A total of 48 paired human glioma tissues and matched normal tissues were collected from patients who underwent surgical resection at the Department of Neurosurgery, The Second Affiliated Hospital of Nanchang University (Nanchang, China) between June 2014 and January 2016 and their details are presented in Table I. None of these patients were treated with radiotherapy, chemotherapy, gene therapy, immunotherapy or other novel biological therapies. All tissues were flash-frozen in liquid nitrogen immediately following collection and then stored at −80°C until RNA extraction. The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Written informed consent was also acquired from each patient.

A total of five glioma cell lines (U87, U251, U373, LN18, A172) and primary normal human astrocytes (NHA) were purchased from the American Type Culture Collection (Manassas, VA, USA). The origin of the U87 cell line is unknown, but it is a likely glioblastoma cell line (18). U373 cell line, known as a U251 derivative (19), was acquired from the National Infrastructure of Cell line Resource (Beijing, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were cultured in a humidified atmosphere at 37°C with 5% CO₂.

A mature miR-216b mimic and an miRNA mimic negative control (miR-NC) were obtained from Chang Jing Bio-Tech, Ltd. (Changsha, China). The miR-216b mimic sequence was 5′-AAAUCUCUGCAGGCAAAUGUGA-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. Small interfering RNA targeting metadherin (MTDH siRNA) and its NC siRNA were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The MTDH siRNA sequence was 5′-GCTGTTCGAACACCTCAAA-3′ and the NC siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. For transfection, cells were seeded into 6-well plates at a density of 60–70% confluence. Cells were transfected with an miR-216b mimic (100 pmol), miR-NC (100 pmol), MTDH siRNA (100 pmol) or NC siRNA (100 pmol) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. At 48 h post-transfection, cells were collected and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the transfection efficiency.

Tissue samples and cells were subjected to RNA isolation using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The concentration and purity of total RNA was determined using a NanoDrop 1000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). To measure miR-216b expression, complementary DNA (cDNA) was synthesized using a TaqMan^® MicroRNA Reverse Transcription kit and qPCR was conducted with a TaqMan^® MicroRNA Assay kit (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. The primers were designed as follows: miR-216b, 5′-AAATCTCTGCAGGCAAATGTGA-3′ (forward) and 5′-GTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse). To detect MTDH mRNA expression, cDNA was synthesized using Moloney-murine leukemia virus reverse transcription system (Promega Corporation, Madison, WI, USA), followed by qPCR using SYBR Premix Ex Taq (Takara, Dalian, China). The cycling conditions were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. The primers were as follows: MTDH, 5′-TGTTGAAGTGGCTGAGGG-3′ (forward) and 5′-CAGGAAATGATGCGGTTG-3′ (reverse); and GAPDH, 5′-GGTGAAGGTCGGAGTCAACG-3′ (forward) and 5′-CAAAGTTGTCATGGATGHACC-3′ (reverse). The miR-216b and MTDH mRNA expression was normalized to those of U6 and GAPDH, respectively, using the 2^−ΔΔCq method (20).

Cell proliferation was evaluated using an MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Transfected cells were harvested at 24 h post-transfection and then plated into 96-well plates at a density of 2,000 cells/well. Cells were incubated in a humidified atmosphere at 37°C with 5% CO₂ for 1, 2, 3 and 4 days. At specific time points, the MTT assay was performed according to the manufacturer's protocol. A total of 10 µl MTT reagent (5 mg/ml) was added into each well and the plates were incubated at 37°C for an additional 4 h. The culture medium containing the MTT solution was removed and formazan crystals were dissolved in 150 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA). Cellular proliferation was determined using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by measuring the absorbance of the converted dye at 490 nm. All experiments were performed in triplicate.

