vitro, which was consistent with the effects induced by miR-205 overexpression. The results of the present study demonstrated that YAP1 may be a downstream effector of miR-205 in glioma.