Transwell filters (diameter 12 mm; pore size 8-µm; EMD Millipore, Billerica, MA, USA) coated with Matrigel^® (BD Biosciences, San Jose, CA, USA) were utilized to assess the invasive ability of cells. A total of 48 h following transfection, cells were collected and resuspended in FBS-free culture medium. A total of 1×10⁵ cells/400 µl were plated into the upper chamber, while 600 µl culture medium containing 20% FBS was added into the lower chamber. The plates were incubated at 37°C for 48 h. Non-invasive cells were removed using a cotton swab. Invasive cells were fixed with 100% methanol at room temperature for 10 min, stained with 1% crystal violet at room temperature for 10 min, washed at room temperature for three times and dried in air. Images of the stained cells were captured and counted under a microscope (magnification, ×200; IX53; Olympus Corporation, Tokyo, Japan) in three independent fields for each Transwell filter.

The target genes of miR-216b were predicted using PicTar (pictar.mdc-berlin. de/) and TargetScan (www.targetscan.org).

For the luciferase reporter assay, the pMIR-MTDH-3′UTR wild-type (Wt) and pMIR-MTDH-3′UTR mutant-type (Mut) reporter vectors were synthesized by Chang Jing Bio-Tech, Ltd. 293T cells were seeded into 24-well plates at a density of 50–60% confluence and co-transfected with an miR-216b mimic or miR-NC, and the pMIR-MTDH-3′UTR Wt or the pMIR-MTDH-3′UTR Mut, using Lipofectamine 2000. Following incubation for 48 h at 37°C in 5% CO₂ for 48 h, the luciferase activity was detected using the Dual-Luciferase^® Reporter Assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Transfected cells were harvested at 72 h post-transfection and total protein was extracted using a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), which contained a protease inhibitor. The concentration of total protein was measured using the bicinchoninic assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were loaded onto 10% SDS-PAGE gels, transferred onto polyvinylidene difluoride membranes (EMD Millipore), blocked with 5% skimmed milk with TBS containing 0.1% Tween-20 (TBST) at room temperature for 1 h and then incubated with the primary antibodies at 4°C overnight. The primary antibodies used in the present study included mouse anti-human MTDH monoclonal antibody (cat no. sc-517220; 1:1,000) and mouse anti-human GAPDH monoclonal antibody (cat no. sc-32233 1:1,000), rabbit anti-human polyclonal protein kinase B (AKT; cat no. sc-8312; 1:1,000), mouse anti-human monoclonal phosphorylated (p)-AKT (cat no. sc-514032; 1:1,000) and mouse anti-human monoclonal phosphatase and tensin homolog (PTEN; cat no. sc-7974; 1:1,000) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were washed with TBST three times and probed with the corresponding horseradish peroxidase-conjugated secondary antibody (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Finally, the protein bands were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.) and analyzed using AlphaEase™ FC software (version 4.0.1; ProteinSimple; Bio-Techne, Minneapolis, MN, USA). GAPDH was used as an internal control.

Statistical analyses were performed using SPSS (version 18.0; SPSS, Inc., Chicago, IL, USA). All results were expressed as the mean ± standard deviation or box plots. The Student's t-test and one-way analysis of variance (ANOVA) plus multiple comparisons were used to analyze significant differences between groups. The post hoc test used after ANOVA was Student-Newman-Keuls. The correlation between miR-216b expression and the clinicopathological factors was analyzed by the χ² test. Spearman's rank correlation coefficient analysis was adopted to evaluate the association between miR-216b and MTDH mRNA expression. P<0.05 was considered to indicate a statistically significant difference.

miR-216b expression was measured in glioma tissues and matched normal tissues using RT-qPCR. The results demonstrated that the expression level of miR-216b was significantly decreased in the glioma tissues compared with the matched normal tissues (P<0.05; Fig. 1A). miR-216b expression in five glioma cell lines (U87, U251, U373, LN18 and A172) and primary NHA was examined. As presented in Fig. 1B, all glioma cell lines exhibited reduced expression of miR-216b in comparison with NHA (P<0.05). These results supported the hypothesis that reduced miR-216b may be involved in glioma formation and progression.

The association between miR-216b expression and the clinicopathological features of patients with glioma was analyzed. All glioma tissue samples were divided into two subgroups according mean value (0.64); a low miR-216b group (27 cases) and a high miR-216b group (21 cases). As demonstrated in Table I, low expression of miR-216b was associated with a low KPS (P=0.018) and WHO grade (P=0.022) in patients with glioma. However, no association was observed between miR-216b expression and the sex (P=0.658) or age (P=0.715) of the patients.

To investigate the effects of miR-216b on glioma initiation and progression, an miR-216b mimic was introduced into U87 and U373 cells. At 48 h post-transfection, RT-qPCR was performed to evaluate the efficiency of overexpression and it was demonstrated that miR-216b was upregulated in U87 and U373 cells following transfection with an miR-216b mimic compared with cells transfected with miR-NC (P<0.05; Fig. 2A and B). An MTT assay was conducted to investigate the effect of miR-216b on glioma cell proliferation. As demonstrated in Fig. 2C and D, ectopic expression of miR-216b suppressed U87 and U373 cells proliferation (P<0.05). A Transwell invasion assay was used to evaluate the roles of miR-216b on glioma cell metastasis. The results indicated that expression of miR-216b decreased the invasive ability of U87 and U373 cells (Fig. 2E, P<0.05). These results suggested that miR-216b exerts a tumor suppressor role in glioma.

Based on bioinformatic analysis using PicTar and TargetScan, a potential list of target genes was predicated. Among these genes, MTDH was of interest (Fig. 3A). MTDH is upregulated in glioma (21) and involved in glioma occurrence, and development (22) indicating that MTDH may be a direct target of miR-216b in glioma. To confirm whether MTDH was a direct target of miR-216b, a luciferase reporter assay was performed. 293T cells were transfected with luciferase reporter vectors, together with an miR-216b mimic or miR-NC. The results demonstrated that the luciferase activity was reduced by the co-transfection with an miR-216b mimic and pMIR-MTDH-3′UTR Wt (P<0.05; Fig. 3B); however, co-transfection of an miR-216b mimic and pMIR-MTDH-3′UTR Mut did not affect the luciferase activity in 293T cells. To further determine the regulatory effects of miR-216b on endogenous MTDH expression, RT-qPCR and western blotting was performed in U87 and U373 cells following transfection with an miR-216b mimic or miR-NC. As demonstrated in Fig. 3C and D, upregulation of miR-216b suppressed MTDH mRNA and protein expression in U87 and U373 cells (all P<0.05). These results demonstrated that MTDH is a direct target gene of miR-216b in glioma.

As miR-216b was lowly expressed in glioma tissues and miR-216b inhibited glioma cell proliferation and invasion by negative regulation of MTDH, it was hypothesized that the expression level of miR-216b may be inversely correlated with miR-216b expression in glioma tissues. MTDH expression was examined in glioma tissues and matched normal tissues using RT-qPCR. As demonstrated in Fig. 4A, elevated expression of MTDH was observed in glioma tissues compared with the matched normal tissues (P<0.05). Furthermore, Spearman's rank correlation coefficient analysis indicated an inverse correlation between miR-216b and MTDH mRNA expression in glioma tissues (P=0.001; r=-0.4597; Fig. 4B). These results confirmed that MTDH is a direct target of miR-216b in glioma.

As MTDH was identified as a direct target of miR-216b, the roles of MTDH in cell proliferation and invasion in glioma were investigated. MTDH expression was silenced using MTDH siRNA and confirmed by western blotting (P<0.05; Fig. 5A). MTT and Transwell invasion assays were used to assess the effects of MTDH-knockdown on cell proliferation and invasion in glioma. The results demonstrated that the inhibition of MTDH expression by transfection with MTDH siRNA decreased U87 and U373 cell proliferation (P<0.05; Fig. 5B and C) and invasion (P<0.05; Fig. 5D). These results suggest that miR-216b inhibited glioma cell proliferation and invasion, partially through downregulation of MTDH expression.

Previous studies demonstrated that MTDH could negatively regulate PTEN expression via blocking its transcription (23,24). Therefore, PTEN, AKT and p-AKT expression was measured in U87 and U373 cells following transfection with an miR-216b mimic or miR-NC. As demonstrated in Fig. 6, upregulation of miR-216b enhanced PTEN expression and reduced p-AKT expression, whereas miR-216b did not affect AKT expression in U87 and U373 cells (P<0.05). These results indicated that miR-216b is involved in PTEN/AKT signaling pathway through regulation of MTDH.